A MR/PA Dual-Modality Imaging for quantitative evaluation of tumor vascular and immune microenvironment

A MR/PA Dual-Modality Imaging for quantitative evaluation of tumor vascular and immune microenvironment | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A MR/PA Dual-Modality Imaging for quantitative evaluation of tumor vascular and immune microenvironment Nan Xu, Dan Wu, Jingyan Gao, Huabei Jiang, Qinqing Li, Shasha Bao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2222488/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 Backgroud: Tumor radiotherapy combined with immunotherapy for solid tumors has been proposed, but tumor vascular structure abnormalities and immune microenvironment often affect the therapeutic effect of tumor, and there are few reports on multimodal imaging markers in the study of tumor therapeutic response. The purpose of this study was to evaluate the dynamic monitoring of tumor blood vessels and microenvironment induced by radiotherapy by magnetic resonance imaging /photoacoustic imaging (MR/PA) imaging, and to explore the therapeutic effect of tumor radiotherapy combined with PD-L1 immunocheckpoint inhibitor. Methods The tumor-bearing mice (TBM) were randomly allocated to six groups: the low-dose group, high-dose group, low-dose + PD-L1 group, high-dose + PD-L1 group, anti-PD-L1 group and control group. Animals received 2 Gy/14 Fx (low-dose group) or 8 Gy/3 Fx (high-dose group) radiation and the combination treatment groups were given an anti-PD-L1 antibody for two consecutive weeks. MR/PA imaging was used to noninvasively evaluate the response of breast cancer model to different doses of radiotherapy, combined with histopathological techniques to observe the changes of tumor vessels and microenvironment. Results The inhibitory effect of high-dose radiotherapy on tumors was significantly greater than that of low-dose radiotherapy, with the MR images revealing that the signal intensity (SI) decreased significantly (p༜0.05). Compared with those in the other groups, the tumor vascular density decreased significantly, and the vascular maturity index (VMI) increased significantly in the low-dose group (p༜0.05). The PA images showed that the deoxyhemoglobin (HbR) and total hemoglobin (HbT) levels decreased and the SO 2 level increased after radiation treatment. In addition, the high-dose group had an increased number of tumor-infiltrating lymphocytes (CD4 + T and CD8 + T cells) and natural killer (NK) cells and increased PD-L1 expression in the tumors (p༜0.05). The combination of radiotherapy and immunotherapy increased the survival rate of the mice, and a regimen of an 8 Gy dose of radiation combined with immunotherapy inhibited tumor growth and increased the survival rate of the mice to a greater degree than the 2 Gy radiation dose with immunotherapy combination (p = 0. 002). Conclusion Differential fractionation radiotherapy doses exert different effects on tumor vascular remodeling and the immune microenvironment, and MR/PA can be used to evaluate tumor vascular remodeling after radiotherapy, which is of great significance for the clinical applications of radiotherapy combined with immunotherapy. tumor microenvironment immunotherapy tumor vasculature radiotherapy magnetic resonance imaging photoacoustic imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cancer immunotherapy is based on the immunogenicity of tumors, which is achieved through the induction of active or passive immunity for tumor suppression and clearance ( 1 , 2 ). However, low immune response rates and drug resistance remain problems in immunotherapy ( 3 ). During tumor progression, the interactions between the tumor microenvironment and tumor cells mediate tumor immune tolerance, which affects the clinical effect of immunotherapy. The state of the tumor vasculature plays an important role in immunotherapy. For effective immunotherapy, sufficient T cells must sufficiently infiltrate tumor tissue; however, abnormal tumor vessels reduce immune cell infiltration through a variety of mechanisms, thereby limiting immunotherapy effectiveness. Moderate antitumor angiogenic therapy can correct the immature and chaotic blood vessels in the tumor, rendering tumor angiogenesis more dynamically balanced, and this improvement of vascular structure combined with immunotherapy drugs can lead to obvious therapeutic effects ( 4 , 5 ). However, antiangiogenic drugs can lead to many complications and cannot effectively stimulate tumor immunogenicity, which limits their applications in the comprehensive treatment of tumors ( 4 ). Radiotherapy not only can kill tumor cells but can also directly promote immune-mediated tumor rejection by enhancing the total