Noninvasive focused ultrasound non-specifically activating spleen immunological function to suppress tumor proliferation: a new strategy for cancer immunotherapy

Noninvasive focused ultrasound non-specifically activating spleen immunological function to suppress tumor proliferation: a new strategy for cancer immunotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Noninvasive focused ultrasound non-specifically activating spleen immunological function to suppress tumor proliferation: a new strategy for cancer immunotherapy Wei Dong, Guihu Wang, Senyang Li, Qian Wang, Wenjuan Li, Heyuan Liu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4639146/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Splenic immunomodulation triggered by ultrasound shows a significant anti-inflammatory effect against various inflammatory diseases, whose mechanism is mainly attributable to the activation of cholinergic anti-inflammatory pathway (CAP). However, the potential role and underlying mechanism of splenic ultrasound stimulation in cancer management have been rarely reported and superficially defined. This study first assessed the universal therapeutic effect of focused ultrasound precisely stimulating spleen (FUS sti. spleen) on various tumors under specific ultrasonic dose. It fully demonstrated that FUS directly stimulated splenic immune cell proliferation and activation (especially NK and CD8 T cells) rather than CAP excitation to modulate splenic immune function. Particularly, NK cells are much more indispensable and important in responding to FUS stimulation for cancer suppression than CD8 T cells. RNA sequencing of NK and CD8 T cells, as well as in vitro experiments revealed that FUS firstly regulated calcium-related signaling pathways to further modulate other signaling pathways, such as ECM-receptor interaction, PI3K-AKT signaling pathway, Rap1 signaling pathway and Hippo signaling pathway to promote immune cell proliferation, migration and activation to suppress cancer cell deterioration. Particularly, FUS sti. spleen and FUS intervention on the tumor synergistically induced the best tumor suppression than each of the two taken individually. focused ultrasound spleen FUS sti. spleen splenic immunomodulation tumor suppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1 Introduction The spleen is the largest secondary lymphoid organ, and an extensive meshwork of nerve fibers is distributed throughout the splenic compartments including the splenic nodules, periarteriolar lymphoid sheath, marginal zones, trabeculae, white pulp and red pulp [ 1 , 2 ]. Even more striking, the nerve fibers are co-localized with various subsets of dendritic cells (DCs), macrophages (Mφ), and lymphocytes (i.e. B cells and T cells) [ 2 , 3 ]. As early as 30 years ago, some studies demonstrated that sympathetic nerve terminals in the spleen secrete norepinephrine (NE) interacting with T cells in response to stimulation, while the content of splenic norepinephrine significantly decreases following chemical or surgical sympathectomy [ 4 , 5 ]. It was not until around 2000 that the cholinergic anti-inflammatory pathway (CAP) concept was formally proposed and extensively studied because of its vital function in modulating the mammalian immune response [ 6 – 10 ]. The CAP mechanism relies on the parasympathetic vagus nerve transmitting signals from the brain to the adrenergic splenic nerve that interacts with immune cells (Supplementary Fig. 1). Munyaka P, et al. [ 11 ] revealed that CAP is activated after central cholinergic activation by intracerebroventricular infusion of the M1 muscarinic acetylcholine (ACh) receptor agonist in mice but is suppressed by vagotomy or splenic neurectomy. Moreover, Ji H, et al. [ 12 ] proved that central cholinergic activation induced by the acetylcholinesterase inhibitor galantamine or the muscarinic ACh receptor agonist relieves colitis in mice, which is inhibited after vagotomy, splenic neurectomy or splenectomy. These reports indicate that spleen is a vital target of the CAP. In the past decade, many researchers applied physical methods to trigger the splenic neural-immune reflex for regulating the immune response to chronic inflammatory diseases [ 13 ]. Inoue T, et al. [ 14 ] utilized a bipolar silver wire electrode stimulating the isolated left cervical vagus nerve to attenuate kidney ischemia-reperfusion injury (IRI), suggesting that vagus nerve stimulation (VNS)-mediated IRI treatment and systemic inflammation depend on α7 nicotinic acetylcholine receptors (α7nAChR)-positive splenocytes. Koopman F A, et al. [ 15 , 16 ] reported that stimulation of the CAP by VNS to treat rheumatoid arthritis (RA) in experimental models and clinical trials exerts a significant mitigation effect, whilst the symptoms aggravated following unilateral cervical vagotomy or α7nAChR knockout in mice. Besides, Meregnani J and colleagues [ 17 ] demonstrated that the symptoms of colonic colitis in rats induced by trinitrobenzene sulphonic acid was significantly reduced after VNS performed with electrode cuffs for 5 days. Moreover, electroacupuncture intervention eliciting spenic efferent vagus nerve activity not only reduced tumor proliferation in breast tumor-bearing mice by alleviating inflammation and enhancing antitumor immunity [ 18 ], but also could alleviate the severity of chemotherapy-induced nausea and vomiting in patients with advanced cancer [ 19 , 20 ]. In addition, bioelectronic devices for VNS in clinical application have already been approved by the European Medicines Agency and the US Food and Drug Administration (FDA) for the treatment of drug-refractory epilepsy and depression [ 13 , 21 , 22 ]. Unsatisfactorily, nerve stimulation using permanently implanted electrodes, transcutaneous bioelectronic devices, or slightly injurious electroacupuncture was limited to stimulate large nerves or superficial nerves [ 23 ]. Peripheral organs, not just the spleen, are extensively traversed by peripheral nervous system, which makes it difficult to selectively stimulate subsets of axons that terminate in spleen and uniquely modulate the immune response to inflammatory diseases [ 23 ]. For example, cervical nerve stimulation usually activates the extensive efferent and afferent neural pathways that elicit neural responses from non-target organs [ 13 , 16 , 24 – 26 ]. Although scientists are committed to developing more advanced, miniaturized and sophisticated electrode designs for implantation on specific nerves near the target organ [ 27 , 28 ], invasive implantation easily causes damage to the delicate nerve fibers of the target organ and it is difficult to stimulate nerves throughout the organ. Thus, new nonintrusive strategies are imperative to trigger specific nerves, especially the splenic nerve, and mobilize the splenic immune function against diseases. Notably, recent studies showed that noninvasive ultrasound successfully elicits peripheral nerve activation in the spleen to treat chronic inflammatory diseases (Supplementary Fig. 1). Gigliotti JC and co-workers [ 29 ] used an ultrasound imaging probe to successfully irradiate the neural innervation in the spleen mitigating IRI in mice, whose mechanism is mainly attributable to the CAP activation [ 6 , 9 , 16 , 24 ]. Later, Cotero V, et al. [ 30 ] reported that ultrasound modulating splenic nerve reduces endotoxin-induced cytokine production at a level comparable with traditional VNS performed with electrodes. Zachs DP, et al. [ 31 ] applied ultrasound stimulation targeting the spleen, which significantly reduces inflammation severity in RA mouse models. These studies provide ample evidence on the potential of precise splenic ultrasound stimulation to replace bioelectronic devices for the translation of peripheral neuromodulation-based therapies for inflammatory disease. However, the potential roles and underlying mechanisms of splenic ultrasound stimulation in cancer management have been rarely reported and superficially defined. Further investigation is needed to establish whether ultrasound directly regulates the immunomodulatory function of the spleen by interfering with splenic immune cells and microenvironment. Ultrasound targeting the spleen is crucial in achieving conspicuously therapeutic effects, since the stimulation of other body locations or the lack of immune cells in mice is noneffective, which suggests the indispensability and importance of immune cells [ 29 , 31 ]. Cotero V, et al. [ 30 ] and Zachs DP, et al. [ 31 ] concluded that ultrasound activating CAP indirectly exerts an influence on splenic immune cells or cytokines to reduce the severity of lipopolysaccharide acute inflammation and RA, but they mainly focused on the analysis of neuro-immune mechanism and did not perform a systematic research on the changes of splenic immune cells in response to ultrasonic stimulation. Generally, ultrasonic irradiation on cells directly irritates the expression of multiple key genes/signaling pathways or interferes with the secretion of cytokines to regulate cell proliferation, differentiation and migration, such as stimulating the expression of vascular endothelial growth factor in several cells including endothelial cells, neural cells and ischemic cells [ 32 – 35 ]. Therefore, we hypothesized that ultrasound could effectively immunomodulated on the splenic immune cells responding to cancer cell antigens in splenic microenvironment, and enhance the proliferation, activation, migration, and information dissemination of immune cells during cancer immunotherapy. Accordingly, this study was designed to explore whether focused ultrasound precisely stimulating spleen (FUS sti. spleen) was effective in suppressing tumor proliferation, and the underlying mechanisms regulating splenic ultrasound stimulation in cancer management, including the activation of splenic nerve related CAP, and splenic immune cells responding directly to ultrasonic capabilities. The therapeutic efficacy of FUS sti. spleen was first assessed on various tumors under specific parameters screened before. Furthermore, the splenic immunomodulation through FUS sti. spleen was evaluated based on the changes of splenic immune cell population and cytokine levels. Subsequently, nerve blockade, immune cell clearance and RNA sequencing were performed to identify the underlying mechanisms of splenic ultrasound stimulation in cancer management, such as splenic nerve related CAP activation and FUS directly modulating immune cells, and the related molecular mechanism. 2 Methods 2.1 Cell lines and animal models HCC cell lines (H22 and hepa1-6) and 4T1 breast cancer cell line were purchased from the American Type Culture Collection (ATCC) and used for subsequent analysis. H22 cells were reproduced by ascites after intraperitoneal injection in C57black/6 mice (0.5–1.5*10^7 cells/mL; 200 µL per mouse). Hepa1-6 cells and 4T1 cells were cultured in Dulbecco’s modified Eagle’s medium ( Cat. No. 11320033, Gibco, Invitrogen, Carlsbag, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. A5669701, Gibco, Invitrogen, Carlsbag, USA) and 1% penicillin-streptomycin solution (Cat. No. 15140122, Gibco, Invitrogen, Carlsbag, USA), and incubated at 37°C under 5% CO 2 and 100% humidity. All animal experiments were performed in accordance with the institutional guidelines and approved by the Animal Experimentation Ethics Committee of Xi’an Jiaotong University. Seven-week (weighted ~ 17 g) C57black/6 mice, purchased from the Beijing Animal Experiment Center (Chinese Academy of Sciences, Beijing, China), were housed at 24 ± 2°C under a 12-h light/dark cycle and acclimatized for at least 1 week before the experiments. All animals had access to sterilized food and water ad libitum. An amount of 0.2 mL H22 cancer cells (approximately 1–3*10^6 cells/mL) or 4T1 cancer cells (1–3*10^6 cells/mL) was hypodermically injected into the dorsal hindlimb region of the mouse to establish a subcutaneous tumor model. As regards the in situ xenograft HCC models, an incision of approximately 1 cm in length was made at the upper end of the midabdominal line after the mouse was anesthetized with isohalothane and fixed on the anatomic stage. A total of 25 µL H22 cancer cells (approximately 0.5–1.5*10^7 cells/mL) or 50 µL hepa1-6 cancer cells (approximately 4–6*10^6 cells/mL) was injected into left hepatic lobe with an insulin syringe, and the incision was closed after applying pressure on the pinhole with a medical cotton swab for 3–4 minutes. The tumor volume was calculated as follows: volume = 0.5*L*W^2, where L and W were the length and width of the tumor, respectively, measured by a caliper. The weight of spleen, tumor and mice was assessed by electronic scales. The splenic index was calculated by the ratio of spleen weight to mouse weight. 2.2 Ultrasonic platform set-up and application for spleen stimulation A diagram of the FUS system is shown in Supplementary Fig. 2A. A function generator (Cat. No. 33120A, Agilent, Santa Clara, USA) produced a pulsed sinusoidal waveform triggering the power amplifier (Cat. No. AG1019, California, USA; or RPR-4000, RITEC Inc., Warwick, RI, USA) to drive a 1.04 MHz FUS transducer with a 100 mm aperture and 65 mm focus, whose focal region was ~ 1.4*1.4*8.6 mm 3 (Supplementary Fig. 2B-C). The acoustic pressure and spatial beam profile of the FUS transducer were measured using a hydrophone (Cat. No. HNR-1000, ONDA, Videlles, France). The FUS transducer was mounted on a XYZ motorized positioning stage to control ultrasonic duration, and the position of the FUS focus relative to the mice was adjusted under a B-mode imaging guidance with a 3.2 MHz phased array positioned at the center of the FUS transducer. The mice were anesthetized with 2–3% inhaled isoflurane and placed on a manual translation stage equipped with heating pad. Then, a centrifuged coupling gel was immediately applied to the shaved skin, and the manual translation stage was adjusted to allow the spleen (or tumor) site to be in tight contact with the bottom of the water tank under the B-mode imaging guidance (Supplementary Fig. 2D). The FUS transducer was moved across the whole spleen (or tumor) through the XYZ programmable logical controller (Supplementary Fig. 2E). 2.3 Flow cytometry Single-cell suspensions from the spleen, peripheral blood, tumor, and para-carcinoma tissue were obtained after red blood cells were lysed using lysis buffer (Cat. No. 555899, BD Bioscience, USA) according to the manufacturer’s instructions. Cell suspensions were washed with sterilized PBS, and then incubated with the antibodies listed in Supplementary Table 1, and the scheme of antibody labeling for each immune cell is shown in Supplementary Table 2. Next, the immune cells were counted by flow cytometry (FCM) using CytoFLEX LX (Beckman Coulter Life Sciences, USA) after filtration through a 70-µm nylon cell strainer (Cat. No. 352350, Corning, USA). Data were analyzed using the FlowJo software (FlowJo 10, LLC, Ashland, OR). 2.4 Cytokine measurement 2.4.1 Luminex assay Cytokine/chemokine quantification in plasma was performed by Luminex xMAP technology using a magnetic Luminex assay (R&D Systems, Minneapolis, MN, USA). The quantification was carried out using a Luminex® 200 Flow Cytometry System (Cat. No. x-200, Thermo Fisher Scientific, MA, USA) and Milliplex Analyst software (Version 5.1, Merck Millipore, MA, USA). 2.4.2 ELISA The concentration of NE, ACh, granzyme and perforin in plasma, respectively, detected by enzyme-linked immunosorbent assay (ELISA). The operation processes were carried out in strict accordance with the instruction manual from Mouse Perforin ELISA Kit (Cat. No. F30718-A, FANKEW, Shanghai, China), Mouse Granzyme-B ELISA Kit (Cat. No. F3214-A, FANKEW, Shanghai, China), Mouse ACh ELISA Kit (Cat. No. MU30072, Bioswamp, Wuhan, China) and Mouse NE ELISA Kit (Cat. No. MU30372, Bioswamp, Wuhan, China), respectively. 2.5 Histological and immunohistochemical staining Hematoxylin eosin (HE) staining was applied to evaluate the pathological changes of the spleen after FUS irradiation. Transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL) staining was performed to observe splenic cell apoptosis through the colorimetric TUNEL Apoptosis Assay Kit (Cat. No. C1091, Beyotime, Shanghai, China). The primary antibodies of anti-Ki67 (Cat. No. RM9106S1, Thermo Fisher Scientific, MA, USA; dilution 1:200), anti-NK1.1 (Cat. No. 108759, BioLegend, California, USA), anti-CD8a (Cat. No. ab4055, Abcam, Cambridge, UK; dilution 1:1000) and anti-c-Fos (Cat. No. ab222699, Abcam, Cambridge, UK; dilution 1:2000) were used for immunohistochemical staining to detect the protein expression of cyclin-D1, Ki67, NK1.1, CD8a and c-Fos respectively. At least 5–10 different regions in each section were randomly selected for image acquisition using a fluorescence microscopy (observer3, Carl Zeiss, Jena, Germany), and the positive area ratio was quantified using the Image Pro Plus 6.0 software (Media Cybernetics, CA, United States). 2.6 RNA sequencing CD8 T cells (CD45 + CD3 + CD8a + ) and NK cells (CD45 + CD3 − NK1.1 + ) were sorted from the spleen using the Beckman Kurt MoFlo Astrios ultra high speed flow cytometry sorting system (MoFlo Astrios EQ, Beckman Coulter, lnc, USA). Subsequently, general transcriptome sequencing was performed when the positive cell rate was greater than 90% detected by flow cytometry (CytoFLEX LX, Beckman Coulter Life Sciences, USA). The cDNA library construction and sequencing of all RNA samples were performed by Shanghai OE Biotech Co., LTD. Transcriptome sequencing was performed to screen differentially expressed genes (DEGs). Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to annotate the function of the DEGs. The DEG screening criteria were as follows: P < 0.05 and differential fold change |logFC| ≥ 1. 2.7 Nerve signal blockade 2.7.1 Atomoxetine hydrochloride blocking CAP Atomoxetine hydrochloride (Cat. No. abs813490, Absin, Shanghai, China) was intraperitoneally administered at a dose of 3 mg/kg to directly inhibit the NE transporter at 1.5 hours before FUS exposure to spleen according to previous reports [ 36 , 37 ], which ensured FUS sti. spleen at the optimal time point of CAP blockade. 2.7.2 Absolute ethanol-induced splenic nerve block Absolute ethanol was used for splenic nerve block according to previous studies [ 38 – 43 ]. Briefly, the mice were anesthetized with the isoflurane, and disinfection with iodophor was performed on the shaved skin. A 7–10 mm incision was made next to the left side of the spleen. The spleen was isolated with tweezers to keep it away from the peritoneal cavity for a clear exposure of the three main blood supply vascular trees. A wet cotton was used to protect the peritoneal cavity and other organs. Subsequently, absolute ethanol was repeatedly applied to those vascular trees with a cotton tip for 5–10 seconds each time, with an interval of five seconds, for a total of seven times to block the splenic nerve fibers. Excessive ethanol dripping from the cotton tip was carefully avoided, to avoid significant vasospasm, which could lead to permanent damage to blood vessels, consequently leading to spleen necrosis and organ complete absorption. As regards the sham-operated mice, the same entire surgical procedure was performed except for the repeated application of anhydrous ethanol that was replaced by normal saline (pH = 7.4). 2.8 In vivo immune cell depletion The experimental C57BL/6 mice were treated with an intravenous injection of Ultra-LEAF™ purified anti-mouse NK1.1 antibody (Cat. No. 108759, BioLegend, California, USA) or Ultra-LEAF™ purified anti-mouse CD8a antibody (Cat. No. 100763, BioLegend, California, USA) at a dose of 50 µg/mouse on day 0, 3, 7, 12 and 17 to deplete NK cell or CD8 T cell. As regards MDSC depletion, the Ultra-LEAF™ Purified anti-mouse Ly-6G antibody (Cat. No. 127650; BioLegend, California, USA) was intraperitoneally administered to C57BL/6 mice at a dose of 0.25 mg/mouse on day 0, 6, 12, and 18. The cell clearance efficacy was assessed by FCM. 2.9 Calcium detection and in vitro anticancer experiment of FUS stimulating splenic CD8 T cells or NK cells enhanced by calcium 2.9.1 Von Kossa staining The splenic specimens were cut into 5 µm-thick sections and subjected to VON KOSSA Calcium Staining Kit (Cat. No. JM1519; HPBIO, Shanghai, China) for the histological visualization of calcium deposits (mineralization). Calcium deposition in splenic cells was assessed by fluorescence microscopy (Zeiss observer3), where mass deposits appeared black, while dispersed deposits appeared gray. The positive area ratio was quantified using the Image Pro Plus 6.0 software. 2.9.2 FCM detection of Fluo-4 AM labeled cells The splenic cell suspensions were washed twice with sterilized PBS, and then incubated with Fluo-4 AM dye (1 mL dye/10 6 cells; Cat. No. S1061M, Beyotime, Shanghai, China) for 30 min at 37 ℃ according to the manufacturer’s instructions. Next, the fluorescent cells were monitored by FCM using the CytoFLEX LX (Ex/Em = 490/525 nm). 2.9.3 Calcium strengthened FUS stimulating splenic NK cells against cancer The NK cells were sorted from the spleen using the Beckman Kurt MoFlo Astrios ultra high speed flow cytometry sorting system, and then subjected to FUS stimulation with or without high calcium concentration (300 nM). Then, NK cells were co-cultured with pEGFP-C1 plasmid-transfected Hepa1-6 cancer cells at a ratio of 1:1, where GFP fluorescent protein was used to distinguish cancer cells from splenic cells. Transwell devices (Corning Incorporated, NY, USA) with 3-µm diameter holes were used to distinguish immune cells in the superstratum and cancer cells in the substratum. The cells were co-cultured for 48 h before GFP fluorescence observation with the Zeiss observer3, CCK-8 assay (Cat. No. ABS50003-500T, Univ, China), and crystal violet (CV) staining to assess cancer cell suppression and immune cell proliferation. The tumoral background was mimicked by adding ultrasound shattered cancer cell suspension to the NK cells solution during FUS stimulation. 2.10 Statistical Analysis Statistical analysis was performed using the statistical product and service solutions software (SPSS, USA). Statistical significance was analyzed using Student’s t -test with Bonferroni’s correction, the mean was calculated from at least four independent experiments, and error bars represent the standard error of mean. A value of p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, and ***p < 0.001). 3 Results 3.1 Screening and optimization of ultrasonic parameters for spleen stimulation In view of the different ultrasonic parameters and platforms applied in previous studies of splenic ultrasound stimulation, it is pseudoscientific to indiscriminately imitate their ultrasonic parameters or methods to this study for regulating splenic immunotherapy to anti-cancer. Based on the pre-experiment results, 11 processing groups were set-up: G1-G11 group (detailed in Table 1), for the screening and optimization of ultrasonic parameters, and FUS sti. spleen in subsequent experiments was performed once every other day starting from the 2nd day after cancer cell implantation (Figure 1 A). The degree of subcutaneous H22 tumor suppression and spleen injury (assessed by HE and TUNEL staining) was used as the decisive criterion to establish the appropriate ultrasonic parameters. The phenotypic results showed that FUS sti. spleen significantly suppressed the proliferation of subcutaneous H22 tumor, especially in the G4, G5, G8 and G11 group (Figure 1 B-D). The weight of the mice subjected to FUS sti. spleen was lower than that of the non-FUS mice (Figure 1 E), and FUS sti. spleen showed a tendency to decrease splenomegaly (Figure 1 F-G). By comparison, the increase of the duty cycle or the FUS exposure time at 1.8 MPa did not improve the anti-tumor effect (G3, G6, G9 and G10 group; Figure 1 B-D). However, the high peak negative pressure of 2.8 MPa induced a severe spleen injury, easily leading to the death of mice (G5 group; Figure 1 B, H-I). In contrast, FUS sti. spleen effectively suppressed tumor proliferation without damaging the splenic cells at 2.3 MPa (Figure 1 B-D, H-I). However, the antitumor effect was significantly reduced when the FUS exposure time of 20 s was shortened to 10 s (G4 and G7 group; Figure 1 B-D). FUS damaged a few splenic cells once increasing the exposure time of 20 s to 40 s or the duty cycle of 1% to 10% (G4, G8 and G11 group; Figure 1 B-D, H-I). The above splenic stimulation results demonstrated a clear “dose response” to ultrasound with a distinct power level required for the specific regulation of biophysical effects on splenic immune cells, which was similar to previous studies [23, 29, 31]. In conclusion, the ultrasonic parameters of the G4 group (2.3 MPa, 1%, 20 s) were selected for subsequent experiments of splenic immunomodulation for cancer therapy. Table 1 Scheme of different ultrasonic parameters for FUS sti. spleen. Groups Tumor Ultrasonic parameters Peak negative pressure (MPa) Duty cycle (%) Exposure time (s) G1 × × × × G2 √ × × × G3 √ 1.8 1 20 G4 √ 2.3 1 20 G5 √ 2.8 1 20 G6 √ 1.8 1 40 G7 √ 2.3 1 10 G8 √ 2.3 1 40 G9 √ 1.8 10 20 G10 √ 1.8 20 20 G11 √ 2.3 10 20 3.2 FUS universally activated spleen immunological function to suppress the proliferation of various tumors 3.2.1 FUS activated splenic immune cells to suppress the proliferation of the xenograft HCC in situ The mouse model of HCC in situ , which was better to resemble the situation of primary liver cancer, was used to assess the anticancer efficiency of FUS sti. spleen. The orthotopic HCC mice were subjected to FUS sti. spleen from day 0 after cancer cell implantation (Figure 2 A). Twenty orthotopic H22 HCC mice were randomly divided into two groups: the S1 group was used as the control group, and the S2 group was subjected to FUS sti. spleen. The results of visual observation, tumor volume and tumor weight demonstrated that FUS sti. spleen significantly suppressed the proliferation of xenograft H22 carcinoma in situ , with a tumor inhibition rate of up to ~70% as compared with the control group (S2 vs . S1, p ＜0.001; Figure 2 B-D). Although the mean value of liver weight (total weight of the tumor and liver parenchyma) showed a decreasing trend in the S2 group compared to the S1 group, no significant difference was observed between the two groups (Figure 2 E). In addition, the weight of the mice in the S2 group was lower than that of the mice in the S1 group ( p ＜0.001; Figure 2 F). The spleen weight and spleen index showed a decreasing trend of splenomegaly in the S2 