effect of tumor antigen processing and cross presentation, making tumor cells sensitive to immune recognition and attack, and increasing the immunosuppressive state of the tumor microenvironment, which benefits the recruitment, activation and action of T cells ( 6 , 7 ). In addition, tumor vessels are properly pruned but not completely destroyed by radiotherapy ( 8 ), the distribution of blood vessels is more uniform, the interstitial pressure in the tumor tissue is decreased, and the oxygen content in the tumor tissue and the distribution of combined therapy drugs are maximized ( 9 ). Therefore, accurate evaluation of tumor vascular repair and microenvironmental changes is the key to the success of tumor combined therapy ( 10 ). To date, imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging (MRI), computed tomography (CT) perfusion imaging, sonography and nuclear medicine are often used to evaluate tumor vessels ( 11 – 13 ). Adequate detection of complex vascular changes is difficult with a single imaging method, although MRI and ultrasound are the measurements most reported in the clinical literature, and their reliability and stability are worthy of further observation. Photoacoustic imaging (PAI) is an emerging imaging technique that combines optics with ultrasound and can provide tissue structure and functional information. PAI has higher resolution than traditional ultrasonic imaging, and its image resolution can reach the order of microns, which can achieve high-resolution tissue imaging ( 14 ). Second, PAI uses different tissues and components in the body to reflect tissue structure and functional information by different light absorption of different wavelengths ( 15 ). In addition, PAI can achieve noninvasive physiological and pathological imaging at the molecular level by combining molecular imaging probe technology and contrast-enhanced imaging technology ( 16 ). Finally, through MRI to provide tumor structure information, perfusion information and tumor vascular information, hypoxia status and other functional information provided by PAI, we can accurately identify the changes in tumor blood vessels and the corresponding microenvironment ( 17 , 18 ). Hence, we conducted this study to evaluate whether MR/PA can potentially be used to assess tumor vascular remodeling after radiotherapy and to observe changes in the immune-related tumor microenvironment at the molecular biological level with the aim of optimizing combination immunotherapy. Methods And Materials Cell Line and Animals Mouse 4T1 breast cancer cells were provided by the Chinese Academy of Sciences Stem Cell Bank and cultured in RPMI-1640 containing 10% fetal bovine serum and penicillin–streptomycin. Female BALB/c mice (5–6 weeks old, weighing approximately 18–20 g) were purchased from the Department of Experimental Animals, Kunming Medical University. The Experimental Animal Ethics Committee of Kunming Medical University approved this study, and all experimental protocols were conducted in accordance with the committee’s guidelines. Approximately 1×10 6 cells in 200 µL of phosphate-buffered saline (PBS) were inoculated subcutaneously in the right lower limb of the mice. Twelve days after implantation, these 4T1 mouse breast cancer models (V tumor ≥200 mm 3 ) were used for treatment experiments. The tumor volume calculation formula was V = 0.5×a×b 2 , where a is the vertical long diameter measured by calipers and b is the vertical short diameter. Tumor Treatment The tumor-bearing mice (TBM) were randomly allocated to six groups: the low-dose group, high-dose group, low-dose + PD-L1 group, high-dose + PD-L1 group, anti-PD-L1 group and control group. Animals received 2 Gy/14 Fx (low-dose group) or 8 Gy/3 Fx (high-dose group) radiation for two consecutive weeks. The combination treatment groups were given an anti-PD-L1 antibody (clone 10F.9G2, isotype rat IgG2b, Bio-Xcell) by intraperitoneal injection (200 µg/3 Fx, on days 12, 18, and 25), and the control group was injected with the same amount of normal saline and evaluated in a survival analysis. The TBM were exposed to X-ray radiation from an RS2000 Biological X-ray Biological Irradiator with an accelerating voltage of 225 kV and a dose rate of 1.8 Gy/min (Georgia, USA). To prevent radiation exposure of normal tissue, the mice were immobilized in the irradiator, the area away from the tumor was covered by a lead screen, and the other parts of the body were covered with lead screens to minimize the radiation to normal tissues. Tumor volume and body weight were measured every 2 days. MR/PA Bimodal Imaging and Analysis The mice from the radiotherapy group and the control group were scanned on days 12 and 26 with a 