group as compared with the S1 group, but without statistically significant difference (Figure 2 G-H). More importantly, the survival statistics proved that the orthotopic H22 HCC mice treated with FUS sti. spleen had a longer survival time than the control group (Figure 2 I). Sixteen orthotopic Hepa1-6 HCC mice were randomly divided into two groups: L1 group was performed as the control group, and L2 group was administered to FUS sti. spleen. The results of visual observation, tumor volume and tumor weight showed that FUS sti. spleen remarkablely inhibited the proliferation of xenograft Hepa1-6 carcinoma in situ , with an antitumor ratio of up to ~80% (L2 vs . L1, p ＜0.001; Figure 2 J-L). Additionally, the weight of the liver and mice in the L2 group was lower than that of the mice in the L1 group ( p ＜0.05; Figure 2 M-N). No significant difference in spleen weight and spleen index was observed, although there was a decreasing trend shown in the L2 group as compared with the L1 group (Figure 2 O-P). It was noteworthy that the survival time of the mice in the L2 group was significantly longer than that of the mice in the L1 group (Figure 2 Q). Thereafter, FCM results revealed that the proportion of tumor suppressor related immune cells in the spleen, blood, tumor and para-carcinoma tissue was significantly increased in the S2 group as compared with the S1 group, such as Th2 cells, NK cells, CD8 T cells, Mφ (Mφ2) and DC1 in the spleen; B cells, NK cells, CD8 T cells, Mφ1 and DC1 in the blood; CD4 T cells, NK cells, CD8 T cells, Mφ (Mφ1) and DC1 in the tumors; Th1 cells, NK cells, CD8 T cells and Mφ (Mφ1) in the para-carcinoma tissue (Table 2; Supplementary Figure 3-6). Moreover, the proportion of negative immune cells was significantly reduced, such as Treg cells and MDSCs (M-MDSCs and PMN-MDSCs) in both spleen and tumor; Th17, PMN-MDSCs in the blood; and PMN-MDSCs in the para-carcinoma tissue (Table 2; Supplementary Figure 3-6). As regards to orthotopic Hepa1-6 HCC mice, FCM results showed that the number of positive anticancer immune cells in the spleen, blood, tumor and para-carcinoma tissue was significantly increased in the L2 group than in the L1 group, such as Th2 cells, NK cells, CD8 T cells, Mφ and DCs in the spleen; CD4 T cells, Th2 cells, NK cells, CD8 T cells, Mφ1, Mφ2 and DCs in the blood; NK cells, CD8 T cells, Mφ and DCs in the tumor; and NK cells, CD8 T cells and Mφ (Mφ1 and Mφ2) in the para-carcinoma tissue (Table 2; Supplementary Figure 7-10). The number of negative immune cells was significantly reduced, such as Treg cells and PMN-MDSCs in the spleen; Th17 and MDSCs in the blood; Th17 and PMN-MDSCs in the tumor; and PMN-MDSC in the para-carcinoma tissue (Table 2; Supplementary Figure 7-10). These results fully demonstrated the significant effect of FUS on splenic immunomodulation for cancer immunotherapy. Particularly, FUS sti. spleen remarkably increased the proportion of NK cells and CD8 T cells in the spleen, blood, tumor and para-carcinoma tissue, followed by Mφ and DCs, whilst it reduced the number of PMN-MDSCs in all tissues with one accord, and then Treg cells. Table 2 Statistical significance of FCM results of immune cells in the spleen, blood, tumor and para-carcinoma tissue from orthotopic HCC mice subjected to FUS sti. spleen or non-treated mice. S1, control group of xenograft H22 HCC in situ ; S2, FUS sti. Spleen-treated orthotopic H22 HCC. L1, control group of xenograft Hepa1-6 HCC in situ ; L2, FUS sti. Spleen-treated orthotopic Hepa1-6 HCC. The red asterisk indicates a significant increase in the proportion of immune cells; the blue asterisk indicates a significant decrease in the proportion of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the S1 and S2 group are shown in Supplementary Figure 3-6, and those in the L1 and L2 group are shown in Supplementary Figure 7-10. Analysis CD4 T cells Th1 cells Th2 cells Th17 cells B cells NK cells CD8 T cells Mφ Mφ1 Mφ2 DC1 DC2 Treg cells M- MDSCs PMN- MDSCs Spleen S 2 vs. S1 — ** * — — * * * * * * — *** * * B lood S2 vs. S1 — — — * * ** * — ** — ** — — — ** Tumor S2 vs. S1 ** — — — — ** * ** * — * — * — * Liver S2 vs. S1 — * — — — * ** *** *** — — — — — * Spleen L2 vs. L1 — — ** * — ** * ** — — *** *** * — * B lood L2 vs. L1 * — * * — * ** — ** * ** — — * * Tumor L2 vs. L1 — — — ** — ** * * * * * ** * — ** Liver L2 vs. L1 — — — — — * ** * ** * — — — — * 3.2.2 FUS activated spleen immunological function to suppress proliferation of the subcutaneous 4T1 breast tumor To further verify the universality of FUS sti. spleen as a non-specific strategy for modulating splenic immune function to suppress tumor progression, another model of subcutaneous 4T1 breast tumor was also used to confirm the anti-cancer effect of FUS sti. spleen. The subcutaneous 4T1 tumor mice were subjected to FUS sti. spleen also from day 0 after cancer cell implantation as described above (Figure 1 A; Figure 2 A), except that the experimental period was set to 34 days. Next, twelve subcutaneous 4T1 breast tumor mice were randomly divided into two groups: the P1 group was used as the control group, and the P2 group was subjected to FUS sti. spleen. The results of visual observation, tumor volume and tumor weight demonstrated that FUS sti spleen significantly suppressed the proliferation of 4T1 breast tumor, with a ~70% of tumor inhibition rate (P2 vs . P1, p ＜0.001; Figure 3 A-C). Moreover, a significant pathological change in the lungs of the P1 group was observed, but the degree of pulmonary lesions in the P2 group was significantly reduced ( p ＜0.01; Figure 3 D). During the experimental period, the P1 group showed a decrease in body weight, while that of the P2 group slightly increased without significant difference compared to the P1 group (Figure 3 E), which was most likely due to the poor health status caused by the metastasis of 4T1 cancer cells to the lungs. Notably, the spleen weight and splenic index in the P2 group showed that FUS sti spleen significantly reduced splenomegaly compared to the P1 group ( p ＜0.01; Figure 3 F-G). Importantly, the survival time of subcutaneous 4T1 breast tumor mice in the P2 group was significantly longer than that in the P1 group (Figure 3 H). FCM results showed that the change of various immune cell proportion was basically consistent with that in the orthotopic HCC mice experiment. In detail, the proportion of NK cells and CD8 T cells in the P2 group was significantly increased in the spleen, blood, tumor, and lung as compared with the P1 group, and PMN-MDSCs was decreased except in the tumor (Table 3; Supplementary Figure 11-14). Moreover, the number of other tumor suppressor related immune cells was significantly increased in the spleen, blood, tumor or lung of the P2 group compared to that in the P1 group (such as Mφ (Mφ1/2) and DC1 in the spleen and blood; Mφ (Mφ2) and DCs in the tumor; and Mφ (Mφ1) and DC1 in the lung (Table 3; Supplementary Figure 11-14). These results further confirmed that FUS sti. spleen universally activated spleen immunological function by upregulating tumor suppressor-related immune cells and inhibiting negative immune cells, especially in the significant modulation of NK cells and CD8 T cells, to suppress the proliferation of various tumors. While FCM confirmed the proliferation of NK cells and CD8 T cells in the spleen following FUS stimulation, immunohistochemical staining of Ki67, NK1.1, and CD8a also indicated enhanced cell proliferation and especially an increase in the number of NK cells and CD8 T cells (Figure 3 I). Table 3 FCM results of various immune cells in the spleen, blood, tumor, and lung tissues from subcutaneous 4T1 tumor-bearing mice subjected to FUS sti. spleen or non-treated mice. P1, control group of subcutaneous 4T1 breast tumor; P2, FUS sti. spleen on day 2 after 4T1 cancer cell subcutaneous implantation. The red asterisk indicates a significant increase in the proportion of immune cells; the blue asterisk indicates a significant decrease in the proportion of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the P1 and P2 group are shown in Supplementary Figure 11-14. Analysis NK cells CD8 T cells Mφ Mφ1 Mφ2 DC1 DC2 Tregcells M- MDSCs PMN-MDSCs S pleen P2 vs. P1 * *** ** *** ** *** * — — ** B lood P2 vs. P1 ** * * * ** ** * * — * T umor P2 vs. P1 ** * ** — * * ** — — — L ung P2 vs. P1 ** * ** ** — — — — — ** 3.2.3 FUS sti. spleen altered the cytokine levels In addition to the modulation of cellular immunity by FUS sti. spleen, humoral immunity may also be significantly regulated, which represents one dominant factor in the anticancer process during FUS irradiation. Herein, we selectively detected the cytokine levels of GM-CSF, TNF-alpha, IL-12, CCL2, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17, IFN-gamma, CXCL10, M-CSF, IL-1alpha, CCL4, CXCL12, IL-27, perforin and granzyme in the peripheral blood using the Luminex xMAP technology. Interestingly, the concentration levels of TNF-alpha, IFN-γ, perforin and granzyme were significantly increased in the FUS sti. spleen group as compared with the control group in both H22 and Hepa1-6 HCC in situ models (Figure 4 A-C), which indicated the activation of cytotoxic CD8 T cells and NK cells against tumor. Usually, the naive CD8 T cells in the immune process undergo activation and clone expansion, which in turn produce the effector cytokine TNF-alpha, IFN-gamma, perforin and granzyme [44, 45]. In addition, the secretion level of IFN-gamma, perforin and granzyme specifically indicated the activation of NK cells to fight cancer [46]. The concentration levels of the pro-inflammatory chemokines CCL2, CCL4 and CXCL10 were significantly elevated in the FUS sti. spleen group of both orthotopic H22 and Hepa1-6 HCC models compared with those in the control group (Figure 4 A-C). These pro-inflammatory chemokines promote immune cells especially effector T cells successfully migrating into metastatic tumor sites to perform an anti-tumor activity [47-49]. CXCL12 is a homeostatic chemokine involved in physiological processes like embryogenesis, neurogenesis, cardiogenesis, hematopoiesis, leukocyte homing, and angiogenesis, by inducing the migration and activation of hematopoietic progenitor and stem cells, endothelial cells, and most leukocytes [50, 51]. In this study, the CXCL12 level was significantly increased in the FUS sti. spleen group (Figure 4 A-C). As a final level of activity regulation, CXCL12 synergizes with CXCL8 but also with CXCL9, CXCL10, CXCL11, and multiple CC chemokines to attract B- and T-lymphocytes, DCs, monocytes, and CD34+ progenitor cells [52]. Moreover, CXCL12 binding to its receptor CXCR4 mediates the biological behavior of tumor cells such as movement, chemotaxis, adhesion, secretion (such as MMPs and VEGF), angiogenesis, growth, and proliferation by activating various signaling pathways [53, 54]. Besides, GM-CSF and M-CSF are 2 important cytokines that regulate macrophage population and function. Many experimental loads of evidence suggest that the aberrant expression of GM-CSF and its receptors are present in many cancers such as glioblastoma, small cell carcinoma, skin carcinoma, meningiomas, colon cancer, lung cancer, and particularly HCC, promoting cancer progression by regulating the tumor microenvironment involving Mφ and MDSCs, and promoting epithelial to mesenchymal transition, angiogenesis, and expression of immune checkpoint molecules [55-58]. The concentration level of GM-CSF in this study was significantly decreased as both orthotopic H22 and Hepa1-6 HCC mouse models underwent FUS sti. spleen (Figure 4 A-C). In addition, the M-CSF level was increased after orthotopic HCC mice were subjected to FUS sti. spleen (Figure 4 A-C), which regulated the survival, proliferation and differentiation of the monocyte-macrophage lineage from progenitors to mature cells and activated several important functions of mature tissular Mφ [59, 60]. The proinflammatory cytokine IL-1beta showed an association with tumor progression [61], but interestingly, although the inhibition of IL-1beta signaling has potent anti-metastatic effects, the inhibition of the activity of this cytokine has contradictory effects on primary tumors, sometimes reducing but often promoting their growth [62]. Moreover, anti-cancer treatments are able to promote IL-1beta production by cancer or immune cells, with opposite effects on cancer progression [63], which resembles the tumor-suppressing effect of FUS sti. spleen. However, beyond that, the cytokine levels of IL-1alpha, IL-17 and IL-27 showed no significant differences in consistency as orthotopic H22 and Hepa1-6 HCC mice subjected to FUS sti. spleen (Figure 4 A-C). In addition, IL-2 is a pleiotropic cytokine required for both effector lymphocyte proliferation/differentiation and regulatory T cell expansion/survival [64], which was significantly secreted as spleen responded to FUS stimulation (Figure 4 A-C). Additionally, IL-4 mainly secreted by T cells and NK cells induces Mφ2 activation, and regulates the expression of IL-6, IL-10, IL-12 and IL-13 [65]. Some studies also reported that polarized Th1 cells produced IL-2, IL-12 and IFN-gamma, and polarized Th2 cells secreted IL-4, IL-6, IL-10 and IL-13 that promoted tumor proliferation [66] (Figure 4 A-C). In this study, IL-6 level was increased and IL-13 level was decreased when the orthotopic HCC mice were subjected to FUS sti. spleen (Figure 4 A-C). Nevertheless, the cytokine levels of IL-4, IL-10 and IL-12 were altered between orthotopic H22 and Hepa1-6 HCC mice models subjected to FUS sti. spleen (Figure 4 A-C). Protein-protein networks of these cytokines and target genes, and KEGG pathway enrichment analysis showed that FUS stimulating splenic immune cells to regulate cytokines against tumor was associated with several signaling pathways, especially JAK-STAT signaling pathway and PI3K-AKT signaling pathway (Figure 4 D-E). It was evident that these cytokines characterized the tumor cytotoxicity of NK cells and CD8 T cells, mainly through cytokine-cytokine receptor interaction pathway to obtain an anticancer effect (Figure 4 D-E). 3.3 FUS directly activated splenic immune cells for cancer immunotherapy 3.3.1 FUS promoted splenic positive immune cell proliferation and activation to combat tumor According to previous studies, splenic ultrasound stimulation activated CAP to inhibit arthritis, colitis, hyperglycemia and other inflammatory diseases, but they paid more attention to the modulation of inflammatory factors such as TNF-α, IL-6 and IL-10, as well as CAP validation, but not to the changes of immune cells [30, 67, 68]. In this study, FUS sti spleen significantly altered the proportion of immune cells in the spleen, blood, and tumor, especially NK cells and CD8 T cells that increased uniformly in various tumor models (Table 2-3), thus arousing our special attention. Inexplicably, the previous studies based on inflammatory disease models (e.g., pneumonia, myocarditis, and colitis) proved that splenic ultrasound stimulation activates CAP to reduce Mφ and inhibits the secretion of pro-inflammatory cytokines (e.g. TNF-α), but in this study based on tumor models, FUS sti. spleen significantly increased Mφ and promoted TNF-α secretion. Therefore, our speculation was that FUS directly stimulated splenic immune cells stressing tumor signals, which promoted the proliferation and activation of positive immune cells (i.e. NK cells and CD8+ T cells) to suppress cancer cell proliferation. To further confirm this hypothesis, splenic nerve blockade was performed with atomoxetine hydrochloride targeting CAP to explore the impact of CAP deficiency on tumor suppression during FUS sti. spleen. Subsequently, NK cells and CD8 T cells were depleted with Ultra-LEAF™ purified anti-mouse NK-1.1 antibody and Ultra-LEAF™ purified anti-mouse CD8a antibody, respectively, to further explore their role and importance in the tumor-suppressing process of FUS sti. spleen. Forty-eight orthotopic H22 HCC mice were randomly divided into eight groups: the Q1 group was used as the control group; the Q2 group was subjected to FUS sti. spleen; the Q3 group was treated with an intraperitoneal injection of atomoxetine hydrochloride to block CAP; the Q4 group was subjected to FUS sti. spleen after an intraperitoneal injection of atomoxetine hydrochloride; the Q5 group was subjected to NK cell clearance; the Q6 group was subjected to FUS sti. spleen after NK cell depletion; the Q7 group was subjected to CD8 T cell clearance; the Q8 group was subjected to FUS sti. spleen after CD8 T cell depletion. The therapeutic results once again demonstrated the significant tumor suppression of FUS sti. spleen (Q2 vs . Q1, p < 0.001; Figure 5 A-C). It is noteworthy that specifically CAP blockage did not significantly attenuate the therapeutic efficacy of FUS sti. spleen on the tumor (Q4 vs . Q3, p < 0.001; Figure 5 A-C), although the anti-tumor rate was slightly reduced in the Q4 group as compared to the Q2 group. It confirmed our previous speculation that CAP did not play a dominant role in the process of FUS sti. spleen for tumor suppression. We supposed that cancer is different from inflammatory diseases such as arthritis, colitis, and pneumonia, and FUS sti spleen to inhibit inflammatory diseases mainly relied on CAP affecting humoral immunity, while it suppressed tumor proliferation mainly depending on the direct modulation of cellular immunity. Furthermore, the tumor proliferation was significantly promoted after NK cell depletion (Q5 vs . Q1, p < 0.001; Figure 5 A-C). Interestingly, FUS sti. spleen did not show any tumor suppressive effect after depleting NK cells (Q6 vs . Q5; Figure 5 A-C). Moreover, CD8 T cell clearance promoted tumor proliferation (Q7 vs . Q1, p ＜0.05; Figure 5 A-C), but as opposite as NK cell clearance, did not reduce the therapeutic efficacy of FUS sti. spleen on tumor (Q8 vs . Q2; Figure 5 A-C). These results proved that NK cells were much more indispensable and important in cancer suppression in response to FUS stimulation than CD8 T cells. The statistical results of liver weight, mice weight, spleen weight and spleen index also showed that NK cell depletion had a significant effect on tumor proliferation and splenomegaly, and elucidated the attenuation of FUS sti. spleen on tumor suppression and splenomegaly (Figure 5 D-G). The concentration of NE and ACh in the plasma was significantly increased after FUS sti. spleen (Q2 vs . Q1, Q6 vs . Q5, and Q8 vs . Q7, p < 0.05; Figure 5 H-I), which indicated that FUS sti. spleen indeed activated splenic CAP. In addition, atomoxetine hydrochloride significantly blocked the binding of NE to adrenergic receptors, that is, effectively blocking CAP, thus increasing the NE content (Q3 vs . Q1, p ＜0.01; Figure 5 H) but no significant changes in ACh (Q3 vs . Q1; Figure 5 I). Therefore, NE content increased after FUS sti. spleen (Q4 vs . Q3; Figure 5H), while ACh did not significantly change (Q4 vs . Q3; Figure 5I). Nevertheless, it is important to note that the results of granzyme and perforin detection by ELISA further confirmed the significance of splenic NK cells activation and their secreting cytotoxic molecules in response to FUS stimulation for an anti-cancer effect, but not affected by CAP (Figure 5 J-K). FCM results proved that Ultra-LEAF™ purified anti-mouse NK-1.1 antibody and Ultra-LEAF™ purified anti-mouse CD8a antibody effectively eliminated NK cells and CD8 T cells in the body, respectively (Q5/6 and Q7/8 vs . Q1/2; Table 4-7; Supplementary Figure 15-18). In the case of CAP blockage and NK or CD8 T cell clearance, the proportion of Mφ, DCs and MDSCs in the body was also affected, especially the number of Mφ and DCs that was generally reduced after depleting NK cells and CD8 T cells (Q3/4, Q5/6 and Q7/8 vs . Q1/2; Table 4-7; Supplementary Figure 15-18). However, FUS sti. spleen consistently increased the number of NK cells and CD8 T cells in the body regardless of CAP blockade and NK or CD8 T cell clearance, followed by Mφ1 and DC1 (Q4 vs . Q3, Q6 vs . Q5, and Q8 vs . Q7; Table 4-7; Supplementary Figure 15-18), while the variation tendency of other immune cells (including PMN-MDSCs) was not consistent in the spleen, blood, tumor and para-carcinoma tissue (Table 4-7; Supplementary Figure 15-18). Clearly, changes in the proportion of immune cells (especially NK cells) with or without modulation by splenic ultrasound stimulation were closely related to the anti-tumor effect. Table 4 FCM results of immune cells in the spleen. Q1, control group of xenograft H22 HCC in situ ; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the Q1-8 groups are shown in Supplementary Figure 15. Analysis NK cells CD8+ T cells Mφ Mφ1 Mφ2 DC1 DC2 Treg cells M-MDSC PMN-MDSC Q2 vs. Q1 ** * ** * ** ** — — ** ** Q4 vs. Q3 ** * * * *** * ** — ** — Q6 vs. Q5 — — — — — ** — — ** — Q8 vs. Q7 ** * — * ** ** * — * — Q3 vs. Q1 — — * — — * — — — — Q4 vs. Q2 — — * — — * * — — — Q5 vs. Q1 *** * *** — ** * — * — — Q6 vs. Q2 *** * *** — — * — — — — Q7 vs. Q1 — ** — — — ** * — * — Q8 vs. Q2 — ** * — — ** — — — — Table 5 FCM results of immune cells in the blood. Q1, control group of xenograft H22 HCC in situ ; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the Q1-8 groups are shown in Supplementary Figure 16. Analysis NK cells CD8+ T cells Mφ Mφ1 Mφ2 DC1 DC2 Treg cells M-MDSC PMN-MDSC Q2 vs. Q1 ** ** — * — * ** ** — * Q4 vs. Q3 ** — ** — — * *** — * ** Q6 vs. Q5 — ** — — — ** * — — — Q8 vs. Q7 ** *** * — * *** — ** — ** Q3 vs. Q1 — — — — — — — — ** — Q4 vs. Q2 — * ** — — * — *** * ** Q5 vs. Q1 *** — — ** — ** *** — * *** Q6 vs. Q2 *** — — ** — — — ** — ** Q7 vs. Q1 — *** — — — *** — — ** — Q8 vs. Q2 — ** * — ** — * — — *** Table 6 FCM results of immune cells in the tumor. Q1, control group of xenograft H22 HCC in situ; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in Q1-8 groups are shown in Supplementary Figure 17. Analysis NK cells CD8+ T cells Mφ Mφ1 Mφ2 DC1 DC2 Treg cells M-MDSC PMN-MDSC Q2 vs. Q1 * ** * — — — — — ** * Q4 vs. Q3 ** ** * — ** — — * — — Q6 vs. Q5 — * — — — * ** — — — Q8 vs. Q7 * *** * ** * * — — — ** Q3 vs. Q1 — — — — — — — — ** — Q4 vs. Q2 — — — — ** ** — — ** — Q5 vs. Q1 *** — — ** *** ** *** *** *** — Q6 vs. Q2 *** — * ** ** — — *** *** — Q7 vs. Q1 — * — * — — — — — * Q8 vs. Q2 — — — — — — — — ** — Table 7 FCM results of immune cells in para-carcinoma tissue. Q1, control group of xenograft H22 HCC in situ; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in Q1-8 groups are shown in Supplementary Figure 18. Analysis NK cells CD8+ T cells Mφ Mφ1 Mφ2 DC1 DC2 Treg cells M-MDSC PMN-MDSC Q2 vs. Q1 * ** * ** — * — * — * Q4 vs. Q3 * * * — — — — * * — Q6 vs. Q5 — — * — — * — — — — Q8 vs. Q7 * *** — * *** — — *** — ** Q3 vs. Q1 — — — — *** — — — — — Q4 vs. Q2 — — — ** *** — — — ** — Q5 vs. Q1 *** — — — *** — ** — — — Q6 vs. Q2 *** — — ** *** — ** * ** — Q7 vs. Q1 — *** * * — *** — — — — Q8 vs. Q2 — * — * * *** — * — ** The results of the above CAP blockage experiments demonstrated that CAP did not play a dominant role in tumor suppression by FUS sti. spleen; however, it could not be excluded the impact of splenic nerve or other nerve signals. Thereafter, we applied absolute ethanol to denervate the splenic nerve to explore the role of the splenic nerve on spleen immunomodulation and anti-cancer effect by FUS sti. spleen. Moreover, the spleen was removed to exclude the possibility of immune regulation and tumor suppression by FUS irradiation on non-splenic organs or tissues. Thus, forty-two orthotopic H22 HCC mice were randomly divided into six groups: H1 group was used as the control group; H2 group was subjected to FUS sti. spleen; H3 group was subjected to splenic nerve denervation by absolute ethanol; H4 group was subjected to FUS sti. spleen after splenic nerve denervation; H5 group was subjected to the removal of the spleen; and H6 group was exposed to FUS on spleen position after splenectomy. The results showed that splenic nerve denervation promoted tumor proliferation to some extent, although not significant (H3 vs . H1, p ＞0.05), suggesting that the splenic nerve had a certain role in the anti-tumor effect. However, more importantly, splenic nerve denervation did not attenuate the tumor suppression efficacy of FUS sti. spleen, which was up to ~75% (H4 vs . H3, p ＜0.001; Figure 6 A-C) that was superior to or consistent with the tumor inhibition rate of FUS sti. spleen without splenic nerve denervation (~70%, H2 vs . H1, p ＜0.001; Figure 6 A-C). It further ruled out the indispensable role of splenic nerve on tumor suppression by FUS sti. spleen, which was also direct evidence that FUS directly stimulated splenic immune cells to regulate immunity against cancer. Liver weight and mice weight also indirectly reflected the anti-cancer effect of FUS sti. spleen after splenic nerve denervation (Figure 6 D-E). However, it must be noted that spleen weight and spleen index clearly indicated that splenic nerve denervation dramatically promoted tumor-induced splenomegaly (H3 vs . H1, p < 0.01; Figure 6 F-G), while FUS sti. spleen significantly reduced splenomegaly (H4 vs . H3, p < 0.01; Figure 6 F-G). The comprehensive analysis of the changes in splenic characteristics and tumor proliferation showed that the splenic nerve played an important role in the anti-cancer effect of the spleen under the natural state but not the splenic ultrasound stimulation. ELISA results of NE and ACh showed that splenic neurons were not activated by FUS under splenic nerve denervation; thus, there was no significant change in the concentration of CAP-related NE and Ach (H4 vs . H3; Figure 6 H-I). The c-Fos immunohistochemical staining also proved that few splenic neurons responded to FUS stimulation after splenic nerve denervation (Figure 6 J). However, splenic nerve denervation did not affect the response of splenic immune cells to FUS stimulation, especially the NK cells that secreted significantly higher levels of perforin and granzyme (H4 vs . H3, p ＜0.01; Figure 6 K-L). Particularly, after splenectomy, FUS irradiation in splenic position did not show any anti-tumor effect (H6 vs . H5; Figure 6 A-E, K-L), which demonstrated that the spleen irradiated with FUS was the only cause of the anti-cancer effect, and directly showed that the integrity of spleen was critical during FUS sti. spleen for tumor suppression. In summary, comprehensive analysis of splenectomy and splenic nerve denervation experiments clearly confirmed that FUS activated splenic anticancer immune function through the direct FUS stimulation of immune cells, thus further suppressing tumor proliferation. FCM results proved that splenic nerve denervation did not affect cell proportion (H3 vs . H1; Supplementary Figure 19-22), particularly not weaken the modulation properties of FUS sti. spleen on the increase of the positive immune cells (especially NK cells, CD8 T cells, Mφ1 and DC1) in the body, and decrease of the negative immune cells PMN-MDSCs (H4 vs . H2/3; Supplementary Figure 19-22). It fully demonstrated that FUS directly stimulated the splenic immune cells to promote their proliferation and activated the anti-cancer capability. After the removal of the spleen, the proportion of most immune cells in the peripheral blood was significantly decreased (H5/6 vs . H1/2; Supplementary Figure 20), which indicated the importance of spleen for the body's immune system. However, after splenectomy, the spleen location irradiated by FUS did not show any impact on the number of immune cells (H6 vs . H5; Supplementary Figure 20-22), which demonstrated that the spleen integrity or the presence of immune cells was essential for responding to FUS stimulation in tumor immunotherapy. 3.3.2 Association of splenic positive and negative immune cells responding to FUS irradiation Our previous study demonstrated that the proportion of PMN-MDSCs in the spleen and blood from tumor-bearing mice was significantly increased, which suppressed NK cells and CD8 T cells [69]. After FUS sti. spleen, the proportion of positive immune cells such as NK cells and CD8 T cells in the body was significantly increased, while the proportion of negative immune cells such as PMN-MDSCs was significantly decreased (Table 2-3). Why did FUS, as a non-specific physical method, cause the number of positive and negative immune cells to change in two directions? In Section 3.3.1, the depletion of NK cells and CD8 T cells did not show consistent changes of PMN-MDSCs in the spleen and blood (Q5/6 and Q7/8 vs . Q1/2; Table 4-5); thus we proposed to reversely demonstrate the association of NK cells and CD8 T cells to PMN-MDSCs by depleting PMN-MDSCs. Forty orthotopic H22 HCC mice were randomly divided into four groups: R1 group was used as the control group; R2 group was subjected to FUS sti. spleen; R3 group was subjected to PMN-MDSCs depletion; and R4 group was subjected to FUS sti. spleen after PMN-MDSCs clearance. PMN-MDSCs depletion inhibited tumor proliferation to a certain extent (R3 vs . R1; Figure 7 A-D) and the subsequent FUS sti. spleen further enhanced tumor suppression (R4 vs . R3; Figure 7 A-D). However, the tumor suppressive effect of FUS sti. spleen after PMN-MDSCs clearance was slightly weaker than that without PMN-MDSCs depletion (R4 vs . R2; Figure 7 A-C). Mice weight did not clearly show a significant difference among the four groups (Figure 7 E), but spleen weight and spleen index showed a decreased trend in the R4 group as compared to the R3 group (Figure 7 F-G). FCM results showed that Ultra-LEAF™ purified anti-mouse Ly-6G antibody effectively eliminated PMN-MDSCs from the spleen, blood, tumor, and para-carcinoma tissue (R3 vs . R1; Figure 7 H-K; Supplementary Figure 23-26). Interestingly, it significantly increased the proportion of Mφ, especially Mφ2, in the spleen and blood (R3 vs . R1; Figure 7 H-I; Supplementary Figure 23-24). Moreover, PMN-MDSC depletion significantly increased NK cells and CD8 T cells only in the blood (R3 vs . R1; Figure 7 I; Supplementary Figure 24). These results explained the reason that PMN-MDSCs clearance suppressed tumor proliferation to a certain extent (Figure 7 A-D). Even more important, PMN-MDSC depletion in spleen did not promote the proliferation of splenic NK cells and CD8 T cells (R3 vs . R1; Figure 7 H; Supplementary Figure 23). Combined with the observation that the depletion of NK cells and CD8 T cells in the spleen did not increase the proportion of splenic PMN-MDSCs (Q5 and Q7 vs . Q1; Table 4), it proved that there was no direct interaction between PMN-MDSCs and NK cells or CD8 T cells. Accordingly, it is reasonable to deduce that FUS sti. spleen increased the number of NK cells and CD8 T cells while the decrease of PMN-MDSCs was not directly correlated. PMN-MDSC decrease may be attributed to the increase of other immune cells in the spleen such as Mφ, especially Mφ2 mentioned above, or even to the rise of total tumor suppressor related immune cells in the spleen and blood. Of course, this mechanism has yet to be explored in the light of more direct and conclusive evidence. 3.4 FUS regulating calcium-related signaling pathways to modulate splenic immunity The above results revealed that splenic NK cells and CD8 T cells were critical for suppressing tumor growth in response to FUS stimulation. Thereafter, RNA sequencing was performed to verify their changes at the molecular level and investigate the biological mechanism underlying this phenomenon. According to the differential gene expression clustering heatmap and volcano plot, 655 upregulated genes and 344 downregulated genes were found in NK cells (Supplementary Figure 27 A-C), as well as 1869 upregulated genes and 877 downregulated genes in CD8 T cells (Supplementary Figure 27 A-C) in the FUS sti. spleen group compared with the control group (Supplementary Figure 27 D-F). Both GO and KEGG enrichment analysis showed that the DEGs were significantly enriched in pathways of cell adhesion, cell migration, immune process (i.e. ECM-receptor interaction, antigen processing and presentation, and intestinal immune network for IgA production) and signal transduction (i.e. PI3K-AKT signaling pathway, Rap1 signaling pathway, Hippo signaling pathway and calcium signaling pathway) in the FUS sti. spleen group compared with the control group (Supplementary Figure 27 G-N). Further analysis revealed that genes related to NK cell immune function, especially activation, proliferation, and motility, were significantly up-regulated after FUS sti. spleen (Figure 8 A-C). Genes involved in calcium-related signaling pathways were also significantly highly expressed (Figure 8 D). According to GO functional annotation analysis, these upregulated genes were not only involved in the positive regulation of cell proliferation, migration and kinase activity, but also mainly involved in integrin-mediated signaling pathway and epidermal growth factor receptor signaling pathway (Figure 8 E). Moreover, MHC class II protein complex was suppressed (Figure 8 F), which promoted NK cell activation in mice as reported by Li Y. et al. [70]. Particularly, KEGG enrichment analysis of these upregulated genes showed the upregulation of PI3K-Akt signaling pathway, Hippo signaling pathway, ErbB signaling pathway, and Ras signaling pathway (Figure 8 G), which were closely associated with cell proliferation, differentiation and activation, especially under ultrasound stimulation [71-73]. Downregulated genes were significantly enriched in the intestinal immune network for IgA production (Figure 8 H), which might be beneficial to tumor suppression as previous studies reported that IgA inhibits NK activity of NK cell-enriched lymphoid cells and gamma-interferon-treated effector cells [74, 75]. More importantly, protein-protein networks revealed that among these significantly different signaling pathways, it is likely that the calcium signaling pathway played a central role and affected the expression of other signals to promote NK cell proliferation, migration and activation (Figure 8 I). Furthermore, GSEA analysis confirmed that FUS stimulation significantly regulated various signaling pathways of NK cells, such as up-regulating ECM-receptor interaction, PI3K-AKT signaling pathway, Hippo signaling pathway and calcium signaling pathways (Figure 8 J). Additionally, the genes related to CD8 T cell activation, proliferation, and motility were significantly up-regulated in the FUS sti. spleen group compared to the control group (Figure 9 A-C). Genes involved in calcium-related signaling pathways were also highly expressed (Figure 9 D). GO and KEGG enrichment and network analysis of the differential genes showed significant enrichment of pathways in signal transduction (i.e. Rap1 signaling pathway, MAPK/ERK signaling pathway, and calcium-related signaling pathways) and immune process (i.e. IL-6 production, IgA production, Fc epsilon RI signaling pathway and Fc gamma R-mediated phagocytosis) (Figure 9 E-H). Particularly, the calcium signaling pathway was involved in regulating most of the others and it was probably the initial response to FUS stimulation (Figure 9 I). GSEA analysis proved that these signaling pathways involving CD8 T cells proliferation, activation and migration, such as ECM-receptor interaction, PI3K-AKT signaling pathway, Rap1 signaling pathway and calcium signaling pathways were significantly upregulated (Figure 9 J). Although the GO and KEGG enrichment analysis of the downregulated genes showed the downregulation of MAPK signaling pathway, TNF signaling pathway, intestinal immune network for IgA production and T cell receptor signaling pathway (Figure 9 H), GSEA analysis revealed that these signaling pathways were highly expressed when the tumor-bearing mice were subjected to FUS sti. spleen (Figure 9 K). These functional annotations suggested that FUS first activated calcium-related signaling pathways and then regulated other signaling pathways to promote the proliferation, activation, and migration of NK cells and CD8 T cells. Many previous studies confirmed that ultrasound affects the proliferation, activation, and differentiation of various cells by altering intracellular calcium-related signaling pathways to achieve specific biological effects [76-78]. The further exploration of splenic calcium changes in response to FUS stimulation by the Von Kossa staining and FCM detecting Fluo-4 AM labeled cells revealed that the splenic cell population of Von Kossa stained and Fluo-4 AM labeled were observably increased in the FUS sti. spleen group as compared with the control group (Figure 10 A-B). Particularly, the results of in vitro experiments (Figure 10 C) fully demonstrated that FUS stimulation promoted NK cell proliferation and activation to suppress cancer cell deterioration (note: H22 cancer cells were unable to adhere to the bottom and difficult to separate from NK cells that migrated from the upper layer of the transwell to the bottom layer, which led to a significant error in the subsequent detection results. Thus, Hepa1-6 cancer cells were chosen for the in vitro study.). Moreover, calcium strengthened the impact of FUS stimulation on splenic NK cells to suppress the proliferation of cancer cells in vitro (Figure 10 C). Besides, ultrasound shattered cancer cell suspension further enhanced the anticancer effect of calcium-involved FUS simulation on splenic NK cells. Additionally, we also verified that calcium reinforced the effect of FUS stimulation on splenic CD8 T cells to suppress cancer cell deterioration in vitro (not supplying the data). Overall, the above experimental results succinctly proved that FUS altered calcium-related signaling pathways in NK cells and CD8 T cells to reinforce the antitumor effect. However, in the future, it is worthwhile comprehensively and systematically analyzing the specific mechanism of splenic immune cells in response to FUS stimulation by spatial region-resolved proteome and spatial single cell sequencing. 4 Discussion 4.1 Therapeutic application of FUS sti. spleen on intermediate and advanced tumors The above studies in which FUS sti. spleen was performed on the second day after cancer cells implanted into the mice were equivalent to the start of the treatment in the early stage of cancer (before tumor formation). However, the anticancer effect of FUS sti. spleen after tumor formation is one of the worthiest explorations in clinical transformation and application. Therefore, a comparative study was performed to investigate the therapeutic application of FUS sti. spleen on 5th and 9th day after H22 cancer cell transplantation into the liver deemed to be tumorigenesis (Fig. 11 A). Four experimental groups were set as follows: K1 group was used as the control group of xenograft H22 HCC in situ ; K2-K4 groups were subjected to FUS sti. spleen on day 0, 4 and 8 after H22 cancer cell implantation (Fig. 11 A). The results showed that FUS sti. spleen on day 0, 4 and 8 significantly suppressed the proliferation of xenograft H22 HCC carcinoma in situ , with an anti-tumor rate of up to ~ 70%, ~ 67% and ~ 48% respectively (K2/3/4 vs . K1; Fig. 11 B-D). The results tautologically demonstrated that FUS sti. spleen exerted an evident tumor suppression, regardless of being performed before or after tumorigenesis, but the later ultrasound irradiation on the spleen was performed after the onset of the tumor, with a smaller effect on tumor suppression. Additionally, the statistical results of liver weight and mice weight showed a decreasing trend in the K2-K4 groups compared to the K1 group (Fig. 11 E-F). The spleen weight and spleen index also demonstrated that FUS sti. spleen attenuated the symptoms of splenomegaly caused by cancer, as same as in Sections 3.1 – 3.3 (Fig. 11 G-H). Statistic FCM results showed that the proportion of NK cells and CD8 T cells was significantly increased in the spleen, blood, tumor, and para-carcinoma tissue of the K2-K4 groups compared with the K1 group, and the proportion of PMN-MDSCs was significantly reduced (Fig. 11 I-L; Supplementary Fig. 28–31). However, no significant difference was found in the number of immune cells among the K2-K4 groups except that NK cells were significantly higher in the K2 group (Fig. 11 I-L; Supplementary Fig. 28–31). In addition, the proportion of Mφ1 and DC1 in four tissues of the K2-K4 groups was significantly increased (Supplementary Fig. 28–31). What needs to be highlighted is the inconsistency in the changes in the cell proportion of Mφ2, DC2 or other immune cells in the spleen, blood, tumor, and para-carcinoma tissue (Supplementary Fig. 28–31). Our hypothesis was that this phenomenon was mainly attributed to the migration of splenic immune cells to tumor lesions after responding to ultrasonic stimulation and stressing the tumor microenvironment, which was fully demonstrated in our previous work that CD11b + CD43 hi Ly6C lo splenocyte-derived Mφ migrate into the lesion in a liver fibrosis model [ 79 ]. Special attention should be paid to the fact that the biological effect of ultrasound on spleen varies in different disease models. As reported by Nunes NS et al. [ 67 ] and Liu T et al. [ 80 ], FUS sti. spleen mainly affects Mφ to inhibit the secretion of inflammatory cytokines in colitis and myocarditis. Also related to RA, CD 4 T cell, CD8 T cell, B cell and myeloid cell populations were significantly changed in response to ultrasound, specifically the CD8 T cells show an evident correlation with FUS sti. spleen based therapy [ 81 ]. However, this study fully confirmed that FUS sti. spleen significantly promoted the proliferation of NK cells, CD8 T cells, Mφ1 and DC1 in various tumor models, and reduced PMN-MDSCs, but mainly suppressed cancer progression by stimulating NK cells. Additionally, the biological effects of ultrasound on different types of cells are also varied, including enhancing MC3T3-E1 osteoblasts and hematopoietic stem cell ingrowth, proliferation, and early differentiation [ 82 , 83 ], inducing neural progenitor cell polarization [ 71 ], promoting proliferation and migration of HaCaT keratinocytes [ 72 ], facilitating the extracellular matrix synthesis of degenerative human nucleus pulposus cells [ 84 ], and motivating the expression of brain-derived neurotrophic factor in astrocyte [ 76 ]. However, the difference in biological effects of ultrasound irradiation on various immune cells enriched in the spleen is not yet known and needs to be detailedly studied in the future. In conclusion, although FUS sti. spleen non-specifically activated spleen immunological function to universally suppress tumor proliferation, it was effective on the early stage of cancer or cancer prevention. Nevertheless, it might be necessary to combine FUS sti. spleen with other treatments in patients with advanced cancer, such as applying FUS qualitatively and quantitatively intervention on the tumor (FUS int. tumor). 4.2 FUS sti. spleen & FUS int. tumor synergistically suppress tumor proliferation Previous studies reported that ultrasonic intervention on tumor lesions enhances tumor suppression by promoting the proliferation, activation, and infiltration of immune cells [ 85 , 86 ]. In this study, comparing and discussing FUS sti. spleen and FUS int. tumors-two strategies for immunomodulation to counteract the swelling by interfering from immune organs and tumor foci, respectively-is crucial. Five experimental groups were considered: #1 group, normal mice (mice without any treatment); #2 group, control group of subcutaneous H22 tumor mice; #3 group, FUS sti. spleen on day 0 after H22 cancer cell implantation; #4 group, FUS int. tumor; #5 group, FUS sti. spleen and FUS int. tumor synergistic treatment. According to pre-experiments and our previous results [ 87 ], FUS int. tumor was performed every 4 days from day 9 after the subcutaneous inoculation of H22 cancer cells (Fig. 12 A), and the ultrasonic parameters were set to 3.3 MPa, 1%, 20 s. The results of #3 vs . #2 group again confirmed the ability and application prospect of FUS sti. spleen in suppressing tumor proliferation (Fig. 12 B-D). Moreover, FUS int. tumor showed an effective tumor suppression (#4 vs. #2 group; Fig. 12 B-D), but it did not show any significant difference as compared with the #3 group (Fig. 12 B-D). It was noteworthy that FUS sti. spleen and FUS int. tumor synergistically induced the best tumor suppression (Fig. 12 B-D). In addition, FUS sti. spleen and FUS int. tumor synergistically or either alone reduced the mice weight (Fig. 12 E), but the combined application significantly exacerbated splenomegaly (Fig. 12 F-G). FCM results revealed that the proportion of NK cells and CD8 T cells in the spleen, blood and tumor was significantly and synergistically increased compared to the effect of each of the two taken individually (Fig. 12 H-J). The comparison of the #3-#5 groups revealed that the number of NK cells and CD8 T cells in the spleen and blood was significantly higher after H22 tumor-bearing mice were subjected to the synergistic effect of FUS sti. spleen and FUS int. tumor compared to the effect of each of the two taken individually (Fig. 12 H-I). However, such a trend in the number of immune cells was not present in tumor tissues, which was mainly attributed to the fact that FUS int. tumor not only disrupted the structure of tumor stromal barrier and cancer cells, but also killed immune cells. Additionally, FUS sti. spleen, FUS int. tumor or both synergistically significantly increased the proportion of Mφ1 or DC1 in the spleen, blood, and tumor (Supplementary Fig. 32–34). However, consistent with the FCM results in sections 3.2.1 – 3.2.2 , FUS sti. spleen had no significant consistent effect on CD4 T cells or their subtype in the spleen, blood, and tumor from subcutaneous H22 tumor-bearing mice. FUS has been gradually developed into a clinical strategy approach for targeted tumor disruption through thermal mechanisms [ 88 – 90 ]. In essence, FUS at high intensity generated focal temperatures exceeding 60 ℃ that instantaneously caused coagulative necrosis of the target soft tissue (Fig. 12 K) [ 91 , 92 ]. In contrast, non-thermomechanical disruption may have a more relevant role in low intensity FUS ablation, particularly in terms of immunomodulatory effects. As opposite to thermal ablation causing protein coagulation necrosis, the mechanical disruption of FUS int. tumor converts immunologically “cold” (poorly infiltrated) tumors into immunologically “hot” (well-infiltrated) ones by damaging cancer cells to release antigens, collapsing the tumor matrix barrier to enhance antigens captured by immune cells, and especially intensifying the infiltration of immune cells and cytokines into the tumor lesions, which strengthened the immunotherapeutic responses to cancer (Fig. 12 K-L) [ 85 , 93 ]. Although FUS int. tumor alone exerted an anti-tumor effect, to a certain extent, the tumor matrix damage would increase the risk of cancer cell spread and metastasis [ 94 ]. However, in this study, the synergistic use of FUS sti. spleen and FUS int. tumor was much more effective, since tumor disruption by FUS provided an antigen source for activating splenic immune cells during the process of FUS sti. spleen to suppress tumor proliferation (Fig. 12 L). 4.3 Optimization and unification of ultrasound system In Section 3.1 , the comparison of tumor inhibition and spleen injury under different ultrasonic parameters of FUS sti. spleen revealed that the spleen (or splenic cells) had significant ultrasound dose dependence and tolerance. This was also reported in previous studies; for example, Liu T, et al. [ 80 ] successfully screened an effective ultrasonic parameter of 0.35 MPa with a 1 s on /5 s off duty cycle to significantly alleviate autoimmune myocarditis, and regulate the proportion and function of Tregs and Mφ by activating CAP. Besides, Cotero V, et al. [ 30 ] discovered that non-invasive splenic ultrasound stimulation at 0.83 MPa effectively alleviates the severity of arthritis and reduces cytokine response to endotoxin through CAP modulation based on CD4 + T cells. However, according to a summary of studies (Table 8 ), although splenic ultrasound stimulation can effectively suppress various inflammatory diseases, the ultrasonic parameters applied for spleen stimulation are extremely variable. Some studies only referred to the ultrasonic parameters mentioned in previous reports but lacked screening and optimization; for example, Hu X, et al. [ 81 ] and Morton C, et al. [ 95 ] used the parameters of 0.35 MPa and 1-s on/5-s off reported by Zachs D, et al. [ 31 ] for splenic immunomodulation to treat inflammatory diseases. However, in practical applications, different ultrasound platforms lead to significant differences in ultrasonic focal region. More importantly, although some studies proved that the mechanism of splenic ultrasound stimulation for inflammatory disease therapy is based on CAP activation [ 29 , 30 , 68 ], some other studies demonstrated the direct action of ultrasound on splenic immune cells [ 31 , 81 ]. Therefore, the dependence, specificity, and mechanism of spleen response to ultrasonic stimulation is also different, which may be one of the reasons for the different ultrasonic parameters applied in the above studies. This study also demonstrated that FUS sti. spleen to modulate immune function for tumor suppression was not mainly by activating CAP but depended on the direct stimulation of the proliferation and activation of NK cells, as well as the biological effects of ultrasonic stimulation on CD8 T cells, Mφ, DCs and MDSCs. Table 8 Summary of the literature related to splenic ultrasound stimulation for modulating immunotherapy on various diseases. No. Summary of the Experimental Content Ultrasonic parameters Therapeutic effect and mechanism Reference 1 Acute colitis was induced by 2% Dextran Sulphate Sodium (DSS) in drinking water for 7 days, and therapeutic ultrasound was performed to the abdominal area. A 5 cm2 transducer (Mettler 740×; Anaheim, CA), 1 MHz, at 10% duty cycle and intensity of 2 w/cm2 for 7 min at day 4–10. Ultrasound improves DSS-induced colitis through the stimulation of the splenic nerve along with the contribution by CAP activation to decrease the levels of colonic F4/80 + α7nAChR + Mφ. [ 1 ] 2 Autoimmune myocarditis was established by a cardiac-specific peptide, and low-intensity pulsed ultrasound stimulated the splenic nerve. A concave transducer (OLYMPUS V302), 1 MHz, at 0.1 MPa, 0.35 MPa, 0.473 MPa, and 1 s on/5 s off bursts for 6 min or 12 min stimulation during day 0 to 21 or day 7 to 21. Splenic ultrasound reduces heart inflammatory injury and improves cardiac remodeling by alleviating the immune response, as well as regulating the proportion and function of CD4 + Treg and Mφ by activating CAP. [ 2 ] 3 A pneumonia model was induced by lung instillation of 10 5 CFU of Streptococcus pneumoniae, and splenic ultrasound stimulation was performed at 4-, 16-, or 48-hours after bacteria inoculation. A transducer (Sonic Concept H106), 1.1 MHz, at 200 mVpp, burst cycles of 150, and a burst period of 200 ms. Non-invasive ultrasound stimulation targeted to the spleen demonstrates a time-dependent effect of CAP activation on the cytokine response during infection progression. [ 3 ] 4 Full thickness cutaneous excisional wounds were made on a rodent model of type II diabetes, and FUS pulses were used externally to the spleen area for 3 min over the course of 15 days. A transducer (Sonic Concept H106), 1.1 MHz, at 300 mVpp, and a pulse repetition period of 0.5 ms. Non-invasive splenic pFUS accelerates wound closure in type II diabetes rodents by up to 4.5 days vs . sham controls. [ 4 ] 5 Acute inflammation and metabolic dysfunction were induced by LPS injection, and FUS was used for splenic nerve stimulation. A transducer (Sonic Concept H106), 1.1 MHz, at 0.27 duty cycle, 136.36 us pulse length, 0.83 MPa peak positive pressure, and a single stimulus not surpassing a single 1 min pulse. Ultrasound applied to spleen reduces cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation by modulating the CAP. [ 5 ] 6 Inflammatory arthritis was induced through arthritogenic donor serum, and the spleen was irradiated by ultrasound one day prior to serum transfer. A single element ultrasound transducer, 1 MHz, at 350 kPa, 1 s on/5 s off pulse pattern, and 2 min per day for 7 consecutive days. Ultrasound stimulation targeting the spleen significantly reduces disease severity in a mouse model of inflammatory arthritis mainly attributed to splenic T and B cells playing an anti-inflammatory effect. [ 6 ] 7 Bilateral renal ischemia-reperfusion injury (IRI) was performed by flank incisions by clamping the renal pedicle for 26 minutes, and ultrasound was applied 3–7 days before IRI. A Sequoia 512 US machine with a small-animal 15L8w transducer (Acuson, Malvern, PA), 7 MHz. mechanical index of 1.2, duration of 1 second to destroy microbubbles, and once every 6 seconds for 2 minutes. Ultrasound prevents renal ischemia-reperfusion injury by stimulating the splenic cholinergic anti-inflammatory pathway. [ 7 ] 8 RA was induced by type II collagen, and FUS irradiation on the spleen was performed one day prior to collagen injection. A FUS concave transducer (Panametrics-NDT, Waltham, MA, USA), 1 MHz, at 350 kPa, 1 s on/5 s off bursts, and 12 min per day. Spleen­targeted low­frequency pulsed FUS effectively improved the severity of arthritis by regulating various cell subpopulations, especially CD8 + T cell subsets. [ 8 ] At present, there is a lack of studies on splenic ultrasound stimulation to regulate immunotherapy for various diseases; thus, ultrasonic parameters are not objectively consistent, which seriously restricts the translational progress of clinical application. Even if the ultrasonic parameters were screened and optimized in this study, there are still some shortcomings. For example, when the ultrasonic intensity is less than 2.3 MPa, the effect of a longer duration of ultrasound irradiation on splenic immune modulation for tumor suppression needs to be further studied. Fortunately, there are two studies of splenic ultrasound stimulation for immunotherapy on inflammatory diseases registered in http://www.clinicaltrials.gov (NCT03690466, and NCT03548116), but their clinical results are not yet officially published. We look forward to their positive news, and expect that FUS sti. spleen can be widely employed in clinical treatment to alleviate patient suffering. Abbreviations IRI, ischemia-reperfusion injury; VNS, vagus nerve stimulation; α7nAChR, α7 nicotinic acetylcholine receptors; FUS, focused ultrasound; DC, dendritic cell; NK, natural killer; MDSC, myeloid-derived suppressor cell; M-MDSC, monocytic myeloid-derived suppressor cell; PMN-MDSC, polymorphonucler myeloid-derived suppressor cell; Mφ, macrophage; Treg, regulatory T cells; FCM, flow cytometry; HCC, hepatocellular carcinoma; TNF-α, tumor necrosis factor α; CAP, cholinergic anti-inflammatory pathway; NE, norepinephrine; ACh, acetylcholine; IgA, immunoglobulin A. Declarations 6 Financial supports This work was supported by the National Natural Science Foundation of China (No. 12204370), the Innovation Ability Supporting Program of Shaanxi Province (No. 2023WGZJ-ZD-09), and the Basic-Clinical Integration Innovation Project of Xi 'an Jiaotong University (No. YXJLRH2022092). 