3.0 T MRI scanner (Ingenia, Philips Medical Systems, Netherlands). The mice were injected intraperitoneally with 200 µL of Mn-Au contrast agent (APN-AU-40, 50 mmol/L MnCI 2 , and 7x10 10 N/mL) 24 hours before imaging. During imaging, the mice in the prone position were anesthetized with 10% chloral hydrate administered intraperitoneally, and eight-channel phased array coils were used for scanning the animals. The tumor was placed in the center of the coil, and the Mn-Au contrast agent was placed in the opposite lower limb of the tumor for image signal intensity (SI) comparison. TSE-T1WI was performed with the following parameters: a repetition time (TR)/echo time (TE) of 510 ms/27 ms, an average signal number (NSA) of 8, a slice thickness/slice spacing of 1.5 mm/0.15 mm, a slice number equal to 14, a flip angle of 90°, a scanning field of view (FOV) equal to 5 cm × 5 cm, a matrix of 200 × 198, and a total imaging time of 588 s. The image data were analyzed using software on multifunction workstations (Syngo.via, version VB10B, Siemens, Erlangen, Germany). The regions of interest (ROIs) were drawn manually on the tumor (including the periphery and center of the tumor) and the contrast agent on the image of one slice, and each region ROI consisted of approximately the same size (approximately 0.5 mm 2 ) and shape. The SI produced by the Mn-Au contrast medium was obtained. The mean signal of the contrast agent was standardized in each slice. The enhanced Mn 2+ intensity in the peripheral and central tumor ROIs was calculated by the following formula: SI = SMn (Con)-SMn (IP (per/c) )/SMn (air), in which SMn (IP per ), SMn (IP c ) and SMn (air) represented the SI of the peripheral and central Mn 2+ of the tumor and the SI of the peripheral air, respectively. The raw PAI data in this study were obtained with an in vivo laboratory-built PAI image acquisition system (Fig. 1 ). A laser (Surelite OPO, Continuum USA) emitted pulses with a width of 6–7 ns and a wavelength of 690–960 nm at a repetition rate of 20 Hz to excite the target and thus produce PA signals. The energy of the pulsed light was controlled to extend 10 mJ/cm 2 below the surface of the animal’s skin, which was well beneath the safety standard (20 mJ/cm 2 ). A 128 element half-ring ultrasonic array probe (with a center frequency of 5 MHz and a bandwidth of 75%, Japan Probe Co., Ltd.) was used to receive the PA signal. The received PA signal was amplified with a preamplifier (gain of 54 dB and frequency range from 460 kHz to 9 MHz) made in house, and the signal was collected through data acquisition cards (NI 5105). Ten-time averaging of the signal can minimize laser energy instability, and thus, the image quality is improved, making the calculation of the tumor area more accurate. During the acquisition process, real-time imaging was realized with a LabVIEW panel. The PAI tumor images were reconstructed from the PA signals using a delay and sum reconstruction algorithm. The PAI tumor images were reconstructed from photoacoustic signals using a delay and sum reconstruction algorithm. According to the algorithm, we identified changes in the cerebral deoxyhemoglobin (HbR), oxyhemoglobin (HbO) and oxygen saturation (SO 2 ), levels in tumor regions at two different wavelengths (760 nm and 840 nm). The absorption coefficient of HbR is higher than that of HbO at 760 nm, while the absorption coefficient of HbO is higher than that of HbR at 840 nm. The HbO, HbR and SO 2 changes were calculated pixel by pixel via Eqs. 1 and 2, and the pixels were chosen according to the calculated total hemoglobin (HbT) concentration ( \({C}_{HbT}={C}_{HbO}+{C}_{HbR}\) ), which was greater than 0. Tumor Immunofluorescence All mice were sacrificed by spinal dislocation. Tumor tissues were fixed with 4% paraformaldehyde and cut into 4-µm-thick sections after dehydration and embedment. In brief, specimens were incubated with anti-CD31 (1:500, Abcam, ab182981), anti-α-SMA (1:200, Servicebio, GB13044), anti-ki67 (1:200, Servicebio, GB111141), anti-HIF-1α (1:200, Cell Signaling Technology, 36169T), and anti-PD-L1 (1:200, Servicebio, GB11339A) antibodies for staining. The slices were imaged with a NIKON ECLIPSE C1 fluorescence microscope and scanned and analyzed with a PANNORAMIC panoramic slice scanner and Image-Pro Plus 6.0 analysis software. Tumor slices were divided into three equal areas, and the microvessel density (MVD) of each area was detected by CD31 staining of perivascular cells. The area was quantified on the basis of the total number of microvessels per unit area; similarly, the coverage of the perivascular cells stained for α-SMA in each area was calculated. The vascular maturity index (VMI) refers to the