7 Conflicts of interest The authors declare that they have no competing financial interest or interest otherwise. 8 Authors’ contributions Conception and design of the study: Zongfang Li, Jing Geng, Wei Dong. Experiments and procedures: Wei Dong, Guihu Wang, Senyang Li, Qian Wang, Wenjuan Li, Heyuan Liu, Yingxue Liang, Zhe Zhou, Xinrui He, Wenlei Guo, Jianing Yuan, and Yichao Chai. Writing of article: Wei Dong, Guihu Wang, Wenjuan Li, Jing Geng, Zongfang Li. 9 Acknowledgements We thank Hongwei Tian, Gaixia He and Haiyan Chen for providing technical support in preparation of animal models. We thank Pro. Pengfei Liu, Pro. Guangyao Kong, Pro. Yujin Zong, Pro. Shemin Lv and Pro. 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Focused ultrasound activates voltage-gated calcium channels through depolarizing TRPC1 sodium currents in kidney and skeletal muscle. Theranostics. 2019;9(19):5517–31. Yoon CW, Lee NS, Koo KM, Moon S, Goo K, Jung H, Yoon C, Lim HG, Shung KK. Investigation of Ultrasound-Mediated Intracellular Ca(2+) Oscillations in HIT-T15 Pancreatic β-Cell Line. Cells 9(5) (2020). Zhang S, Wan D, Zhu M, Wang G, Zhang X, Huang N, Zhang J, Zhang C, Shang Q, Zhang C, Liu X, Liang F, Zhang C, Kong G, Geng J, Yao L, Lu S, Chen Y, Li Z. CD11b + CD43 hi Ly6C lo splenocyte-derived macrophages exacerbate liver fibrosis via spleen-liver axis. Hepatology. 2023;77(5):1612–29. Liu T, Fu Y, Shi J, He S, Chen D, Li W, Chen Y, Zhang L, Lv Q, Yang Y, Jin Q, Wang J, Xie M. Noninvasive ultrasound stimulation to treat myocarditis through splenic neuro-immune regulation. J Neuroinflammation. 2023;20(1):94. Hu X, Li F, Zeng J, Zhou Z, Wang Z, Chen J, Cao D, Hong Y, Huang L, Chen Y, Xu J, Dong F, Yu R, Zheng H. Noninvasive Low-Frequency Pulsed Focused Ultrasound Therapy for Rheumatoid Arthritis in Mice, Research 2022 (2022) 0013. Wu L, Lin L, Qin YX. Enhancement of cell ingrowth, proliferation, and early differentiation in a three-dimensional silicon carbide scaffold using low-intensity pulsed ultrasound. Tissue Eng Part A. 2015;21(1–2):53–61. Xu P, Gul-Uludag H, Ang WT, Yang X, Huang M, Marquez-Curtis L, McGann L, Janowska-Wieczorek A, Xing J, Swanson E, Chen J. Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro. Biotechnol Lett. 2012;34(10):1965–73. Zhang X, Hu Z, Hao J, Shen J. Low Intensity Pulsed Ultrasound Promotes the Extracellular Matrix Synthesis of Degenerative Human Nucleus Pulposus Cells Through FAK/PI3K/Akt Pathway, SpinePhila Pa (1976) 41(5) (2016) E248-54. Qu S, Worlikar T, Felsted AE, Ganguly A, Beems MV, Hubbard R, Pepple AL, Kevelin AA, Garavaglia H, Dib J, Toma M, Huang H, Tsung A, Xu Z, Cho CS. Non-thermal histotripsy tumor ablation promotes abscopal immune responses that enhance cancer immunotherapy. J Immunother Cancer 8(1) (2020). Mauri G, Nicosia L, Xu Z, Di Pietro S, Monfardini L, Bonomo G, Varano GM, Prada F, Della Vigna P, Orsi F. Focused ultrasound: tumour ablation and its potential to enhance immunological therapy to cancer. Br J Radiol. 2018;91(1083):20170641. Dong W, Wu P, Zhou D, Huang J, Qin M, Yang X, Wan M, Zong Y. Ultrasound-Mediated Gene Therapy of Hepatocellular Carcinoma Using Pre-microRNA Plasmid-Loaded Nanodroplets. Ultrasound Med Biol. 2020;46(1):90–107. Hoogenboom M, Eikelenboom D, den Brok MH, Heerschap A, Fütterer JJ, Adema GJ. Mechanical high-intensity focused ultrasound destruction of soft tissue: working mechanisms and physiologic effects. Ultrasound Med Biol. 2015;41(6):1500–17. Izadifar Z, Babyn P, Chapman D. Mechanical and Biological Effects of Ultrasound: A Review of Present Knowledge. Ultrasound Med Biol. 2017;43(6):1085–104. Cheung TT, Chu FS, Jenkins CR, Tsang DS, Chok KS, Chan AC, Yau TC, Chan SC, Poon RT, Lo CM, Fan ST. Tolerance of high-intensity focused ultrasound ablation in patients with hepatocellular carcinoma. World J Surg. 2012;36(10):2420–7. Hoogenboom M, Eikelenboom D, den Brok MH, Veltien A, Wassink M, Wesseling P, Dumont E, Fütterer JJ, Adema GJ, Heerschap A. In vivo MR guided boiling histotripsy in a mouse tumor model evaluated by MRI and histopathology. NMR Biomed. 2016;29(6):721–31. Zhou YF. High intensity focused ultrasound in clinical tumor ablation. World J Clin Oncol. 2011;2(1):8–27. Sheybani ND, Price RJ. Perspectives on Recent Progress in Focused Ultrasound Immunotherapy. Theranostics. 2019;9(25):7749–58. Curley CT, Sheybani ND, Bullock TN, Price RJ. Focused Ultrasound Immunotherapy for Central Nervous System Pathologies: Challenges and Opportunities. Theranostics. 2017;7(15):3608–23. Morton C, Cotero V, Ashe J, Ginty F, Puleo C. Accelerating cutaneous healing in a rodent model of type II diabetes utilizing non-invasive focused ultrasound targeted at the spleen. Front Neurosci. 2022;16:1039960. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresTables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4639146","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":327767230,"identity":"c7dcb298-2c97-4b3d-8ade-5378e2fbadcb","order_by":0,"name":"Wei 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University","correspondingAuthor":false,"prefix":"","firstName":"Jianing","middleName":"","lastName":"Yuan","suffix":""},{"id":327767241,"identity":"6cc3eeeb-5bec-4d82-979a-bfdbe877d92c","order_by":11,"name":"Yichao Chai","email":"","orcid":"","institution":"National and Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital, Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yichao","middleName":"","lastName":"Chai","suffix":""},{"id":327767242,"identity":"578313f6-14b1-4f46-a935-562640cbc002","order_by":12,"name":"Jing Geng","email":"","orcid":"","institution":"National and Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital, Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Geng","suffix":""},{"id":327767243,"identity":"6f70d6d6-5ed2-4537-bd7c-bef125964071","order_by":13,"name":"Zongfang Li","email":"","orcid":"","institution":"National and Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, The Second Affiliated Hospital, Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Zongfang","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-26 01:27:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4639146/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4639146/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60556325,"identity":"4354dc8b-73e0-4583-9a47-f8551f284b5e","added_by":"auto","created_at":"2024-07-18 06:35:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1677287,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of FUS sti. spleen on subcutaneous H22 tumor under various ultrasonic parameters. A, experimental flow diagram. B, images of spleen and tumor in G1-G11 groups. C, tumor growth curves. D, tumor weight. E, mice weight. F, spleen weight. G, spleen index. H, HE and TUNEL staining of the spleen irradiated with FUS under different ultrasonic parameters; the red arrow indicates the area of the magnified image, and the green arrow indicates the TUNEL-positive cells. I, analysis of TUNEL positive area. G1-G11 group details are shown in Table 1.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/d784dabc8e6476eb02f9eb47.jpg"},{"id":60557162,"identity":"db5bbf89-6fa6-4a2c-b92d-d5c381693f74","added_by":"auto","created_at":"2024-07-18 06:43:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":889478,"visible":true,"origin":"","legend":"\u003cp\u003eFUS sti. spleen suppressed the proliferation of the xenograft HCC \u003cem\u003ein situ\u003c/em\u003e. B-I, results of FUS sti. spleen suppressing the proliferation of the xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e. A, experimental flow diagram of FUS sti. spleen after HCC cancer cell implantation. B, images of spleen and xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e. C-D, tumor volume and tumor weight, respectively. E-G, liver weight, mice weight and spleen weight, respectively. H, spleen index calculated by the ratio of spleen weight to mouse weight. I, survival curves. S1, control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; S2, FUS sti. spleen started on day 0 after H22 cancer cell implantation. J-Q, results of FUS sti. spleen suppressing the proliferation of the xenograft Hepa1-6 HCC \u003cem\u003ein situ\u003c/em\u003e.J, images of spleen and xenograft Hepa1-6 HCC \u003cem\u003ein situ\u003c/em\u003e. K-L, tumor volume and tumor weight, respectively. M-O, liver weight, mice weight and spleen weight, respectively. P, spleen index calculated by the ratio of spleen weight to mouse weight. Q, survival curves. L1, control group of xenograft Hepa1-6 HCC \u003cem\u003ein situ\u003c/em\u003e; L2, FUS sti. spleen started on day 0 after Hepa1-6 cancer cell implantation.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/ce2a5155ba0a0593d5b87472.jpg"},{"id":60556328,"identity":"70b67d92-d690-481e-a857-661819beded7","added_by":"auto","created_at":"2024-07-18 06:35:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1328295,"visible":true,"origin":"","legend":"\u003cp\u003eFUS sti. spleen suppressed the proliferation of the subcutaneous4T1 breast tumor. A, images of spleen and 4T1 tumor. B-C, tumor growth curves and tumor weight, respectively. D, pulmonary variation score to evaluate lung metastasis of 4T1 cancer cells, where the color area of the lung tissue was scored from 0 to 10; the larger the black-brown area, the higher the score. E-F, Mouse weight and spleen weight, respectively. G, splenic index calculated by the ratio of spleen weight to mouse weight. H, survival curves. I, immunohistochemical staining of Ki67, NK1.1, and CD8a, and the corresponded statistical diagrams. P1, control group of subcutaneous 4T1 breast tumor; P2, FUS sti. spleen on day 2 after 4T1 cancer cell implantation.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/3b9088c41629f1831df2492a.jpg"},{"id":60557166,"identity":"c6af6de8-468d-4dfb-8d0e-f9fee2bd66e6","added_by":"auto","created_at":"2024-07-18 06:43:28","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1866333,"visible":true,"origin":"","legend":"\u003cp\u003eCytokine detection and bioinformatics analysis. A-B, heatmap of cytokine levels in orthotopic H22 and Hepa1-6 HCC micesubjected to FUS sti. spleen. C, Venn diagram of up- and down-regulated cytokines in both orthotopic H22 and Hepa1-6 HCC mice subjected to FUS sti. spleen. D, protein-protein \u003ca href=\"https://www.sciencedirect.com/topics/engineering/electric-network-analysis\" title=\"Learn more about network analysis from ScienceDirect's AI-generated Topic Pages\"\u003enetwork analysis\u003c/a\u003e of cytokines and target genes based on STRING database. E, KEGG pathway enrichment analysis of cytokines and target genes.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/b87c461a780ee3a3019a5953.jpg"},{"id":60557159,"identity":"3665ce72-7e75-48f0-9c05-43dc7fd7e4c3","added_by":"auto","created_at":"2024-07-18 06:43:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2114015,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effect of FUS sti. spleen on xenograft H22 carcinoma \u003cem\u003ein situ\u003c/em\u003e after CAP blockage, NK cells depletion and CD8 T cell clearance. A, images of spleen and orthotopic H22 tumor. B-C, tumor volume and tumor weight, respectively. D-F, liver weight, mice weight and spleen weight, respectively. G, splenic index calculated by the ratio of spleen weight to mouse weight. H-K, concentration of NE, ACh, granzyme and perforin, respectively, detected by ELISA. Q1, control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; Q2, FUS sti. spleen on day 2 after H22 cancer cell implantation; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/b499d3bedae93c3f852684ac.jpg"},{"id":60557920,"identity":"33ad314f-8ce2-4ec8-a426-7e06fe86eef9","added_by":"auto","created_at":"2024-07-18 06:51:28","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1806688,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effect of FUS sti. spleen on tumor suppression after splenic nerve denervation and splenectomy. A, images of spleen and orthotopic H22 tumor. B-C, tumor volume and tumor weight, respectively. D-F, liver weight, mice weight and spleen weight respectively. G, splenic index calculated by the ratio of spleen weight to mouse weight. H-I, concentration of NE and ACh, respectively, detected by ELISA. J, c-Fos immunohistochemical staining and statistical result of positive area in section. K-L, concentration of granzyme and perforin, respectively, detected by ELISA. H1, control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; H2, FUS sti. spleen on day 2 after H22 cancer cell implantation; H3, splenic nerve denervation by absolute ethanol; H4, FUS sti. spleen after splenic nerve denervation; H5, removal of the spleen; H6, FUS stimulated the splenic position aftersplenectomy.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/e1373e4ac75bb3be4a934e98.jpg"},{"id":60557161,"identity":"6752ab9a-2209-4067-88d8-6f0d20920856","added_by":"auto","created_at":"2024-07-18 06:43:28","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2464332,"visible":true,"origin":"","legend":"\u003cp\u003eFUS sti. spleen for tumor suppression after depleting PMN-MDSCs. A, images of spleen and orthotopic H22 tumor. B, tumor volume. C, tumor weight. D, liver weight. E, mice weight. F, spleen weight. G, splenic index. H, FCM results of PMN-MDSCs, NK cells, CD8 T cells, Mφ and Mφ2 in the spleen; I, FCM results of PMN-MDSCs, NK cells, CD8 T cells, Mφ and Mφ2 in the blood; J, FCM results of PMN-MDSCs, NK cells, CD8 T cells, Mφ and Mφ2 in the tumor; K, FCM results of PMN-MDSCs, NK cells, CD8 T cells, Mφ and Mφ2 in para-carcinoma tissue. R1, control group of xenograft H22 carcinoma \u003cem\u003ein situ\u003c/em\u003e; R2, groupsubjected to FUS sti. spleen; R3, group subjected to PMN-MDSCs depletion; R4, group subjected to FUS sti. spleen after PMN-MDSCs clearance. Representative FCM plots and statistical diagram of immune cells in the R1-4 groups are shown in Supplementary Figure 23-26.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/2caa8bfddee34a89674327b8.jpg"},{"id":60557160,"identity":"0501959e-7741-4974-b753-4e9f0c6f7f40","added_by":"auto","created_at":"2024-07-18 06:43:28","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2140110,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatic analysis of the DEGs related to NK cell activation, migration, and proliferation, and involved in calcium-related signaling pathways. A-D, heatmap diagram of the DEGs related to NK cell activation, migration, and proliferation, and involved in calcium-related signaling pathways, respectively. E-F, GO pathway enrichment analysis of the upregulated and downregulated genes in A-D. G-H, KEGG pathway enrichment analysis of the upregulated and downregulated genes in A-D. I, protein-protein \u003ca href=\"https://www.sciencedirect.com/topics/engineering/electric-network-analysis\" title=\"Learn more about network analysis from ScienceDirect's AI-generated Topic Pages\"\u003enetwork analysis\u003c/a\u003eof the DEGs in A-D based on STRING database. J, GSEA snapshots of KEGG pathway enrichment analysis: ECM-receptor interaction, PI3K-AKT signaling pathway, Hippo signaling pathway and calcium signaling pathways.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/5e36399c1aab7676865894c7.jpg"},{"id":60556336,"identity":"4932fa58-63c8-48e9-b030-8189b7240f17","added_by":"auto","created_at":"2024-07-18 06:35:28","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":11694867,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatic analysis of DEGs related to CD8 T cells activation, proliferation, and migration, and involved in calcium-related signaling pathways. A-D, heatmap diagram of the DEGs related to CD8 T cell activation, proliferation, and migration, and involved in calcium-related signaling pathways, respectively. E-F, GO pathway enrichment of the upregulated genes and downregulated genes in A-D, respectively. G-H, KEGG pathway enrichment analysis of the upregulated genes and downregulated genes in A-D, respectively. I, protein-protein \u003ca href=\"https://www.sciencedirect.com/topics/engineering/electric-network-analysis\" title=\"Learn more about network analysis from ScienceDirect's AI-generated Topic Pages\"\u003enetwork analysis\u003c/a\u003e of the DEGs in A-D based on STRING database. J, GSEA snapshots of KEGG pathway enrichment analysis: calcium signaling pathways, Rap1 signaling pathway, PI3K-AKT signaling pathway and ECM-receptor interaction. K, GSEA snapshots of KEGG pathway enrichment analysis: MAPK signaling pathways, TNF signaling pathway, intestinal immune network for IgA production and T cell receptor signaling pathway.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/63e7161472d5bbc74b9a7811.jpg"},{"id":60557164,"identity":"4e3535a2-08e1-4aa9-a531-02996dde1b67","added_by":"auto","created_at":"2024-07-18 06:43:28","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2482258,"visible":true,"origin":"","legend":"\u003cp\u003eCalcium strengthened FUS-stimulated splenic NK cells against Hepa1-6 cancer cells. A, Von Kossa staining of calcium deposition in the spleen with or without FUS stimulation. B, FCM detected Fluo-4 AM labeled cells in the spleen with or without FUS stimulation. C, results of calcium strengthened impact of FUS stimulation on splenic NK cells to suppress cancer cells \u003cem\u003ein vitro\u003c/em\u003e, including GFP fluorescence imaging, CV staining and CCK-8 assay to analyze the proliferation and activity of Hepa1-6 cancer cells, and CCK-8 assay to characterize the proliferation and cytoactivity of NK cells. T1, Hepa1-6 cancer cells; T2, NK cells; T3, NK cells cocultured with Hepa1-6 cancer cells in the transwell; T4, FUS stimulated NK cells; T5, NK cells subjected to FUS irradiation, and then cocultured with Hepa1-6 cancer cells in the transwell; T6, NK cells mixed with Hepa1-6 lysate and cocultured with Hepa1-6 cancer cells in the transwell; T7, NK cells mixed with Hepa1-6 lysate, subjected to FUS irradiation, and then cocultured with Hepa1-6 cancer cells in the transwell; T8, NK cells mixed with Hepa1-6 lysate in 300 nM calcium culture medium and cocultured with Hepa1-6 cancer cells in the transwell; T9, NK cells in 300 nM calcium culture medium subjected to FUS irradiation, and then cocultured with Hepa1-6 cancer cells in the transwell; T10, NK cells mixed with Hepa1-6 lysate in 300 nM calcium culture medium, subjected to FUS irradiation, and then cocultured with Hepa1-6 cancer cells in the transwell.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/652aa3b479c5781db2bbc73f.jpg"},{"id":60556332,"identity":"fed8ecea-b98f-4662-846b-7d02398dd1b0","added_by":"auto","created_at":"2024-07-18 06:35:28","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1687323,"visible":true,"origin":"","legend":"\u003cp\u003eFUS sti. spleen suppressed the proliferation of the xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e. A, experimental flow diagram of FUS sti. spleen on day 0, 4 and 8 after H22 cancer cell implantation. B, images of spleen and xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e. C, tumor volume. D, tumor weight. E, liver weight. F, curve of mice weight. G, spleen weight. H, spleen index calculated by the ratio of spleen weight to mouse weight. I-L, FCM results of NK cells, CD8 T cells and PMN-MDSCs in the spleen, blood, tumor, and para-carcinoma tissues, respectively. K1, control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; K2-K4, FUS sti. spleen on day 0, 4 and 8, respectively, after H22 cancer cell implantation. Representative FCM plots and statistical diagram of immune cells in K1-K4 groups are shown in Supplementary Figure 28-31.The results shown in Figure 11 and Figure 6 were from the same batch of experiments. With animal welfare in mind, only one control group and FUS sti. spleen group were set up; thus, the K1 and K2 group were the same as the H1 and H2 group respectively.\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/b9d2755bfe277fd04cc7b516.jpg"},{"id":60557921,"identity":"602f5e84-4401-4b17-8e30-71b5c423f0eb","added_by":"auto","created_at":"2024-07-18 06:51:28","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1003249,"visible":true,"origin":"","legend":"\u003cp\u003eFUS sti. spleen and FUS int. tumor synergistically or individually suppressed the proliferation of the subcutaneous H22 HCC tumor. A, experimental flow diagram of FUS int. tumor every four days started on day 8 after H22 cancer cell implantation. B, image of spleen and tumor. C, tumor volume. D, tumor weight. E, curve of mice weight. F, spleen weight. G, spleen index, the ratio of spleen weight to mouse weight. H-J, FCM results of NK cells, CD8 T cells and PMN-MDSCs in the spleen, blood, and tumor, respectively. K-L, schematic illustration of the mechanism during FUS int. tumor to enhance anticancer immune response of FUS sti. spleen. #1, normal mice group; #2, control group of subcutaneousH22 tumor mice; #3, FUS sti. spleen on the second day after H22 cancer cell implantation; #4, FUS int.tumor; #5, FUS sti. spleen and FUS int. tumor synergistic effect.\u003c/p\u003e","description":"","filename":"Figure12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/da005c99998e811f5086fb7e.jpg"},{"id":74178167,"identity":"dc206d5a-03ab-4fa0-85e2-a3420d3d7c5d","added_by":"auto","created_at":"2025-01-19 13:16:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33407101,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/d6f11c5d-f177-4531-a0f0-7b1260e5dbab.pdf"},{"id":60556342,"identity":"e02920d2-0c24-4c00-8e89-a2da2c436124","added_by":"auto","created_at":"2024-07-18 06:35:28","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":39546677,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4639146/v1/db886b8d334ae3e4ea77a33c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Noninvasive focused ultrasound non-specifically activating spleen immunological function to suppress tumor proliferation: a new strategy for cancer immunotherapy","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe spleen is the largest secondary lymphoid organ, and an extensive meshwork of nerve fibers is distributed throughout the splenic compartments including the splenic nodules, periarteriolar lymphoid sheath, marginal zones, trabeculae, white pulp and red pulp [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Even more striking, the nerve fibers are co-localized with various subsets of dendritic cells (DCs), macrophages (Mφ), and lymphocytes (i.e. B cells and T cells) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As early as 30 years ago, some studies demonstrated that sympathetic nerve terminals in the spleen secrete norepinephrine (NE) interacting with T cells in response to stimulation, while the content of splenic norepinephrine significantly decreases following chemical or surgical sympathectomy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It was not until around 2000 that the cholinergic anti-inflammatory pathway (CAP) concept was formally proposed and extensively studied because of its vital function in modulating the mammalian immune response [\u003cspan additionalcitationids=\"CR7 CR8 CR9\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The CAP mechanism relies on the parasympathetic vagus nerve transmitting signals from the brain to the adrenergic splenic nerve that interacts with immune cells (Supplementary Fig.\u0026nbsp;1). Munyaka P, et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] revealed that CAP is activated after central cholinergic activation by intracerebroventricular infusion of the M1 muscarinic acetylcholine (ACh) receptor agonist in mice but is suppressed by vagotomy or splenic neurectomy. Moreover, Ji H, et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] proved that central cholinergic activation induced by the acetylcholinesterase inhibitor galantamine or the muscarinic ACh receptor agonist relieves colitis in mice, which is inhibited after vagotomy, splenic neurectomy or splenectomy. These reports indicate that spleen is a vital target of the CAP.\u003c/p\u003e \u003cp\u003eIn the past decade, many researchers applied physical methods to trigger the splenic neural-immune reflex for regulating the immune response to chronic inflammatory diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Inoue T, et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] utilized a bipolar silver wire electrode stimulating the isolated left cervical vagus nerve to attenuate kidney ischemia-reperfusion injury (IRI), suggesting that vagus nerve stimulation (VNS)-mediated IRI treatment and systemic inflammation depend on α7 nicotinic acetylcholine receptors (α7nAChR)-positive splenocytes. Koopman F A, et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] reported that stimulation of the CAP by VNS to treat rheumatoid arthritis (RA) in experimental models and clinical trials exerts a significant mitigation effect, whilst the symptoms aggravated following unilateral cervical vagotomy or α7nAChR knockout in mice. Besides, Meregnani J and colleagues [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] demonstrated that the symptoms of colonic colitis in rats induced by trinitrobenzene sulphonic acid was significantly reduced after VNS performed with electrode cuffs for 5 days. Moreover, electroacupuncture intervention eliciting spenic efferent vagus nerve activity not only reduced tumor proliferation in breast tumor-bearing mice by alleviating inflammation and enhancing antitumor immunity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but also could alleviate the severity of chemotherapy-induced nausea and vomiting in patients with advanced cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In addition, bioelectronic devices for VNS in clinical application have already been approved by the European Medicines Agency and the US Food and Drug Administration (FDA) for the treatment of drug-refractory epilepsy and depression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnsatisfactorily, nerve stimulation using permanently implanted electrodes, transcutaneous bioelectronic devices, or slightly injurious electroacupuncture was limited to stimulate large nerves or superficial nerves [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Peripheral organs, not just the spleen, are extensively traversed by peripheral nervous system, which makes it difficult to selectively stimulate subsets of axons that terminate in spleen and uniquely modulate the immune response to inflammatory diseases [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For example, cervical nerve stimulation usually activates the extensive efferent and afferent neural pathways that elicit neural responses from non-target organs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although scientists are committed to developing more advanced, miniaturized and sophisticated electrode designs for implantation on specific nerves near the target organ [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], invasive implantation easily causes damage to the delicate nerve fibers of the target organ and it is difficult to stimulate nerves throughout the organ. Thus, new nonintrusive strategies are imperative to trigger specific nerves, especially the splenic nerve, and mobilize the splenic immune function against diseases.\u003c/p\u003e \u003cp\u003eNotably, recent studies showed that noninvasive ultrasound successfully elicits peripheral nerve activation in the spleen to treat chronic inflammatory diseases (Supplementary Fig.\u0026nbsp;1). Gigliotti JC and co-workers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] used an ultrasound imaging probe to successfully irradiate the neural innervation in the spleen mitigating IRI in mice, whose mechanism is mainly attributable to the CAP activation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Later, Cotero V, et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] reported that ultrasound modulating splenic nerve reduces endotoxin-induced cytokine production at a level comparable with traditional VNS performed with electrodes. Zachs DP, et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] applied ultrasound stimulation targeting the spleen, which significantly reduces inflammation severity in RA mouse models. These studies provide ample evidence on the potential of precise splenic ultrasound stimulation to replace bioelectronic devices for the translation of peripheral neuromodulation-based therapies for inflammatory disease. However, the potential roles and underlying mechanisms of splenic ultrasound stimulation in cancer management have been rarely reported and superficially defined.\u003c/p\u003e \u003cp\u003eFurther investigation is needed to establish whether ultrasound directly regulates the immunomodulatory function of the spleen by interfering with splenic immune cells and microenvironment. Ultrasound targeting the spleen is crucial in achieving conspicuously therapeutic effects, since the stimulation of other body locations or the lack of immune cells in mice is noneffective, which suggests the indispensability and importance of immune cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Cotero V, et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and Zachs DP, et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] concluded that ultrasound activating CAP indirectly exerts an influence on splenic immune cells or cytokines to reduce the severity of lipopolysaccharide acute inflammation and RA, but they mainly focused on the analysis of neuro-immune mechanism and did not perform a systematic research on the changes of splenic immune cells in response to ultrasonic stimulation. Generally, ultrasonic irradiation on cells directly irritates the expression of multiple key genes/signaling pathways or interferes with the secretion of cytokines to regulate cell proliferation, differentiation and migration, such as stimulating the expression of vascular endothelial growth factor in several cells including endothelial cells, neural cells and ischemic cells [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, we hypothesized that ultrasound could effectively immunomodulated on the splenic immune cells responding to cancer cell antigens in splenic microenvironment, and enhance the proliferation, activation, migration, and information dissemination of immune cells during cancer immunotherapy.\u003c/p\u003e \u003cp\u003eAccordingly, this study was designed to explore whether focused ultrasound precisely stimulating spleen (FUS sti. spleen) was effective in suppressing tumor proliferation, and the underlying mechanisms regulating splenic ultrasound stimulation in cancer management, including the activation of splenic nerve related CAP, and splenic immune cells responding directly to ultrasonic capabilities. The therapeutic efficacy of FUS sti. spleen was first assessed on various tumors under specific parameters screened before. Furthermore, the splenic immunomodulation through FUS sti. spleen was evaluated based on the changes of splenic immune cell population and cytokine levels. Subsequently, nerve blockade, immune cell clearance and RNA sequencing were performed to identify the underlying mechanisms of splenic ultrasound stimulation in cancer management, such as splenic nerve related CAP activation and FUS directly modulating immune cells, and the related molecular mechanism.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell lines and animal models\u003c/h2\u003e \u003cp\u003eHCC cell lines (H22 and hepa1-6) and 4T1 breast cancer cell line were purchased from the American Type Culture Collection (ATCC) and used for subsequent analysis. H22 cells were reproduced by ascites after intraperitoneal injection in C57black/6 mice (0.5\u0026ndash;1.5*10^7 cells/mL; 200 \u0026micro;L per mouse). Hepa1-6 cells and 4T1 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium ( Cat. No. 11320033, Gibco, Invitrogen, Carlsbag, USA) supplemented with 10% fetal bovine serum (FBS; Cat. No. A5669701, Gibco, Invitrogen, Carlsbag, USA) and 1% penicillin-streptomycin solution (Cat. No. 15140122, Gibco, Invitrogen, Carlsbag, USA), and incubated at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e and 100% humidity.\u003c/p\u003e \u003cp\u003e All animal experiments were performed in accordance with the institutional guidelines and approved by the Animal Experimentation Ethics Committee of Xi\u0026rsquo;an Jiaotong University. Seven-week (weighted\u0026thinsp;~\u0026thinsp;17 g) C57black/6 mice, purchased from the Beijing Animal Experiment Center (Chinese Academy of Sciences, Beijing, China), were housed at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 12-h light/dark cycle and acclimatized for at least 1 week before the experiments. All animals had access to sterilized food and water ad libitum. An amount of 0.2 mL H22 cancer cells (approximately 1\u0026ndash;3*10^6 cells/mL) or 4T1 cancer cells (1\u0026ndash;3*10^6 cells/mL) was hypodermically injected into the dorsal hindlimb region of the mouse to establish a subcutaneous tumor model. As regards the \u003cem\u003ein situ\u003c/em\u003e xenograft HCC models, an incision of approximately 1 cm in length was made at the upper end of the midabdominal line after the mouse was anesthetized with isohalothane and fixed on the anatomic stage. A total of 25 \u0026micro;L H22 cancer cells (approximately 0.5\u0026ndash;1.5*10^7 cells/mL) or 50 \u0026micro;L hepa1-6 cancer cells (approximately 4\u0026ndash;6*10^6 cells/mL) was injected into left hepatic lobe with an insulin syringe, and the incision was closed after applying pressure on the pinhole with a medical cotton swab for 3\u0026ndash;4 minutes.\u003c/p\u003e \u003cp\u003eThe tumor volume was calculated as follows: volume\u0026thinsp;=\u0026thinsp;0.5*L*W^2, where L and W were the length and width of the tumor, respectively, measured by a caliper. The weight of spleen, tumor and mice was assessed by electronic scales. The splenic index was calculated by the ratio of spleen weight to mouse weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Ultrasonic platform set-up and application for spleen stimulation\u003c/h2\u003e \u003cp\u003eA diagram of the FUS system is shown in Supplementary Fig.\u0026nbsp;2A. A function generator (Cat. No. 33120A, Agilent, Santa Clara, USA) produced a pulsed sinusoidal waveform triggering the power amplifier (Cat. No. AG1019, California, USA; or RPR-4000, RITEC Inc., Warwick, RI, USA) to drive a 1.04 MHz FUS transducer with a 100 mm aperture and 65 mm focus, whose focal region was ~\u0026thinsp;1.4*1.4*8.6 mm\u003csup\u003e3\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2B-C). The acoustic pressure and spatial beam profile of the FUS transducer were measured using a hydrophone (Cat. No. HNR-1000, ONDA, Videlles, France). The FUS transducer was mounted on a XYZ motorized positioning stage to control ultrasonic duration, and the position of the FUS focus relative to the mice was adjusted under a B-mode imaging guidance with a 3.2 MHz phased array positioned at the center of the FUS transducer.\u003c/p\u003e \u003cp\u003eThe mice were anesthetized with 2\u0026ndash;3% inhaled isoflurane and placed on a manual translation stage equipped with heating pad. Then, a centrifuged coupling gel was immediately applied to the shaved skin, and the manual translation stage was adjusted to allow the spleen (or tumor) site to be in tight contact with the bottom of the water tank under the B-mode imaging guidance (Supplementary Fig.\u0026nbsp;2D). The FUS transducer was moved across the whole spleen (or tumor) through the XYZ programmable logical controller (Supplementary Fig.\u0026nbsp;2E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Flow cytometry\u003c/h2\u003e \u003cp\u003eSingle-cell suspensions from the spleen, peripheral blood, tumor, and para-carcinoma tissue were obtained after red blood cells were lysed using lysis buffer (Cat. No. 555899, BD Bioscience, USA) according to the manufacturer\u0026rsquo;s instructions. Cell suspensions were washed with sterilized PBS, and then incubated with the antibodies listed in Supplementary Table\u0026nbsp;1, and the scheme of antibody labeling for each immune cell is shown in Supplementary Table\u0026nbsp;2. Next, the immune cells were counted by flow cytometry (FCM) using CytoFLEX LX (Beckman Coulter Life Sciences, USA) after filtration through a 70-\u0026micro;m nylon cell strainer (Cat. No. 352350, Corning, USA). Data were analyzed using the FlowJo software (FlowJo 10, LLC, Ashland, OR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cytokine measurement\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Luminex assay\u003c/h2\u003e \u003cp\u003eCytokine/chemokine quantification in plasma was performed by Luminex xMAP technology using a magnetic Luminex assay (R\u0026amp;D Systems, Minneapolis, MN, USA). The quantification was carried out using a Luminex\u0026reg; 200 Flow Cytometry System (Cat. No. x-200, Thermo Fisher Scientific, MA, USA) and Milliplex Analyst software (Version 5.1, Merck Millipore, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 ELISA\u003c/h2\u003e \u003cp\u003eThe concentration of NE, ACh, granzyme and perforin in plasma, respectively, detected by enzyme-linked immunosorbent assay (ELISA). The operation processes were carried out in strict accordance with the instruction manual from Mouse Perforin ELISA Kit (Cat. No. F30718-A, FANKEW, Shanghai, China), Mouse Granzyme-B ELISA Kit (Cat. No. F3214-A, FANKEW, Shanghai, China), Mouse ACh ELISA Kit (Cat. No. MU30072, Bioswamp, Wuhan, China) and Mouse NE ELISA Kit (Cat. No. MU30372, Bioswamp, Wuhan, China), respectively.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histological and immunohistochemical staining\u003c/h2\u003e \u003cp\u003eHematoxylin eosin (HE) staining was applied to evaluate the pathological changes of the spleen after FUS irradiation. Transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL) staining was performed to observe splenic cell apoptosis through the colorimetric TUNEL Apoptosis Assay Kit (Cat. No. C1091, Beyotime, Shanghai, China). The primary antibodies of anti-Ki67 (Cat. No. RM9106S1, Thermo Fisher Scientific, MA, USA; dilution 1:200), anti-NK1.1 (Cat. No. 108759, BioLegend, California, USA), anti-CD8a (Cat. No. ab4055, Abcam, Cambridge, UK; dilution 1:1000) and anti-c-Fos (Cat. No. ab222699, Abcam, Cambridge, UK; dilution 1:2000) were used for immunohistochemical staining to detect the protein expression of cyclin-D1, Ki67, NK1.1, CD8a and c-Fos respectively. At least 5\u0026ndash;10 different regions in each section were randomly selected for image acquisition using a fluorescence microscopy (observer3, Carl Zeiss, Jena, Germany), and the positive area ratio was quantified using the Image Pro Plus 6.0 software (Media Cybernetics, CA, United States).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 RNA sequencing\u003c/h2\u003e \u003cp\u003eCD8 T cells (CD45\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e CD8a\u003csup\u003e+\u003c/sup\u003e) and NK cells (CD45\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e\u0026minus;\u003c/sup\u003e NK1.1\u003csup\u003e+\u003c/sup\u003e) were sorted from the spleen using the Beckman Kurt MoFlo Astrios ultra high speed flow cytometry sorting system (MoFlo Astrios EQ, Beckman Coulter, lnc, USA). Subsequently, general transcriptome sequencing was performed when the positive cell rate was greater than 90% detected by flow cytometry (CytoFLEX LX, Beckman Coulter Life Sciences, USA). The cDNA library construction and sequencing of all RNA samples were performed by Shanghai OE Biotech Co., LTD. Transcriptome sequencing was performed to screen differentially expressed genes (DEGs). Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied to annotate the function of the DEGs. The DEG screening criteria were as follows: \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and differential fold change |logFC| \u0026ge; 1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Nerve signal blockade\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Atomoxetine hydrochloride blocking CAP\u003c/h2\u003e \u003cp\u003eAtomoxetine hydrochloride (Cat. No. abs813490, Absin, Shanghai, China) was intraperitoneally administered at a dose of 3 mg/kg to directly inhibit the NE transporter at 1.5 hours before FUS exposure to spleen according to previous reports [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which ensured FUS sti. spleen at the optimal time point of CAP blockade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Absolute ethanol-induced splenic nerve block\u003c/h2\u003e \u003cp\u003eAbsolute ethanol was used for splenic nerve block according to previous studies [\u003cspan additionalcitationids=\"CR39 CR40 CR41 CR42\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, the mice were anesthetized with the isoflurane, and disinfection with iodophor was performed on the shaved skin. A 7\u0026ndash;10 mm incision was made next to the left side of the spleen. The spleen was isolated with tweezers to keep it away from the peritoneal cavity for a clear exposure of the three main blood supply vascular trees. A wet cotton was used to protect the peritoneal cavity and other organs. Subsequently, absolute ethanol was repeatedly applied to those vascular trees with a cotton tip for 5\u0026ndash;10 seconds each time, with an interval of five seconds, for a total of seven times to block the splenic nerve fibers. Excessive ethanol dripping from the cotton tip was carefully avoided, to avoid significant vasospasm, which could lead to permanent damage to blood vessels, consequently leading to spleen necrosis and organ complete absorption. As regards the sham-operated mice, the same entire surgical procedure was performed except for the repeated application of anhydrous ethanol that was replaced by normal saline (pH\u0026thinsp;=\u0026thinsp;7.4).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.8 \u003cem\u003eIn vivo\u003c/em\u003e immune cell depletion\u003c/h2\u003e \u003cp\u003eThe experimental C57BL/6 mice were treated with an intravenous injection of Ultra-LEAF\u0026trade; purified anti-mouse NK1.1 antibody (Cat. No. 108759, BioLegend, California, USA) or Ultra-LEAF\u0026trade; purified anti-mouse CD8a antibody (Cat. No. 100763, BioLegend, California, USA) at a dose of 50 \u0026micro;g/mouse on day 0, 3, 7, 12 and 17 to deplete NK cell or CD8 T cell. As regards MDSC depletion, the Ultra-LEAF\u0026trade; Purified anti-mouse Ly-6G antibody (Cat. No. 127650; BioLegend, California, USA) was intraperitoneally administered to C57BL/6 mice at a dose of 0.25 mg/mouse on day 0, 6, 12, and 18. The cell clearance efficacy was assessed by FCM.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.9 Calcium detection and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eanticancer experiment of FUS stimulating splenic CD8 T cells or NK cells enhanced by calcium\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.9.1 Von Kossa staining\u003c/h2\u003e \u003cp\u003eThe splenic specimens were cut into 5 \u0026micro;m-thick sections and subjected to VON KOSSA Calcium Staining Kit (Cat. No. JM1519; HPBIO, Shanghai, China) for the histological visualization of calcium deposits (mineralization). Calcium deposition in splenic cells was assessed by fluorescence microscopy (Zeiss observer3), where mass deposits appeared black, while dispersed deposits appeared gray. The positive area ratio was quantified using the Image Pro Plus 6.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.9.2 FCM detection of Fluo-4 AM labeled cells\u003c/h2\u003e \u003cp\u003eThe splenic cell suspensions were washed twice with sterilized PBS, and then incubated with Fluo-4 AM dye (1 mL dye/10\u003csup\u003e6\u003c/sup\u003e cells; Cat. No. S1061M, Beyotime, Shanghai, China) for 30 min at 37 ℃ according to the manufacturer\u0026rsquo;s instructions. Next, the fluorescent cells were monitored by FCM using the CytoFLEX LX (Ex/Em\u0026thinsp;=\u0026thinsp;490/525 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.9.3 Calcium strengthened FUS stimulating splenic NK cells against cancer\u003c/h2\u003e \u003cp\u003eThe NK cells were sorted from the spleen using the Beckman Kurt MoFlo Astrios ultra high speed flow cytometry sorting system, and then subjected to FUS stimulation with or without high calcium concentration (300 nM). Then, NK cells were co-cultured with pEGFP-C1 plasmid-transfected Hepa1-6 cancer cells at a ratio of 1:1, where GFP fluorescent protein was used to distinguish cancer cells from splenic cells. Transwell devices (Corning Incorporated, NY, USA) with 3-\u0026micro;m diameter holes were used to distinguish immune cells in the superstratum and cancer cells in the substratum. The cells were co-cultured for 48 h before GFP fluorescence observation with the Zeiss observer3, CCK-8 assay (Cat. No. ABS50003-500T, Univ, China), and crystal violet (CV) staining to assess cancer cell suppression and immune cell proliferation. The tumoral background was mimicked by adding ultrasound shattered cancer cell suspension to the NK cells solution during FUS stimulation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using the statistical product and service solutions software (SPSS, USA). Statistical significance was analyzed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test with Bonferroni\u0026rsquo;s correction, the mean was calculated from at least four independent experiments, and error bars represent the standard error of mean. A value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003ch2\u003e\u003cstrong\u003e3.1 Screening and optimization of ultrasonic parameters for spleen stimulation\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eIn view of the different ultrasonic parameters and platforms applied in previous studies of splenic ultrasound stimulation, it is pseudoscientific to indiscriminately imitate their ultrasonic parameters or methods to this study for regulating splenic immunotherapy to anti-cancer.\u0026nbsp;Based on the pre-experiment results,\u0026nbsp;11 processing groups were set-up: G1-G11 group (detailed in Table 1), for the screening and optimization of ultrasonic parameters, and\u0026nbsp;FUS\u0026nbsp;sti.\u0026nbsp;spleen in subsequent experiments was performed once every other day starting from the 2nd day after cancer cell implantation (Figure 1 A). The degree of subcutaneous H22 tumor suppression and spleen injury (assessed by HE and TUNEL staining) was used as the decisive criterion to establish the appropriate ultrasonic parameters.\u003c/p\u003e\n\u003cp\u003eThe phenotypic results showed that FUS sti. spleen significantly suppressed the proliferation of subcutaneous H22 tumor, especially in the G4, G5, G8 and G11 group (Figure 1 B-D). The weight of the mice subjected to FUS sti. spleen was lower than that of the non-FUS mice (Figure 1 E), and FUS sti. spleen showed a tendency to decrease splenomegaly (Figure 1 F-G). By comparison, the increase of the duty cycle or the FUS exposure time at 1.8 MPa did not improve the anti-tumor effect (G3, G6, G9 and G10 group; Figure 1 B-D). However, the high peak negative pressure of 2.8 MPa induced a severe spleen injury, easily leading to the death of mice (G5 group; Figure 1 B, H-I). In contrast, FUS sti. spleen effectively suppressed tumor proliferation without damaging the splenic cells at 2.3 MPa (Figure 1 B-D, H-I). However, the antitumor effect was significantly reduced when the FUS exposure time of 20 s was shortened to 10 s (G4 and G7 group; Figure 1 B-D). FUS damaged a few splenic cells once increasing the exposure time of 20 s to 40 s or the duty cycle of 1% to 10% (G4, G8 and G11 group; Figure 1 B-D, H-I). The above splenic stimulation results demonstrated a clear \u0026ldquo;dose response\u0026rdquo; to ultrasound with a distinct power level required for the specific regulation of biophysical effects on splenic immune cells, which was similar to previous studies [23, 29, 31]. In conclusion, the ultrasonic parameters of the G4 group (2.3 MPa, 1%, 20 s) were selected for subsequent experiments of splenic immunomodulation for cancer therapy.\u003c/p\u003e\n\u003cp\u003eTable 1\u0026nbsp;Scheme of different ultrasonic parameters for FUS sti. spleen.\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"481\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.187110187110187%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.76091476091476%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"75.05197505197505%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eUltrasonic parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"40.331491712707184%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePeak negative pressure\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;(MPa)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.900552486187845%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDuty\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ecycle\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.76795580110497%\"\u003e\n \u003cp\u003e\u003cstrong\u003eExposure time\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e\u0026times;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd 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\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.16597510373444%\"\u003e\n \u003cp\u003eG11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.730290456431534%\"\u003e\n \u003cp\u003e\u0026radic;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.29045643153527%\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.95435684647303%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.858921161825727%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2 FUS universally activated spleen immunological function to suppress the proliferation of various tumors\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3\u003e3.2.1 FUS activated splenic immune cells to suppress the proliferation of the xenograft HCC \u003cem\u003ein situ\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eThe mouse model of HCC \u003cem\u003ein situ\u003c/em\u003e,\u0026nbsp;which was better to resemble the situation of\u0026nbsp;primary liver cancer,\u0026nbsp;was used to assess the anticancer\u0026nbsp;efficiency\u0026nbsp;of FUS sti.\u003cem\u003e\u0026nbsp;\u003c/em\u003espleen. The orthotopic HCC mice were subjected to FUS sti. spleen from\u0026nbsp;day 0 after cancer cell implantation (Figure 2 A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwenty orthotopic H22 HCC mice were randomly divided into two groups: the S1 group was used as the control group, and the S2 group was subjected to FUS sti. spleen. The results of visual observation, tumor volume and tumor weight demonstrated that FUS sti. spleen\u0026nbsp;significantly suppressed the proliferation of\u0026nbsp;xenograft\u0026nbsp;H22 carcinoma \u003cem\u003ein situ\u003c/em\u003e, with a tumor inhibition rate of up to ~70% as compared with the control group (S2 \u003cem\u003evs\u003c/em\u003e. S1, \u003cem\u003ep\u003c/em\u003e＜0.001; Figure 2 B-D). Although the mean value of liver weight (total weight of the tumor and liver parenchyma) showed a decreasing trend in the S2 group compared to the S1 group, no significant difference was observed between the two groups (Figure 2 E). In addition, the weight of the mice in the S2 group was lower than that of the mice in the S1 group (\u003cem\u003ep\u003c/em\u003e＜0.001; Figure 2 F).