percentage of blood vessels stained with the anti-α-SMA antibody compared to the total number of blood vessels stained with CD31 ( 19 – 21 ). An apoptosis kit (Servicebio, G1501) was used to perform TUNEL assays and thus evaluate the number of apoptotic tumor cells. Tumor cell proliferation was quantitatively analyzed based on the percentage of the number of positive cells stained for Ki-67 in each tumor area compared to the total number of cells. The ratio of HIF-1α-positive cells to total cells per unit area was calculated to analyze tumor hypoxia, and the proportion of PD-L1-positive cells to total cells was analyzed. Flow Cytometry To obtain a single-cell suspension, the tumor tissue was mechanically cut into small pieces and subjected to cell filtration. The number of single cells in suspension was counted, and the cells were stained with specific antibodies against CD4 (BioLegend, clone GK1.5, 100408), CD8a (BioLegend, clone 53 − 6.7, 100707), CD16 (BioLegend, clone S17014E, 158007), CD3 (BioLegend, clone 17A2, 100273), CD163 (BioLegend, clone S15049I, 155308), FOXP3 (BioLegend, clone 150D, 320011), CD25 (BioLegend, clone PC61, 102015), CD68 (eBioscience, clone FA-11, 25-0681-80), CD80 (eBioscience, clone 16-10A1, 11-0801-81), and CD56 (Santa Cruz, clone 123C3, SC-7326). The expression levels of CD4, CD8, tumor-associated macrophages (TAMs), regulatory T (Treg) cells, and natural killer (NK) cells were detected with an acoustic focus flow cytometer (Invitrogen Attune NxT, AFC2), and the data were analyzed using FlowJo software (Tree Star Inc.). Western Blot Analysis The apoptosis of tumor cells in the tumor microenvironment was detected by Western blotting. As previously described( 22 ), the tumor tissue was mechanically lysed with RIPA cleavage buffer (Solarbio, Beijing, China). After 30 min of incubation on ice, the total protein was extracted from the cells, centrifuged for 15 min at 4°C and 12000 rpm, and quantified with a BCA protein assay kit (Biosharp, Hefei, Anhui, China). The protein in the supernatant was dissolved in 5x electrophoresis sample buffer at a ratio of 1:4, heated to a high temperature and stored at -20 ℃ until use. The isolated proteins were separated by polyacrylamide gel electrophoresis (Biosharp, Hefei, Anhui, China), transferred to a polyvinylidene fluoride membrane (0.22 µm, Merck KGaA, Darmstadt, Germany), soaked in TBST (Solarbio, Beijing, China) blocking solution containing 5% w/v skim milk (Biofroxx, Germany) for 2 hours, and then incubated for 0.5 hours with a primary antibody; horseradish peroxidase-bound secondary antibodies (1:10000, Cell Signaling Technology, #7074) were added to the membrane and incubated for 1 hour at room temperature. Cleaved caspase-3 was visualized with an enhanced chemiluminescence enhancement kit (Biosharp, Hefei, Anhui, China), and the protein expression level was detected with ImageJ software (GE Healthcare Bio-Sciences, Uppsala, Sweden). The image was decolorized into an 8-bit black-and-white image, and its gray value represented the grayscale state of the image. The depth and area of the bands in the image comprehensively represented the amount of protein. The following antibody was used: rabbit anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, 9664). Statistical Analysis All data are expressed as the means ± standard deviations (SD). Independent-samples t test, one-way ANOVA or nonparametric tests were performed to assess the differences between different groups. The correlation between MR/PA parameters and histological results was analyzed on the basis of Pearson correlation coefficients. SPSS 25.0 software (SPSS Company, Chicago, Illinois, USA) was used for the statistical analysis, and the survival data were analyzed by the Kaplan–Meier test; p < 0.05 was considered to be statistically significant. Results Tumor Volume The changes in tumor volume were continuously observed after implantation of the breast cancer cells (Fig. 2 A). No significant difference in tumor volume was observed among the three groups before radiotherapy. On the 10th day, the tumor volume was 205.42 ± 2.69 mm 3 in the control group, 199.08 ± 2.18 mm 3 in the low-dose group and 203.33 ± 1.79 mm 3 in the high-dose group. After fractionation radiotherapy, the percentage of tumor volume increased on the 26th day: in the control group, it increased by 83.60%, in the low-dose group, it increased by 77.59%, and in the high-dose group, it increased by 71.21%. Compared with tumor progression in the control group, radiotherapy significantly inhibited tumor progression (p < 0.001). The tumor growth in the high-dose group was lower than that in the low-dose group (p < 0.001) (Fig. 2 B). MR/PA Imaging MR