\u0026nbsp;The spleen weight and spleen index showed a decreasing trend of splenomegaly in the S2 group as compared with the S1 group, but without statistically significant difference\u0026nbsp;(Figure 2 G-H). More importantly, the survival statistics proved that the\u0026nbsp;orthotopic H22 HCC mice treated with FUS sti. spleen had a longer survival time than the control group\u0026nbsp;(Figure 2 I).\u003c/p\u003e\n\u003cp\u003eSixteen\u0026nbsp;orthotopic Hepa1-6\u0026nbsp;HCC\u0026nbsp;mice were randomly\u0026nbsp;divided into two groups: L1 group was performed as the control group, and L2 group was administered to FUS sti. spleen.\u0026nbsp;The results of\u0026nbsp;visual observation, tumor volume and tumor weight\u0026nbsp;showed that FUS sti. spleen remarkablely inhibited the proliferation of\u0026nbsp;xenograft\u0026nbsp;Hepa1-6 carcinoma \u003cem\u003ein situ\u003c/em\u003e, with an\u0026nbsp;antitumor ratio\u0026nbsp;of up to ~80% (L2 \u003cem\u003evs\u003c/em\u003e. L1, \u003cem\u003ep\u003c/em\u003e＜0.001; Figure 2 J-L). Additionally, the weight of the liver and mice in the L2 group was lower than that of the mice in the L1 group (\u003cem\u003ep\u003c/em\u003e＜0.05; Figure 2 M-N). No significant difference in spleen weight and spleen index was observed, although there was a decreasing trend shown in the L2 group as compared with the L1 group (Figure 2 O-P). It was noteworthy that the survival time of the mice in the L2 group was significantly longer than that of the mice in the L1 group (Figure 2 Q).\u003c/p\u003e\n\u003cp\u003eThereafter, FCM results revealed that the proportion of tumor suppressor related immune cells in the spleen, blood, tumor and para-carcinoma tissue was significantly increased in the S2 group as compared with the S1 group, such as Th2 cells, NK cells, CD8 T cells, M\u0026phi; (M\u0026phi;2) and DC1 in the spleen; B cells, NK cells, CD8 T cells, M\u0026phi;1 and DC1 in the blood; CD4 T cells, NK cells, CD8 T cells, M\u0026phi; (M\u0026phi;1) and DC1 in the tumors; Th1 cells, NK cells, CD8 T cells and M\u0026phi; (M\u0026phi;1) in the para-carcinoma tissue (Table 2; Supplementary Figure 3-6). Moreover, the proportion of negative immune cells was significantly reduced, such as Treg cells and MDSCs (M-MDSCs and PMN-MDSCs) in both spleen and tumor; Th17, PMN-MDSCs in the blood; and PMN-MDSCs in the para-carcinoma tissue (Table 2; Supplementary Figure 3-6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs regards to orthotopic Hepa1-6\u0026nbsp;HCC\u0026nbsp;mice, FCM results\u0026nbsp;showed that the number of positive anticancer\u0026nbsp;immune cells in the spleen, blood, tumor and para-carcinoma tissue was significantly increased in the L2 group than in the L1 group, such as Th2 cells, NK cells, CD8 T cells, M\u0026phi; and DCs in the spleen; CD4 T cells, Th2 cells, NK cells, CD8 T cells, M\u0026phi;1, M\u0026phi;2 and DCs in the blood; NK cells, CD8 T cells, M\u0026phi; and DCs in the tumor; and NK cells, CD8 T cells and M\u0026phi; (M\u0026phi;1 and M\u0026phi;2) in the para-carcinoma tissue (Table 2; Supplementary Figure 7-10). The number of negative immune cells was significantly reduced, such as Treg cells and PMN-MDSCs in the spleen; Th17 and MDSCs in the blood; Th17 and PMN-MDSCs in the tumor; and PMN-MDSC in the para-carcinoma tissue (Table 2; Supplementary Figure 7-10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results fully demonstrated the significant effect of FUS on splenic immunomodulation for cancer immunotherapy. Particularly,\u0026nbsp;FUS sti. spleen remarkably increased the proportion of NK cells and\u0026nbsp;CD8 T cells in the spleen, blood, tumor and para-carcinoma tissue, followed by M\u0026phi; and DCs, whilst it reduced the number of PMN-MDSCs\u0026nbsp;in all tissues\u0026nbsp;with one accord, and then Treg cells.\u003c/p\u003e\n\u003cp\u003eTable 2 Statistical significance of FCM results of immune cells in the spleen, blood, tumor and para-carcinoma tissue from orthotopic HCC mice subjected to FUS sti. spleen or non-treated mice. S1, control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; S2, FUS sti. Spleen-treated orthotopic H22 HCC. L1, control group of xenograft Hepa1-6 HCC \u003cem\u003ein situ\u003c/em\u003e; L2, FUS sti. Spleen-treated orthotopic Hepa1-6 HCC. The red asterisk indicates a significant increase in the proportion of immune cells; the blue asterisk indicates a significant decrease in the proportion of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the S1 and S2 group are shown in Supplementary Figure 3-6, and those in the L1 and L2 group are shown in Supplementary Figure 7-10.\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width:548.6pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.5pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.75pt;border-top:solid windowtext 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTh1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTh2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.1pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.1pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.1pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eLiver S2 vs. S1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:24.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:28.15pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eSpleen L2 vs. L1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.85pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.25pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:24.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:23.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eB\u003c/span\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003elood \u003cspan style=\"color:black;\"\u003eL2 vs. L1\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.85pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.25pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:24.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTumor L2 vs. L1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.85pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.25pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:24.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:21.25pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:80.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eLiver L2 vs. L1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.85pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:37.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:24.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:28.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:25.45pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003e3.2.2 FUS activated spleen immunological function to suppress proliferation of the subcutaneous 4T1 breast tumor\u003c/h3\u003e\n\u003cp\u003eTo further verify the universality of FUS sti. spleen as a non-specific strategy for modulating splenic immune function to suppress tumor progression, another model of\u0026nbsp;subcutaneous\u0026nbsp;4T1 breast tumor was also used to confirm the anti-cancer effect of FUS sti. spleen.\u0026nbsp;The\u0026nbsp;subcutaneous 4T1 tumor\u0026nbsp;mice were subjected to FUS sti. spleen also from\u0026nbsp;day 0 after cancer cell implantation as described above (Figure 1 A; Figure 2 A), except that the experimental period was set to 34 days.\u0026nbsp;Next,\u0026nbsp;twelve\u0026nbsp;subcutaneous\u0026nbsp;4T1 breast\u0026nbsp;tumor\u0026nbsp;mice were randomly divided into two groups: the P1 group was used as the control group, and the P2 group was subjected to FUS sti. spleen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results of\u0026nbsp;visual observation, tumor volume and tumor weight demonstrated that\u0026nbsp;FUS sti spleen significantly suppressed the proliferation of 4T1 breast tumor, with a ~70% of tumor inhibition rate (P2\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. P1,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e＜0.001; Figure 3 A-C). Moreover, a significant pathological change in the lungs of the P1 group was observed, but the degree of pulmonary lesions in the P2 group was significantly reduced (\u003cem\u003ep\u003c/em\u003e＜0.01; Figure 3 D). During the experimental period, the P1 group showed a decrease in body weight, while that of the P2 group slightly increased without significant difference compared to the P1 group (Figure 3 E), which was most likely due to the poor health status caused by the metastasis of 4T1 cancer cells to the lungs. Notably, the spleen weight and splenic index in the P2 group showed that FUS sti spleen significantly reduced splenomegaly compared to the P1 group (\u003cem\u003ep\u003c/em\u003e＜0.01; Figure 3 F-G). Importantly, the survival time of subcutaneous 4T1 breast tumor mice in the P2 group was significantly longer than that in the P1 group (Figure 3 H).\u003c/p\u003e\n\u003cp\u003eFCM results showed that the change of various immune cell proportion was basically consistent with that in the orthotopic HCC mice experiment.\u0026nbsp;In detail, the proportion of NK cells and CD8 T cells in the P2 group was significantly increased in the\u0026nbsp;spleen, blood, tumor,\u0026nbsp;and lung as compared with the P1 group, and PMN-MDSCs was decreased except in the tumor (Table 3; Supplementary Figure 11-14).\u0026nbsp;Moreover, the number of other tumor suppressor related immune cells was significantly increased in the spleen, blood, tumor or lung of the P2 group compared to that in the P1 group (such as M\u0026phi; (M\u0026phi;1/2) and DC1 in the spleen and blood; M\u0026phi; (M\u0026phi;2) and DCs in the tumor; and M\u0026phi; (M\u0026phi;1) and DC1 in the lung (Table 3; Supplementary Figure 11-14). These results further confirmed that FUS sti. spleen universally activated spleen immunological function by upregulating tumor suppressor-related immune cells and inhibiting negative immune cells, especially in the significant modulation of NK cells and CD8 T cells, to suppress the proliferation of various tumors.\u0026nbsp;While FCM confirmed the proliferation of NK cells and CD8 T cells in the spleen following FUS stimulation, immunohistochemical staining of Ki67, NK1.1, and CD8a also indicated enhanced cell proliferation and especially an increase in the number of NK cells and CD8 T cells (Figure 3 I).\u003c/p\u003e\n\u003cp\u003eTable 3 FCM results of various immune cells in the spleen, blood, tumor, and lung tissues from subcutaneous 4T1 tumor-bearing mice subjected to FUS sti. spleen or non-treated mice. P1, control group of subcutaneous 4T1 breast tumor; P2, FUS sti. spleen on day 2 after 4T1 cancer cell subcutaneous implantation. The red asterisk indicates a significant increase in the proportion of immune cells; the blue asterisk indicates a significant decrease in the proportion of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the P1 and P2 group are shown in Supplementary Figure 11-14.\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width: 4.2e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:78.6pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.05pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNK\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eCD8\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eT cells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.05pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.25pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.5pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTregcells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:33.85pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM-\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eMDSCs\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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style=\"width:30.05pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.05pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.25pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.8pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:78.6pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eT\u003c/span\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eumor \u003cspan style=\"color:black;\"\u003eP2 vs. P1\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.05pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.05pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.35pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.25pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.5pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.8pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:20.95pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:78.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eL\u003c/span\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003eung \u003cspan style=\"color:black;\"\u003eP2 vs. P1\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:38.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:30.25pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:29.5pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:44.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:29.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003e3.2.3 FUS sti. spleen altered the cytokine levels\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eIn addition to the modulation of cellular immunity by FUS sti. spleen, humoral immunity may also be significantly regulated, which represents one dominant factor in the anticancer process during FUS irradiation. Herein, we selectively detected the cytokine levels of GM-CSF, TNF-alpha, IL-12, CCL2, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17, IFN-gamma, CXCL10, M-CSF, IL-1alpha, CCL4, CXCL12, IL-27, perforin and granzyme in the peripheral blood using the\u0026nbsp;Luminex xMAP technology.\u0026nbsp;Interestingly, the concentration levels of TNF-alpha, IFN-\u0026gamma;,\u0026nbsp;perforin and granzyme\u0026nbsp;were significantly increased in the FUS sti. spleen group as compared with the control group in both H22 and Hepa1-6\u0026nbsp;HCC \u003cem\u003ein situ\u003c/em\u003e models\u0026nbsp;(Figure 4 A-C), which indicated the activation of cytotoxic CD8 T cells and NK cells against tumor.\u0026nbsp;Usually, the naive CD8 T cells in the immune process undergo activation and clone expansion, which in turn produce the effector cytokine TNF-alpha, IFN-gamma,\u0026nbsp;perforin and granzyme\u0026nbsp;[44, 45]. In addition, the secretion level of IFN-gamma,\u0026nbsp;perforin and granzyme specifically\u0026nbsp;indicated the activation of NK cells to fight cancer\u0026nbsp;[46].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concentration levels of the pro-inflammatory chemokines CCL2, CCL4 and CXCL10 were significantly elevated in the FUS sti. spleen group of both\u0026nbsp;orthotopic H22 and Hepa1-6 HCC models\u0026nbsp;compared with those in the control group (Figure 4 A-C). These pro-inflammatory chemokines\u0026nbsp;promote immune cells especially effector T cells successfully migrating into metastatic tumor sites to perform an anti-tumor activity\u0026nbsp;[47-49]. CXCL12 is a homeostatic chemokine involved in physiological processes like embryogenesis, neurogenesis, cardiogenesis, hematopoiesis, leukocyte homing, and angiogenesis, by inducing the migration and activation of hematopoietic progenitor and stem cells, endothelial cells, and most leukocytes\u0026nbsp;[50, 51]. In this study, the CXCL12 level was significantly increased in the FUS sti. spleen group (Figure 4 A-C). As a final level of activity regulation, CXCL12 synergizes with CXCL8 but also with CXCL9, CXCL10, CXCL11, and multiple CC chemokines to attract B- and T-lymphocytes, DCs, monocytes, and CD34+ progenitor cells\u0026nbsp;[52]. Moreover, CXCL12 binding to its receptor CXCR4 mediates the biological behavior of tumor cells such as movement, chemotaxis, adhesion, secretion (such as MMPs and VEGF), angiogenesis, growth, and proliferation by activating various signaling pathways\u0026nbsp;[53, 54].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides,\u0026nbsp;GM-CSF and M-CSF are 2 important cytokines that regulate macrophage population and function.\u0026nbsp;Many experimental loads of evidence suggest that the aberrant expression of GM-CSF and its receptors are present in many cancers such as glioblastoma, small cell carcinoma, skin carcinoma, meningiomas, colon cancer, lung cancer, and particularly HCC, promoting cancer progression by regulating the tumor microenvironment involving\u0026nbsp;M\u0026phi;\u0026nbsp;and MDSCs, and promoting epithelial to mesenchymal transition, angiogenesis, and expression of immune checkpoint molecules\u0026nbsp;[55-58]. The concentration level of GM-CSF in this study was significantly decreased as both\u0026nbsp;orthotopic H22 and Hepa1-6 HCC mouse models underwent\u0026nbsp;FUS sti. spleen (Figure 4 A-C). In addition, the M-CSF level was increased\u0026nbsp;after\u0026nbsp;orthotopic HCC mice were subjected to FUS sti. spleen\u0026nbsp;(Figure 4 A-C), which\u0026nbsp;regulated the survival, proliferation and differentiation of the monocyte-macrophage lineage from progenitors to mature cells and activated several important functions of mature tissular\u0026nbsp;M\u0026phi;\u0026nbsp;[59, 60].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe proinflammatory cytokine\u0026nbsp;IL-1beta showed\u0026nbsp;an association with tumor progression\u0026nbsp;[61], but interestingly, although the inhibition of IL-1beta\u0026nbsp;signaling has potent anti-metastatic effects, the inhibition of the activity of this cytokine has contradictory effects on primary tumors, sometimes reducing but often promoting their growth\u0026nbsp;[62]. Moreover, anti-cancer treatments are able to promote IL-1beta\u0026nbsp;production by cancer or immune cells, with opposite effects on cancer progression\u0026nbsp;[63], which resembles the tumor-suppressing effect of FUS sti. spleen.\u0026nbsp;However, beyond that, the\u0026nbsp;cytokine levels of\u0026nbsp;IL-1alpha, IL-17 and IL-27 showed no significant differences in consistency as\u0026nbsp;orthotopic H22 and Hepa1-6 HCC mice subjected to FUS sti. spleen\u0026nbsp;(Figure 4 A-C).\u0026nbsp;In addition, IL-2 is a pleiotropic cytokine required for both effector lymphocyte proliferation/differentiation and regulatory T cell expansion/survival\u0026nbsp;[64], which was significantly secreted as spleen responded to FUS stimulation (Figure 4 A-C). Additionally, IL-4 mainly secreted by T cells and NK cells induces\u0026nbsp;M\u0026phi;2\u0026nbsp;activation, and regulates the expression of IL-6, IL-10, IL-12 and IL-13\u0026nbsp;[65]. Some studies also reported that\u0026nbsp;polarized Th1 cells produced IL-2, IL-12 and IFN-gamma, and polarized Th2 cells secreted IL-4, IL-6, IL-10 and IL-13 that promoted tumor proliferation\u0026nbsp;[66]\u0026nbsp;(Figure 4 A-C).\u0026nbsp;In this study, IL-6 level was increased and\u0026nbsp;IL-13 level was decreased when the\u0026nbsp;orthotopic HCC mice were subjected to\u0026nbsp;FUS sti. spleen\u0026nbsp;(Figure 4 A-C). Nevertheless, the\u0026nbsp;cytokine levels of\u0026nbsp;IL-4, IL-10 and IL-12\u0026nbsp;were\u0026nbsp;altered between\u0026nbsp;orthotopic H22 and Hepa1-6 HCC mice models subjected to\u0026nbsp;FUS sti. spleen\u0026nbsp;(Figure 4 A-C).\u003c/p\u003e\n\u003cp\u003eProtein-protein networks of these cytokines and target genes, and KEGG pathway enrichment analysis showed that FUS stimulating splenic immune cells to regulate cytokines against tumor was associated with several signaling pathways, especially JAK-STAT signaling pathway and PI3K-AKT signaling pathway (Figure 4 D-E). It was evident that these cytokines characterized the tumor cytotoxicity of NK cells and CD8 T cells, mainly through cytokine-cytokine receptor interaction pathway to obtain an anticancer effect (Figure 4 D-E).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.3 FUS directly activated splenic immune cells for cancer immunotherapy\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3\u003e3.3.1 FUS promoted splenic positive immune cell proliferation and activation to combat tumor\u003c/h3\u003e\n\u003cp\u003eAccording to previous studies, splenic ultrasound stimulation activated CAP to inhibit arthritis, colitis, hyperglycemia and other inflammatory diseases, but they paid more attention to the modulation of\u0026nbsp;inflammatory factors such as TNF-\u0026alpha;, IL-6 and IL-10, as well as CAP validation, but not to the changes of immune cells\u0026nbsp;[30, 67, 68].\u0026nbsp;In this study, FUS sti spleen significantly altered the proportion of immune cells in the spleen, blood, and tumor, especially NK cells and CD8 T cells that increased uniformly in various tumor models (Table 2-3), thus arousing our special attention. Inexplicably, the previous studies based on inflammatory disease models (e.g., pneumonia, myocarditis, and colitis) proved that splenic ultrasound stimulation activates CAP to reduce M\u0026phi; and inhibits the secretion of pro-inflammatory cytokines (e.g. TNF-\u0026alpha;), but in this study based on tumor models, FUS sti. spleen significantly increased M\u0026phi; and promoted TNF-\u0026alpha; secretion. Therefore, our speculation was that FUS directly stimulated splenic immune cells stressing tumor signals, which promoted the proliferation and activation of positive immune cells (i.e. NK cells and CD8+ T cells) to suppress cancer cell proliferation. To further confirm this hypothesis, splenic nerve blockade was performed with\u0026nbsp;atomoxetine hydrochloride\u0026nbsp;targeting CAP to explore the impact of CAP deficiency on tumor suppression during FUS sti. spleen.\u0026nbsp;Subsequently, NK cells and CD8 T cells were depleted\u0026nbsp;with Ultra-LEAF\u0026trade; purified anti-mouse NK-1.1 antibody and\u0026nbsp;Ultra-LEAF\u0026trade; purified anti-mouse CD8a antibody, respectively, to further explore their role and importance in the tumor-suppressing process of FUS sti. spleen.\u0026nbsp;Forty-eight orthotopic H22 HCC mice were randomly divided into eight groups: the Q1 group was used as the control group; the Q2 group\u0026nbsp;was subjected to FUS sti. spleen; the Q3 group was treated with an\u0026nbsp;intraperitoneal\u0026nbsp;injection of\u0026nbsp;atomoxetine hydrochloride to block CAP; the Q4 group was\u0026nbsp;subjected to FUS sti. spleen after\u0026nbsp;an\u0026nbsp;intraperitoneal\u0026nbsp;injection of\u0026nbsp;atomoxetine hydrochloride; the Q5 group was subjected to NK cell clearance; the Q6 group was subjected to\u0026nbsp;FUS sti. spleen\u0026nbsp;after NK cell depletion; the Q7 group was subjected to CD8 T cell clearance; the Q8 group was subjected to\u0026nbsp;FUS sti. spleen\u0026nbsp;after CD8 T cell depletion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe therapeutic results once again demonstrated the significant tumor suppression of FUS sti. spleen (Q2 \u003cem\u003evs\u003c/em\u003e. Q1, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001; Figure 5 A-C). It is noteworthy that specifically CAP blockage did not significantly attenuate the therapeutic efficacy of FUS sti. spleen on the tumor (Q4 \u003cem\u003evs\u003c/em\u003e. Q3, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Figure 5 A-C), although the anti-tumor rate was slightly reduced in the Q4 group as compared to the Q2 group. It confirmed our previous speculation that CAP did not play a dominant role in the process of FUS sti. spleen for tumor suppression. We supposed that cancer is different from inflammatory diseases such as arthritis, colitis, and pneumonia, and FUS sti spleen to inhibit inflammatory diseases mainly relied on CAP affecting humoral immunity, while it suppressed tumor proliferation mainly depending on the direct modulation of cellular immunity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, the tumor proliferation was significantly promoted after NK cell depletion (Q5 \u003cem\u003evs\u003c/em\u003e. Q1, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Figure 5 A-C). Interestingly, FUS sti. spleen did not show any tumor suppressive effect after depleting NK cells (Q6 \u003cem\u003evs\u003c/em\u003e. Q5; Figure 5 A-C). Moreover, CD8 T cell clearance promoted tumor proliferation (Q7 \u003cem\u003evs\u003c/em\u003e. Q1, \u003cem\u003ep\u003c/em\u003e＜0.05; Figure 5 A-C), but as opposite as NK cell clearance, did not reduce the therapeutic efficacy of FUS sti. spleen on tumor (Q8 \u003cem\u003evs\u003c/em\u003e. Q2; Figure 5 A-C). These results proved that NK cells were much more indispensable and important in cancer suppression in response to FUS stimulation than CD8 T cells. The statistical results of liver weight, mice weight, spleen weight and spleen index also showed that NK cell depletion had a significant effect on tumor proliferation and splenomegaly, and elucidated the attenuation of FUS sti. spleen on tumor suppression and splenomegaly (Figure 5 D-G).\u003c/p\u003e\n\u003cp\u003eThe concentration of NE and ACh in the plasma was significantly increased after FUS sti. spleen (Q2 \u003cem\u003evs\u003c/em\u003e. Q1, Q6 \u003cem\u003evs\u003c/em\u003e. Q5, and Q8 \u003cem\u003evs\u003c/em\u003e. Q7, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05;\u0026nbsp;Figure 5 H-I), which indicated that FUS sti. spleen indeed activated splenic CAP. In addition, atomoxetine hydrochloride significantly blocked the binding of NE to adrenergic receptors, that is, effectively blocking CAP, thus\u0026nbsp;increasing the NE content\u0026nbsp;(Q3 \u003cem\u003evs\u003c/em\u003e. Q1, \u003cem\u003ep\u003c/em\u003e＜0.01;\u0026nbsp;Figure 5 H) but no significant changes in ACh (Q3 \u003cem\u003evs\u003c/em\u003e. Q1; Figure 5 I).\u0026nbsp;Therefore, NE content increased after FUS sti. spleen (Q4 \u003cem\u003evs\u003c/em\u003e. Q3; Figure 5H), while ACh did not significantly change (Q4 \u003cem\u003evs\u003c/em\u003e. Q3; Figure 5I). Nevertheless, it is important to note that\u0026nbsp;the results of granzyme and perforin detection by ELISA further confirmed the significance of splenic NK cells activation and their secreting\u0026nbsp;cytotoxic molecules\u0026nbsp;in response to FUS stimulation for an anti-cancer effect, but not affected by CAP (Figure 5 J-K).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFCM results proved that\u0026nbsp;Ultra-LEAF\u0026trade; purified anti-mouse NK-1.1 antibody and\u0026nbsp;Ultra-LEAF\u0026trade; purified anti-mouse CD8a antibody\u0026nbsp;effectively eliminated\u0026nbsp;NK cells and CD8 T cells in the body, respectively (Q5/6 and Q7/8 \u003cem\u003evs\u003c/em\u003e. Q1/2; Table 4-7; Supplementary Figure 15-18).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eIn the case of CAP blockage and NK or CD8 T cell clearance, the proportion of M\u0026phi;, DCs and MDSCs in the body was also affected, especially the number of M\u0026phi; and DCs that was generally reduced after depleting NK cells and CD8 T cells (Q3/4, Q5/6 and Q7/8 \u003cem\u003evs\u003c/em\u003e. Q1/2; Table 4-7; Supplementary Figure 15-18).\u0026nbsp;However, FUS sti. spleen consistently increased the number of NK cells and CD8 T cells in the body regardless of CAP blockade and NK or CD8 T cell clearance, followed by M\u0026phi;1 and DC1 (Q4 \u003cem\u003evs\u003c/em\u003e. Q3, Q6 \u003cem\u003evs\u003c/em\u003e. Q5, and Q8 \u003cem\u003evs\u003c/em\u003e. Q7; Table 4-7; Supplementary Figure 15-18), while the variation tendency of other immune cells (including PMN-MDSCs) was not consistent in the spleen, blood, tumor and para-carcinoma tissue (Table 4-7; Supplementary Figure 15-18). Clearly, changes in the proportion of immune cells (especially NK cells) with or without modulation by splenic ultrasound stimulation were closely related to the anti-tumor effect.\u003c/p\u003e\n\u003cp\u003eTable 4 FCM results of immune cells in the spleen. Q1, control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the Q1-8 groups are shown in Supplementary Figure 15.\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width:428.75pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNK\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eCD8+\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eT cells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTreg\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ePMN-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ2 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp 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5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ6 vs. Q5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ3 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n 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5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ4 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ5 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ6 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ7 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable 5 FCM results of immune cells in the blood.\u0026nbsp;Q1, control group of xenograft H22 HCC\u0026nbsp;\u003cem\u003ein situ\u003c/em\u003e; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in the Q1-8 groups are shown in Supplementary Figure 16.\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width:428.75pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNK\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eCD8+\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eT cells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTreg\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ePMN-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ2 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp 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style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ4 vs. Q3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 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Roman\",serif;color:black;'\u003eQ8 vs. Q7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New 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Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ3 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ4 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ6 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n 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5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ7 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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\u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eTable 6 FCM results of immune cells in the tumor. Q1, control group of xenograft H22 HCC in situ; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in Q1-8 groups are shown in Supplementary Figure 17.\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width:428.75pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNK\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eCD8+\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eT cells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTreg\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ePMN-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ2 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ4 vs. Q3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n 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0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n 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5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ7 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 7 FCM results of immune cells in para-carcinoma tissue. Q1, control group of xenograft H22 HCC in situ; Q2, FUS sti. spleen; Q3, injection of atomoxetine hydrochloride to block CAP; Q4, FUS sti. spleen after injection of atomoxetine hydrochloride; Q5, NK cell clearance; Q6, FUS sti. spleen after NK cell depletion; Q7, CD8 T cell clearance; Q8, FUS sti. spleen after CD8 T cell depletion. The red asterisk indicates a significant increase in the number of immune cells; the blue asterisk indicates a significant decrease in the number of immune cells; and the dash represents no significant difference. The representative FCM plots and statistical diagram of immune cells in Q1-8 groups are shown in Supplementary Figure 18.\u003c/p\u003e\n\u003cdiv align=\"center\" style=\"margin:0in;text-align:justify;font-size:14px;font-family:DengXian;\"\u003e\n \u003ctable style=\"width:428.75pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnalysis\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eNK\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eCD8+\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eT cells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM\u0026phi;2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eDC2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eTreg\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ecells\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eM-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:31.35pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cstrong\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003ePMN-MDSC\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ2 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n 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5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n 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5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ5 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n 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Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times 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Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ7 vs. Q1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:55.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eQ8 vs. Q2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:35.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:40.55pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.95pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:red;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e***\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:34.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:39.65pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:42.45pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026mdash;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:41.75pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:19.7pt;\"\u003e\n \u003cp style=\"margin:0in;text-align:center;font-size:14px;font-family:DengXian;\"\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:blue;'\u003e**\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe results of the above CAP blockage experiments demonstrated that CAP did not play a dominant role in tumor suppression by FUS sti. spleen; however, it could not be excluded the impact of splenic nerve or other nerve signals. Thereafter, we applied absolute ethanol to denervate the splenic nerve to explore the role of the splenic nerve on spleen immunomodulation and anti-cancer effect by FUS sti. spleen. Moreover, the spleen was removed to exclude the possibility of immune regulation and tumor suppression by FUS irradiation on non-splenic organs or tissues.\u0026nbsp;Thus,\u0026nbsp;forty-two orthotopic H22 HCC mice were randomly divided into six groups:\u0026nbsp;H1 group was used as the control group; H2 group was subjected to FUS sti. spleen; H3 group was subjected to splenic nerve\u0026nbsp;denervation\u0026nbsp;by absolute ethanol; H4 group was subjected to FUS sti. spleen after splenic nerve\u0026nbsp;denervation; H5 group was subjected to the removal of the spleen; and H6 group was exposed to FUS on spleen position after splenectomy.\u003c/p\u003e\n\u003cp\u003eThe results showed that splenic nerve denervation promoted tumor proliferation to some extent, although not significant (H3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H1,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e＞0.05), suggesting that the splenic nerve had a certain role in the anti-tumor effect. However, more importantly, splenic nerve denervation did not attenuate the tumor suppression efficacy of FUS sti. spleen, which was up to ~75% (H4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H3,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e＜0.001;\u0026nbsp;Figure 6 A-C) that was superior to or consistent with the tumor inhibition rate of FUS sti. spleen without splenic nerve denervation (~70%, H2\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H1,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e＜0.001;\u0026nbsp;Figure 6 A-C). It further ruled out the indispensable role of splenic nerve on tumor suppression by FUS sti. spleen, which was also direct evidence that FUS directly stimulated splenic immune cells to regulate immunity against cancer.\u003c/p\u003e\n\u003cp\u003eLiver weight and mice weight also indirectly reflected the anti-cancer effect of FUS sti. spleen after splenic nerve denervation\u0026nbsp;(Figure 6 D-E). However, it must be noted that spleen weight and spleen index clearly indicated that splenic nerve denervation dramatically promoted tumor-induced\u0026nbsp;splenomegaly\u0026nbsp;(H3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H1,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01;\u0026nbsp;Figure 6 F-G), while FUS sti. spleen significantly reduced splenomegaly (H4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H3,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Figure 6 F-G). The comprehensive analysis of the changes in splenic characteristics and tumor proliferation showed that the splenic nerve played an important role in the anti-cancer effect of the spleen under the natural state but not the splenic ultrasound stimulation.\u003c/p\u003e\n\u003cp\u003eELISA results of NE and ACh showed that splenic neurons were not activated by FUS under splenic nerve denervation; thus, there was no significant change in the concentration of CAP-related NE and Ach (H4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H3; Figure 6 H-I). The c-Fos\u0026nbsp;immunohistochemical staining also proved that few splenic neurons responded to FUS stimulation after\u0026nbsp;splenic nerve denervation (Figure 6 J). However, splenic nerve denervation did not affect the response of splenic immune cells to FUS stimulation, especially the NK cells that secreted significantly higher levels of perforin and granzyme (H4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H3,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e＜0.01; Figure 6 K-L). Particularly, after splenectomy, FUS irradiation in splenic position did not show any anti-tumor effect (H6\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H5; Figure 6 A-E, K-L), which demonstrated that the spleen irradiated with FUS was the only cause of the anti-cancer effect, and directly showed that the integrity of spleen was critical during FUS sti. spleen for tumor suppression. In summary, comprehensive analysis of splenectomy and splenic nerve denervation experiments clearly confirmed that FUS activated splenic anticancer immune function through the direct FUS stimulation of immune cells, thus further suppressing tumor proliferation.\u003c/p\u003e\n\u003cp\u003eFCM results proved that\u0026nbsp;splenic nerve denervation\u0026nbsp;did not affect cell proportion (H3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H1; Supplementary Figure 19-22), particularly not weaken the modulation properties of FUS sti. spleen on the increase of the positive immune cells (especially NK cells, CD8 T cells, M\u0026phi;1 and DC1) in the body, and decrease of the negative immune cells PMN-MDSCs (H4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. H2/3; Supplementary Figure 19-22).\u0026nbsp;It fully demonstrated that FUS directly stimulated the splenic immune cells to promote their proliferation and activated the anti-cancer capability.\u0026nbsp;After the removal of the spleen, the proportion of most immune cells in the peripheral blood was significantly decreased (H5/6 \u003cem\u003evs\u003c/em\u003e. H1/2;\u0026nbsp;Supplementary Figure 20), which indicated the importance of spleen for the body\u0026apos;s immune system. However, after splenectomy, the spleen location irradiated by FUS did not show any impact on the number of immune cells (H6 \u003cem\u003evs\u003c/em\u003e. H5;\u0026nbsp;Supplementary Figure 20-22), which demonstrated that the spleen integrity or the presence of immune cells was essential for responding to FUS stimulation in tumor immunotherapy.\u003c/p\u003e\n\u003ch3\u003e3.3.2 Association of splenic positive and negative immune cells responding to FUS irradiation\u003c/h3\u003e\n\u003cp\u003eOur\u0026nbsp;previous study demonstrated that the proportion of PMN-MDSCs in the spleen and blood from tumor-bearing mice\u0026nbsp;was significantly increased, which suppressed NK cells and CD8 T cells\u0026nbsp;[69]. After FUS sti. spleen, the proportion of positive immune cells such as NK cells and CD8 T cells in the body was significantly increased, while the proportion of negative immune cells such as PMN-MDSCs was significantly decreased (Table 2-3). Why did FUS, as a non-specific physical method, cause the number of positive and negative immune cells to change in two directions? In Section 3.3.1, the depletion of NK cells and CD8 T cells did not show consistent changes of PMN-MDSCs in the spleen and blood (Q5/6 and Q7/8\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. Q1/2; Table 4-5); thus we proposed to reversely demonstrate the association of NK cells and CD8 T cells to PMN-MDSCs by depleting PMN-MDSCs. Forty\u0026nbsp;orthotopic H22 HCC mice were randomly divided into four groups: R1 group was used as the control group; R2 group\u0026nbsp;was subjected to FUS sti. spleen; R3 group was subjected to PMN-MDSCs depletion; and R4 group was subjected to\u0026nbsp;FUS sti. spleen\u0026nbsp;after PMN-MDSCs clearance.\u003c/p\u003e\n\u003cp\u003ePMN-MDSCs depletion inhibited tumor proliferation to a certain extent (R3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R1; Figure 7 A-D) and the subsequent FUS sti. spleen further enhanced tumor suppression (R4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R3; Figure 7 A-D). However, the tumor suppressive effect of FUS sti. spleen after PMN-MDSCs clearance was slightly weaker than that without PMN-MDSCs depletion (R4\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R2; Figure 7 A-C). Mice weight did not clearly show a significant difference among the four groups (Figure 7 E), but spleen weight and spleen index showed a decreased trend in the R4 group as compared to the R3 group (Figure 7 F-G).\u003c/p\u003e\n\u003cp\u003eFCM results showed that Ultra-LEAF\u0026trade; purified anti-mouse Ly-6G antibody effectively eliminated PMN-MDSCs from the spleen, blood, tumor, and para-carcinoma tissue (R3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R1; Figure 7 H-K;\u0026nbsp;Supplementary Figure 23-26). Interestingly, it significantly increased the proportion of M\u0026phi;, especially M\u0026phi;2, in the spleen and blood (R3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R1; Figure 7 H-I;\u0026nbsp;Supplementary Figure 23-24). Moreover, PMN-MDSC depletion significantly increased NK cells and CD8 T cells only in the blood (R3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R1; Figure 7 I;\u0026nbsp;Supplementary Figure 24). These results explained the reason that PMN-MDSCs clearance suppressed tumor proliferation to a certain extent (Figure 7 A-D).\u0026nbsp;Even more important, PMN-MDSC depletion in spleen did not promote the proliferation of splenic NK cells and CD8 T cells (R3\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. R1; Figure 7 H;\u0026nbsp;Supplementary Figure 23). Combined with the observation that the depletion of NK cells and CD8 T cells in the spleen did not increase the proportion of splenic PMN-MDSCs (Q5 and Q7\u0026nbsp;\u003cem\u003evs\u003c/em\u003e. Q1; Table 4), it proved that there was no direct interaction between PMN-MDSCs and NK cells or CD8 T cells. Accordingly, it is reasonable to deduce that FUS sti. spleen increased the number of NK cells and CD8 T cells while the decrease of PMN-MDSCs was not directly\u0026nbsp;correlated. PMN-MDSC decrease may be attributed to the increase of other immune cells in the spleen such as M\u0026phi;, especially M\u0026phi;2 mentioned above, or even to the rise of total tumor suppressor related immune cells in the spleen and blood. Of course, this mechanism has yet to be explored in the light of more direct and conclusive evidence.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.4 FUS regulating calcium-related signaling pathways to modulate splenic immunity\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe above results revealed that splenic NK cells and CD8 T cells were critical for suppressing tumor growth in response to FUS stimulation. Thereafter,\u0026nbsp;RNA sequencing was performed to verify their changes at the molecular level\u0026nbsp;and investigate the biological mechanism underlying this phenomenon.\u003c/p\u003e\n\u003cp\u003eAccording to the differential gene expression clustering heatmap and volcano plot, 655 upregulated genes and 344 downregulated genes were found in NK cells (Supplementary Figure 27 A-C), as well as 1869 upregulated genes and 877 downregulated genes in CD8 T cells (Supplementary Figure 27 A-C) in the FUS sti. spleen group compared with the control group (Supplementary Figure 27 D-F). Both GO and KEGG enrichment analysis showed that the DEGs were significantly enriched in pathways of cell adhesion, cell migration, immune process (i.e. ECM-receptor interaction, antigen processing and presentation, and intestinal immune network for IgA production) and signal transduction (i.e. PI3K-AKT signaling pathway, Rap1 signaling pathway, Hippo signaling pathway and calcium signaling pathway) in the FUS sti. spleen group compared with the control group (Supplementary Figure 27 G-N).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurther analysis revealed that genes related to NK cell immune function, especially activation, proliferation, and motility, were significantly up-regulated after FUS sti. spleen (Figure 8 A-C). Genes involved in calcium-related signaling pathways were also significantly highly expressed (Figure 8 D). According to GO functional annotation analysis, these upregulated genes were not only involved in the positive regulation of cell proliferation, migration and kinase activity, but also mainly involved in integrin-mediated signaling pathway and epidermal growth factor receptor signaling pathway (Figure 8 E). Moreover, MHC class II protein complex was suppressed (Figure 8 F), which promoted NK cell activation in mice as reported by Li Y. et al. [70]. Particularly, KEGG enrichment analysis of these upregulated genes showed the upregulation of PI3K-Akt signaling pathway, Hippo signaling pathway, ErbB signaling pathway, and Ras signaling pathway (Figure 8 G), which were closely associated with cell proliferation, differentiation and activation, especially under ultrasound stimulation [71-73]. Downregulated genes were significantly enriched in the intestinal immune network for IgA production (Figure 8 H), which might be beneficial to tumor suppression as previous studies reported that IgA inhibits NK activity of NK cell-enriched \u0026nbsp;lymphoid cells and gamma-interferon-treated effector cells [74, 75].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMore importantly, protein-protein networks revealed that among these significantly different signaling pathways, it is likely that the calcium signaling pathway played a central role and affected the expression of other signals to promote NK cell proliferation, migration and activation (Figure 8 I). Furthermore, GSEA analysis confirmed that FUS stimulation significantly regulated various signaling pathways of NK cells, such as up-regulating ECM-receptor interaction, PI3K-AKT signaling pathway, Hippo signaling pathway and calcium signaling pathways (Figure 8 J).\u003c/p\u003e\n\u003cp\u003eAdditionally, the genes related to CD8 T cell activation, proliferation, and motility were significantly up-regulated in the FUS sti. spleen group compared to the control group (Figure 9 A-C). Genes involved in calcium-related signaling pathways were also highly expressed (Figure 9 D). GO and KEGG enrichment and network analysis of the differential genes showed significant enrichment of pathways in signal transduction (i.e. Rap1 signaling pathway, MAPK/ERK signaling pathway, and calcium-related signaling pathways) and immune process (i.e. IL-6 production, IgA production, Fc epsilon RI signaling pathway and Fc gamma R-mediated phagocytosis) (Figure 9 E-H). Particularly, the calcium signaling pathway was involved in regulating most of the others and it was probably the initial response to FUS stimulation (Figure 9 I). GSEA analysis proved that these signaling pathways involving CD8 T cells proliferation, activation and migration, such as ECM-receptor interaction, PI3K-AKT signaling pathway, Rap1 signaling pathway and calcium signaling pathways were significantly upregulated (Figure 9 J). Although the GO and KEGG enrichment analysis of the downregulated genes showed the downregulation of MAPK signaling pathway, TNF signaling pathway, intestinal immune network for IgA production and T cell receptor signaling pathway (Figure 9 H), GSEA analysis revealed that these signaling pathways were highly expressed when the tumor-bearing mice were subjected to FUS sti. spleen (Figure 9 K).\u003c/p\u003e\n\u003cp\u003eThese functional annotations suggested that FUS\u0026nbsp;first\u0026nbsp;activated\u0026nbsp;calcium-related signaling pathways and then regulated other signaling pathways to promote the proliferation, activation, and migration of NK cells and CD8 T cells. Many previous studies confirmed that ultrasound affects the proliferation, activation, and differentiation of various cells by altering intracellular calcium-related signaling pathways to achieve specific biological effects\u0026nbsp;[76-78]. The further exploration of splenic calcium changes in response to FUS stimulation by the Von Kossa staining and\u0026nbsp;FCM\u0026nbsp;detecting Fluo-4 AM labeled cells revealed that the splenic cell population of Von Kossa stained and Fluo-4 AM labeled were observably increased in the FUS sti. spleen group as compared with the control group (Figure 10 A-B). Particularly, the results of \u003cem\u003ein vitro\u003c/em\u003e experiments (Figure 10 C) fully demonstrated that FUS stimulation promoted\u0026nbsp;NK cell\u0026nbsp;proliferation and activation to suppress cancer cell deterioration (note: H22 cancer cells were unable to adhere to the bottom and difficult to separate from NK cells that migrated from the upper layer of the transwell to the bottom layer, which led to a significant error in the subsequent detection results. Thus, Hepa1-6 cancer cells were chosen for the \u003cem\u003ein vitro\u003c/em\u003e study.). Moreover,\u0026nbsp;calcium strengthened the impact of FUS stimulation on splenic NK cells to suppress the proliferation of cancer cells \u003cem\u003ein vitro\u003c/em\u003e (Figure 10 C). Besides, ultrasound shattered cancer cell suspension further enhanced the anticancer effect of calcium-involved FUS simulation on splenic NK cells. Additionally, we also verified that calcium reinforced the effect of FUS stimulation on splenic CD8 T cells to suppress cancer cell deterioration \u003cem\u003ein vitro\u003c/em\u003e (not supplying the data). Overall, the above experimental results succinctly proved that FUS altered calcium-related signaling pathways in NK cells and CD8 T cells to reinforce the antitumor effect. However, in the future, it is worthwhile comprehensively and systematically analyzing the specific mechanism of splenic immune cells in response to FUS \u0026nbsp;stimulation by spatial region-resolved proteome and spatial single cell sequencing.