images were obtained from each group of mice before and after radiotherapy. The images taken before radiotherapy showed no significant difference in signal intensity among the control group, low-dose group and high-dose group (p > 0.05). The radiotherapy group image showed a low enhancement area (Fig. 3 A), which was significantly different than that taken before radiotherapy. This finding suggested local necrosis inside the tumor. Although the images of the control group showed localized hypointense areas inside the tumor compared with those in the images taken before radiotherapy, the difference in tumor signal change was not significant. Further analysis showed that compared with that in the control group, the signal enhancement of the tumor center was decreased in the radiotherapy groups, and the SI decreased more significantly in the high-dose group (p < 0.05) (Fig. 3 B). The SI in the peripheral region of the tumor was also decreased after radiotherapy (p < 0.05) (Fig. 3 B). In the PAI experiments, 5 representative images were continuously obtained for quantitative analysis, as shown in Fig. 4 A. The levels of HbT and HbR in the high-dose group and low-dose group were significantly lower than those in the control group (p < 0.05) (Fig. 4 B). The levels of HbO 2 and SO 2 in the high-dose group and low-dose group was higher than that in the control group, and there was a significant difference in SO 2 between the high dose group and the control group (p < 0.05). Further analysis showed that HbT (Fig. 4 C) and HbR (Fig. 4 D) levels in different regions of the tumor in the radiotherapy groups were different from those in the control group. In the high-dose and low-dose groups, the HbT levels in the central area of the tumor were lower than those in the peripheral region in the high-dose (p < 0.05), and the HbR levels in the central area of the tumor were lower than those in the peripheral region (p < 0.05). There was no regional difference in HbT and HbR levels in the control group (p < 0.05). Immunofluorescence Assay of the Tumors Compared with that in the control group, the number of Ki67-positive proliferative cells decreased (p < 0.01), and the number of TUNEL-positive apoptotic cells increased in both the low-dose and high-dose groups (Fig. 5 ). Furthermore, in the high-dose group, the number of Ki-67-positive cells was decreased most significantly (p < 0.01), and the number of TUNEL-positive cells increased most significantly (p < 0.05). In the high-dose group, Ki-67 was positively correlated with SI(per) (r = 0.879, p = 0.021) and SI(c) (r = 0.938, p = 0.006), but TUNEL staining was negatively correlated with SI(c) (r=-0.867, p = 0.025). In the low-dose group, Ki-67 was positively correlated with SI(per) (r = 0.826, p = 0.043) and SI(c) (r = 0.907, p = 0.012). Tumor sections were stained with anti-CD31 and anti-α SMA antibodies to detect tumor vessels (Fig. 5 ). Compared with that in the control group, the number of blood vessels decreased; in the low-dose group, it decreased to 48.93% (p < 0.05), and in the high-dose group, it decreased to 14.67%. In addition, compared with the control group, radiotherapy increased the VMI; in the low-dose group, it increased to 55.13% (p < 0.05), and in the high-dose group, it increased to 29.60% (p༞0.05). Tumors were stained with anti-PD-L1 and anti-HIF-1α antibodies (Fig. 5 ), and the effects of radiation on tumor immunity and hypoxia were evaluated. Compared with that in the control group, radiotherapy increased the expression of PD-L1 in the tumor, which increased by 25.69% in the low-dose group and 30.8% in the high-dose group, and the expression of PD-L1 in the high-dose group was significantly higher than that in the control group (p < 0.05). Compared with the control group, radiotherapy slightly reduced the content of HIF-1α in the tumor tissue (p༞0.05). Expression of Cleaved Caspase-3 in the Tumor Tissue Compared with the control group, the expression of cleaved caspase-3 in the high-dose group and low-dose group was significantly increased (p < 0.01) (Fig. 6 ). Effect of Radiotherapy on Immune Cells in Tumors To determine the changes in the number of tumor immune cells after radiotherapy, tumor-associated immune cells were analyzed by flow cytometry. Compared with the control group, the high-dose CD4 + group showed an increase of 73.96% (p = 0.006), CD8 + cells showed an increase of 74.91% (p = 0.015), and NK cells showed an increase of 29.25% (p = 0.001); the low-dose CD4 + group showed an increase of 67.46%, CD8 + cells showed an increase of 3.89%, and NK cells showed an increase of 15.98% (Fig. 7 A, B, E). However, there was no significant difference in the levels of CD4 + , CD8 + , and