\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1 Therapeutic application of FUS sti. spleen on intermediate and advanced tumors\u003c/h2\u003e\n \u003cp\u003eThe above studies in which FUS sti. spleen was performed on the second day after cancer cells implanted into the mice were equivalent to the start of the treatment in the early stage of cancer (before tumor formation). However, the anticancer effect of FUS sti. spleen after tumor formation is one of the worthiest explorations in clinical transformation and application. Therefore, a comparative study was performed to investigate the therapeutic application of FUS sti. spleen on 5th and 9th day after H22 cancer cell transplantation into the liver deemed to be tumorigenesis (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA). Four experimental groups were set as follows: K1 group was used as the control group of xenograft H22 HCC \u003cem\u003ein situ\u003c/em\u003e; K2-K4 groups were subjected to FUS sti. spleen on day 0, 4 and 8 after H22 cancer cell implantation (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eThe results showed that FUS sti. spleen on day 0, 4 and 8 significantly suppressed the proliferation of xenograft H22 HCC carcinoma \u003cem\u003ein situ\u003c/em\u003e, with an anti-tumor rate of up to ~\u0026thinsp;70%, ~\u0026thinsp;67% and ~\u0026thinsp;48% respectively (K2/3/4 \u003cem\u003evs\u003c/em\u003e. K1; Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eB-D). The results tautologically demonstrated that FUS sti. spleen exerted an evident tumor suppression, regardless of being performed before or after tumorigenesis, but the later ultrasound irradiation on the spleen was performed after the onset of the tumor, with a smaller effect on tumor suppression. Additionally, the statistical results of liver weight and mice weight showed a decreasing trend in the K2-K4 groups compared to the K1 group (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eE-F). The spleen weight and spleen index also demonstrated that FUS sti. spleen attenuated the symptoms of splenomegaly caused by cancer, as same as in Sections \u003cspan class=\"InternalRef\"\u003e3.1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3.3\u003c/span\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eG-H).\u003c/p\u003e\n \u003cp\u003eStatistic FCM results showed that the proportion of NK cells and CD8 T cells was significantly increased in the spleen, blood, tumor, and para-carcinoma tissue of the K2-K4 groups compared with the K1 group, and the proportion of PMN-MDSCs was significantly reduced (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eI-L; Supplementary Fig. 28\u0026ndash;31). However, no significant difference was found in the number of immune cells among the K2-K4 groups except that NK cells were significantly higher in the K2 group (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003eI-L; Supplementary Fig.\u0026nbsp;28\u0026ndash;31). In addition, the proportion of M\u0026phi;1 and DC1 in four tissues of the K2-K4 groups was significantly increased (Supplementary Fig.\u0026nbsp;28\u0026ndash;31). What needs to be highlighted is the inconsistency in the changes in the cell proportion of M\u0026phi;2, DC2 or other immune cells in the spleen, blood, tumor, and para-carcinoma tissue (Supplementary Fig.\u0026nbsp;28\u0026ndash;31). Our hypothesis was that this phenomenon was mainly attributed to the migration of splenic immune cells to tumor lesions after responding to ultrasonic stimulation and stressing the tumor microenvironment, which was fully demonstrated in our previous work that CD11b\u0026thinsp;+\u0026thinsp;CD43 hi Ly6C lo splenocyte-derived M\u0026phi; migrate into the lesion in a liver fibrosis model [\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eSpecial attention should be paid to the fact that the biological effect of ultrasound on spleen varies in different disease models. As reported by Nunes NS et al. [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e] and Liu T et al. [\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e], FUS sti. spleen mainly affects M\u0026phi; to inhibit the secretion of inflammatory cytokines in colitis and myocarditis. Also related to RA, CD 4 T cell, CD8 T cell, B cell and myeloid cell populations were significantly changed in response to ultrasound, specifically the CD8 T cells show an evident correlation with FUS sti. spleen based therapy [\u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e]. However, this study fully confirmed that FUS sti. spleen significantly promoted the proliferation of NK cells, CD8 T cells, M\u0026phi;1 and DC1 in various tumor models, and reduced PMN-MDSCs, but mainly suppressed cancer progression by stimulating NK cells. Additionally, the biological effects of ultrasound on different types of cells are also varied, including enhancing MC3T3-E1 osteoblasts and hematopoietic stem cell ingrowth, proliferation, and early differentiation [\u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e83\u003c/span\u003e], inducing neural progenitor cell polarization [\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e], promoting proliferation and migration of HaCaT keratinocytes [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e], facilitating the extracellular matrix synthesis of degenerative human nucleus pulposus cells [\u003cspan class=\"CitationRef\"\u003e84\u003c/span\u003e], and motivating the expression of brain-derived neurotrophic factor in astrocyte [\u003cspan class=\"CitationRef\"\u003e76\u003c/span\u003e]. However, the difference in biological effects of ultrasound irradiation on various immune cells enriched in the spleen is not yet known and needs to be detailedly studied in the future.\u003c/p\u003e\n \u003cp\u003eIn conclusion, although FUS sti. spleen non-specifically activated spleen immunological function to universally suppress tumor proliferation, it was effective on the early stage of cancer or cancer prevention. Nevertheless, it might be necessary to combine FUS sti. spleen with other treatments in patients with advanced cancer, such as applying FUS qualitatively and quantitatively intervention on the tumor (FUS int. tumor).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2 FUS sti. spleen \u0026amp; FUS int. tumor synergistically suppress tumor proliferation\u003c/h2\u003e\n \u003cp\u003ePrevious studies reported that ultrasonic intervention on tumor lesions enhances tumor suppression by promoting the proliferation, activation, and infiltration of immune cells [\u003cspan class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e86\u003c/span\u003e]. In this study, comparing and discussing FUS sti. spleen and FUS int. tumors-two strategies for immunomodulation to counteract the swelling by interfering from immune organs and tumor foci, respectively-is crucial. Five experimental groups were considered: #1 group, normal mice (mice without any treatment); #2 group, control group of subcutaneous H22 tumor mice; #3 group, FUS sti. spleen on day 0 after H22 cancer cell implantation; #4 group, FUS int. tumor; #5 group, FUS sti. spleen and FUS int. tumor synergistic treatment. According to pre-experiments and our previous results [\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e], FUS int. tumor was performed every 4 days from day 9 after the subcutaneous inoculation of H22 cancer cells (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eA), and the ultrasonic parameters were set to 3.3 MPa, 1%, 20 s.\u003c/p\u003e\n \u003cp\u003eThe results of #3 \u003cem\u003evs\u003c/em\u003e. #2 group again confirmed the ability and application prospect of FUS sti. spleen in suppressing tumor proliferation (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB-D). Moreover, FUS int. tumor showed an effective tumor suppression (#4 vs. #2 group; Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB-D), but it did not show any significant difference as compared with the #3 group (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB-D). It was noteworthy that FUS sti. spleen and FUS int. tumor synergistically induced the best tumor suppression (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eB-D). In addition, FUS sti. spleen and FUS int. tumor synergistically or either alone reduced the mice weight (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eE), but the combined application significantly exacerbated splenomegaly (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eF-G).\u003c/p\u003e\n \u003cp\u003eFCM results revealed that the proportion of NK cells and CD8 T cells in the spleen, blood and tumor was significantly and synergistically increased compared to the effect of each of the two taken individually (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eH-J). The comparison of the #3-#5 groups revealed that the number of NK cells and CD8 T cells in the spleen and blood was significantly higher after H22 tumor-bearing mice were subjected to the synergistic effect of FUS sti. spleen and FUS int. tumor compared to the effect of each of the two taken individually (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eH-I). However, such a trend in the number of immune cells was not present in tumor tissues, which was mainly attributed to the fact that FUS int. tumor not only disrupted the structure of tumor stromal barrier and cancer cells, but also killed immune cells. Additionally, FUS sti. spleen, FUS int. tumor or both synergistically significantly increased the proportion of M\u0026phi;1 or DC1 in the spleen, blood, and tumor (Supplementary Fig. 32\u0026ndash;34). However, consistent with the FCM results in sections \u003cspan class=\"InternalRef\"\u003e3.2.1\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e3.2.2\u003c/span\u003e, FUS sti. spleen had no significant consistent effect on CD4 T cells or their subtype in the spleen, blood, and tumor from subcutaneous H22 tumor-bearing mice.\u003c/p\u003e\n \u003cp\u003eFUS has been gradually developed into a clinical strategy approach for targeted tumor disruption through thermal mechanisms [\u003cspan class=\"CitationRef\"\u003e88\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e90\u003c/span\u003e]. In essence, FUS at high intensity generated focal temperatures exceeding 60 ℃ that instantaneously caused coagulative necrosis of the target soft tissue (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eK) [\u003cspan class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e92\u003c/span\u003e]. In contrast, non-thermomechanical disruption may have a more relevant role in low intensity FUS ablation, particularly in terms of immunomodulatory effects. As opposite to thermal ablation causing protein coagulation necrosis, the mechanical disruption of FUS int. tumor converts immunologically \u0026ldquo;cold\u0026rdquo; (poorly infiltrated) tumors into immunologically \u0026ldquo;hot\u0026rdquo; (well-infiltrated) ones by damaging cancer cells to release antigens, collapsing the tumor matrix barrier to enhance antigens captured by immune cells, and especially intensifying the infiltration of immune cells and cytokines into the tumor lesions, which strengthened the immunotherapeutic responses to cancer (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eK-L) [\u003cspan class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e93\u003c/span\u003e]. Although FUS int. tumor alone exerted an anti-tumor effect, to a certain extent, the tumor matrix damage would increase the risk of cancer cell spread and metastasis [\u003cspan class=\"CitationRef\"\u003e94\u003c/span\u003e]. However, in this study, the synergistic use of FUS sti. spleen and FUS int. tumor was much more effective, since tumor disruption by FUS provided an antigen source for activating splenic immune cells during the process of FUS sti. spleen to suppress tumor proliferation (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eL).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3 Optimization and unification of ultrasound system\u003c/h2\u003e\n \u003cp\u003eIn Section \u003cspan class=\"InternalRef\"\u003e3.1\u003c/span\u003e, the comparison of tumor inhibition and spleen injury under different ultrasonic parameters of FUS sti. spleen revealed that the spleen (or splenic cells) had significant ultrasound dose dependence and tolerance. This was also reported in previous studies; for example, Liu T, et al. [\u003cspan class=\"CitationRef\"\u003e80\u003c/span\u003e] successfully screened an effective ultrasonic parameter of 0.35 MPa with a 1 s on /5 s off duty cycle to significantly alleviate autoimmune myocarditis, and regulate the proportion and function of Tregs and M\u0026phi; by activating CAP. Besides, Cotero V, et al. [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] discovered that non-invasive splenic ultrasound stimulation at 0.83 MPa effectively alleviates the severity of arthritis and reduces cytokine response to endotoxin through CAP modulation based on CD4\u0026thinsp;+\u0026thinsp;T cells. However, according to a summary of studies (Table \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e), although splenic ultrasound stimulation can effectively suppress various inflammatory diseases, the ultrasonic parameters applied for spleen stimulation are extremely variable. Some studies only referred to the ultrasonic parameters mentioned in previous reports but lacked screening and optimization; for example, Hu X, et al. [\u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e] and Morton C, et al. [\u003cspan class=\"CitationRef\"\u003e95\u003c/span\u003e] used the parameters of 0.35 MPa and 1-s on/5-s off reported by Zachs D, et al. [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e] for splenic immunomodulation to treat inflammatory diseases. However, in practical applications, different ultrasound platforms lead to significant differences in ultrasonic focal region. More importantly, although some studies proved that the mechanism of splenic ultrasound stimulation for inflammatory disease therapy is based on CAP activation [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e], some other studies demonstrated the direct action of ultrasound on splenic immune cells [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e81\u003c/span\u003e]. Therefore, the dependence, specificity, and mechanism of spleen response to ultrasonic stimulation is also different, which may be one of the reasons for the different ultrasonic parameters applied in the above studies. This study also demonstrated that FUS sti. spleen to modulate immune function for tumor suppression was not mainly by activating CAP but depended on the direct stimulation of the proliferation and activation of NK cells, as well as the biological effects of ultrasonic stimulation on CD8 T cells, M\u0026phi;, DCs and MDSCs.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of the literature related to splenic ultrasound stimulation for modulating immunotherapy on various diseases.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSummary of the Experimental Content\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUltrasonic parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTherapeutic effect and mechanism\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcute colitis was induced by 2% Dextran Sulphate Sodium (DSS) in drinking water for 7 days, and therapeutic ultrasound was performed to the abdominal area.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA 5 cm2 transducer (Mettler 740\u0026times;; Anaheim, CA), 1 MHz, at 10% duty cycle and intensity of 2 w/cm2 for 7 min at day 4\u0026ndash;10.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltrasound improves DSS-induced colitis through the stimulation of the splenic nerve along with the contribution by CAP activation to decrease the levels of colonic F4/80\u0026thinsp;+\u0026thinsp;\u0026alpha;7nAChR\u0026thinsp;+\u0026thinsp;M\u0026phi;.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAutoimmune myocarditis was established by a cardiac-specific peptide, and low-intensity pulsed ultrasound stimulated the splenic nerve.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA concave transducer (OLYMPUS V302), 1 MHz, at 0.1 MPa, 0.35 MPa, 0.473 MPa, and 1 s on/5 s off bursts for 6 min or 12 min stimulation during day 0 to 21 or day 7 to 21.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSplenic ultrasound reduces heart inflammatory injury and improves cardiac remodeling by alleviating the immune response, as well as regulating the proportion and function of CD4\u0026thinsp;+\u0026thinsp;Treg and M\u0026phi; by activating CAP.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA pneumonia model was induced by lung instillation of 10\u003csup\u003e5\u003c/sup\u003e CFU of Streptococcus pneumoniae, and splenic ultrasound stimulation was performed at 4-, 16-, or 48-hours after bacteria inoculation.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA transducer (Sonic Concept H106), 1.1 MHz, at 200 mVpp, burst cycles of 150, and a burst period of 200 ms.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-invasive ultrasound stimulation targeted to the spleen demonstrates a time-dependent effect of CAP activation on the cytokine response during infection progression.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFull thickness cutaneous excisional wounds were made on a rodent model of type II diabetes, and FUS pulses were used externally to the spleen area for 3 min over the course of 15 days.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA transducer (Sonic Concept H106), 1.1 MHz, at 300 mVpp, and a pulse repetition period of 0.5 ms.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNon-invasive splenic pFUS accelerates wound closure in type II diabetes rodents by up to 4.5 days \u003cem\u003evs\u003c/em\u003e. sham controls.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcute inflammation and metabolic dysfunction were induced by LPS injection, and FUS was used for splenic nerve stimulation.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA transducer (Sonic Concept H106), 1.1 MHz, at 0.27 duty cycle, 136.36 us pulse length, 0.83 MPa peak positive pressure, and a single stimulus not surpassing a single 1 min pulse.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltrasound applied to spleen reduces cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation by modulating the CAP.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInflammatory arthritis was induced through arthritogenic donor serum, and the spleen was irradiated by ultrasound one day prior to serum transfer.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA single element ultrasound transducer, 1 MHz, at 350 kPa, 1 s on/5 s off pulse pattern, and 2 min per day for 7 consecutive days.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltrasound stimulation targeting the spleen significantly reduces disease severity in a mouse model of inflammatory arthritis mainly attributed to splenic T and B cells playing an anti-inflammatory effect.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBilateral renal ischemia-reperfusion injury (IRI) was performed by flank incisions by clamping the renal pedicle for 26 minutes, and ultrasound was applied 3\u0026ndash;7 days before IRI.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA Sequoia 512 US machine with a small-animal 15L8w transducer (Acuson, Malvern, PA), 7 MHz. mechanical index of 1.2, duration of 1 second to destroy microbubbles, and once every 6 seconds for 2 minutes.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUltrasound prevents renal ischemia-reperfusion injury by stimulating the splenic cholinergic anti-inflammatory pathway.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRA was induced by type II collagen, and FUS irradiation on the spleen was performed one day prior to collagen injection.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA FUS concave transducer (Panametrics-NDT, Waltham, MA, USA), 1 MHz, at 350 kPa, 1 s on/5 s off bursts, and 12 min per day.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpleen\u0026shy;targeted low\u0026shy;frequency pulsed FUS effectively improved the severity of arthritis by regulating various cell subpopulations, especially CD8\u0026thinsp;+\u0026thinsp;T cell subsets.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAt present, there is a lack of studies on splenic ultrasound stimulation to regulate immunotherapy for various diseases; thus, ultrasonic parameters are not objectively consistent, which seriously restricts the translational progress of clinical application. Even if the ultrasonic parameters were screened and optimized in this study, there are still some shortcomings. For example, when the ultrasonic intensity is less than 2.3 MPa, the effect of a longer duration of ultrasound irradiation on splenic immune modulation for tumor suppression needs to be further studied. Fortunately, there are two studies of splenic ultrasound stimulation for immunotherapy on inflammatory diseases registered in \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.clinicaltrials.gov\u003c/span\u003e\u003c/span\u003e (NCT03690466, and NCT03548116), but their clinical results are not yet officially published. We look forward to their positive news, and expect that FUS sti. spleen can be widely employed in clinical treatment to alleviate patient suffering.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIRI, ischemia-reperfusion injury; VNS, vagus nerve stimulation; \u0026alpha;7nAChR, \u0026alpha;7 nicotinic acetylcholine receptors; FUS, focused ultrasound; DC, dendritic cell; NK, natural killer; MDSC, myeloid-derived suppressor cell; M-MDSC, monocytic myeloid-derived suppressor cell; PMN-MDSC, polymorphonucler myeloid-derived suppressor cell; M\u0026phi;, macrophage; Treg, regulatory T cells; FCM, flow cytometry; HCC, hepatocellular carcinoma; TNF-\u0026alpha;, tumor necrosis factor \u0026alpha;; CAP, cholinergic anti-inflammatory pathway; NE, norepinephrine; ACh, acetylcholine; IgA, immunoglobulin A.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6 Financial supports\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 12204370), the Innovation Ability Supporting Program of Shaanxi Province (No. 2023WGZJ-ZD-09), and the Basic-Clinical Integration Innovation Project of Xi \u0026apos;an Jiaotong University (No. YXJLRH2022092).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7 Conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interest or interest otherwise.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8 Authors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design of the study:\u0026nbsp;Zongfang Li,\u0026nbsp;Jing Geng, Wei Dong.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperiments and procedures:\u0026nbsp;Wei Dong, Guihu Wang, Senyang Li, Qian Wang, Wenjuan Li, Heyuan Liu, Yingxue Liang, Zhe Zhou, Xinrui He, Wenlei Guo, Jianing Yuan, and Yichao Chai.\u003c/p\u003e\n\u003cp\u003eWriting of article:\u0026nbsp;Wei Dong, Guihu Wang, Wenjuan Li, Jing Geng, Zongfang Li.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9 Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Hongwei Tian, Gaixia He and Haiyan Chen for providing technical support in preparation of animal models. We thank Pro. Pengfei Liu, Pro. Guangyao Kong, Pro. Yujin Zong, Pro. Shemin Lv and Pro. Tielin Yang for their careful guidance on the design of experimental scheme. In addition, Wei Dong thanks the care and support from Yinggang Zhang, Shoufei Qu, Yameng Wei, Shaoying Zhang, Pro. Jun Li and Pro. Fanpu Ji in the past 2 years.\u003c/p\u003e\n\u003cp\u003eWe would like to thank MogoEdit (https://www.mogoedit.com) for its English editing during the preparation of this manuscript. And we sincerely thank\u0026nbsp;Shanghai OE Biotech Co., LTD (https://www.oebiotech.com/) for the RNA sequencing and data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10 Supplementary data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary data to this paper can be found in Supplementary Figures \u0026amp; Tables.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11 Data availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw and analyzed sequencing data in this study have been deposited in the NCBI\u0026rsquo;s Gene\u003c/p\u003e\n\u003cp\u003eExpression Omnibus (under series accession code GSE267237 and GSE267445).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVerlinden TJM, van Dijk P, Hikspoors J, Herrler A, Lamers WH, K\u0026ouml;hler SE. Innervation of the human spleen: A complete hilum-embedding approach. 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Front Neurosci. 2022;16:1039960.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"focused ultrasound, spleen, FUS sti. spleen, splenic immunomodulation, tumor suppression","lastPublishedDoi":"10.21203/rs.3.rs-4639146/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4639146/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSplenic immunomodulation triggered by ultrasound shows a significant anti-inflammatory effect against various inflammatory diseases, whose mechanism is mainly attributable to the activation of cholinergic anti-inflammatory pathway (CAP). However, the potential role and underlying mechanism of splenic ultrasound stimulation in cancer management have been rarely reported and superficially defined. This study first assessed the universal therapeutic effect of focused ultrasound precisely stimulating spleen (FUS sti. spleen) on various tumors under specific ultrasonic dose. It fully demonstrated that FUS directly stimulated splenic immune cell proliferation and activation (especially NK and CD8 T cells) rather than CAP excitation to modulate splenic immune function. Particularly, NK cells are much more indispensable and important in responding to FUS stimulation for cancer suppression than CD8 T cells. RNA sequencing of NK and CD8 T cells, as well as \u003cem\u003ein vitro\u003c/em\u003e experiments revealed that FUS firstly regulated calcium-related signaling pathways to further modulate other signaling pathways, such as ECM-receptor interaction, PI3K-AKT signaling pathway, Rap1 signaling pathway and Hippo signaling pathway to promote immune cell proliferation, migration and activation to suppress cancer cell deterioration. Particularly, FUS sti. spleen and FUS intervention on the tumor synergistically induced the best tumor suppression than each of the two taken individually.\u003c/p\u003e","manuscriptTitle":"Noninvasive focused ultrasound non-specifically activating spleen immunological function to suppress tumor proliferation: a new strategy for cancer immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 06:35:23","doi":"10.21203/rs.3.rs-4639146/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"400ca36a-589e-4b81-97c3-65f10ea3e94c","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-19T13:08:28+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 06:35:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4639146","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4639146","identity":"rs-4639146","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}