NK cells between the low-dose group and the control group. In addition, no significant changes in TAMs (p = 0.643) or Treg cells (p = 0.932) were observed in the radiotherapy groups compared to the control group (Fig. 7 C, D). Therapeutic Effects of the Combination Therapy on the Tumors To evaluate the efficacy of combination therapy, the mice were treated with a single anti-PD-L1 antibody, high-dose irradiation + the anti-PD-L1 antibody (8 Gy/3 Fx, 200 µg/3 Fx on days 12, 18, and 25), or low-dose irradiation (2 Gy/14 Fx) + the anti-PD-L1 antibody (200 µg/3 Fx on days 12, 18, and 25) (Fig. 8 A). During the course of treatment, the tumor growth in the three treatment groups was significantly lower than that in the control group (p < 0.01). Compared with anti-PD-L1 therapy alone, low-dose irradiation + anti-PD-L1 antibody and high-dose irradiation + anti-PD-L1 antibody treatment significantly slowed tumor progression (p < 0.01) ( Fig. 8 B ) . Compared with the control group regimen, the dose regimen of combined therapy showed a more significant inhibitory effect on tumor growth (p < 0.01) and a tendency to increase the survival rate (p < 0.01) (Fig. 8 C). Furthermore, compared with anti-PD-L1 antibody therapy alone, the survival of the low-dose irradiation + anti-PD-L1 antibody therapy group was significantly increased (p = 0.035), and this increase in the survival rate was the most obvious in the high-dose irradiation + anti-PD-L1 antibody treatment group (p = 0. 002). Discussion In this study, radiotherapy induced tumor vascular remodeling by changing the vascular density and oxygenation level and increased the infiltration of immune cells and the expression of PD-L1 in the tumors, thus promoting the inhibition of tumor immune environment activation. Compared with histopathological biopsy, noninvasive quantitative imaging is valuable in providing anatomical and vascular information on the tumor response to radiotherapy. To the best of our knowledge, this is the first study to use MR/PA bimodal imaging to observe the response of tumor vessels and the microenvironment to different radiotherapy regimens in tumor-bearing mice. Tumor growth is affected in many ways during radiotherapy, and the differences in a radiotherapy fractionation scheme are important factors in controlling tumor progression and metastasis. In this study, we found that tumor growth was continuously inhibited by radiotherapy, and the effect of high-dose fractionated radiotherapy was more obvious than that of low-dose fractionated radiotherapy. Of course, this effect may have been related to our radiotherapy program. The inhibitory effect of low-dose fractionation radiotherapy on tumor cell growth was short-lived and weak, while interval high-dose fractionation radiotherapy can cause a wide range of DNA double-strand breaks, destroy the ability of cellular self-repair, and thus accelerate tumor cell apoptosis. Therefore, we should not only ensure the inhibitory effect of high-dose radiotherapy on tumor growth but also consider the interval of treatment. Ki-67 expression, the best marker of cell proliferation, and apoptosis marker expression were assessed to compare the effects of dose differences on tumor growth. The apoptosis index significantly increased, indicating that high-dose irradiation caused tumor cell death and effectively prevented tumor cell proliferation. In addition to analyzing the growth status of tumors, we also used manganese-enhanced MRI to monitor the changes in apoptosis of tumor cells after radiotherapy. Manganese ions are actively transported to biologically active cells through calcium channels, so T1-weighted positive signals of magnetic resonance imaging were used to reflect the survival status of cells, especially to observe the response of tumor cells to different doses of radiotherapy ( 23 , 24 ). Compared with that of low-dose radiotherapy, the killing effect of high-dose fraction radiation on tumor cells was more direct and persistent. After high-dose radiotherapy, the degree of signal enhancement in the tumor center was decreased significantly, and the peripheral area of the tumor was more sensitive to high-dose radiotherapy and showed little enhancement, suggesting that the tumor cells in the peripheral region underwent apoptosis at a significant rate. More importantly, through MRI, we found that the radiosensitivity in different regions of the tumor differed. This finding may have been a result of phenotypic and genetically diverse tumor cell populations, and the proliferation rate and therapeutic sensitivity of these cells may differ ( 25 ). In addition, when tumor cells recruit stromal cell groups, such as immune cells and endothelial cells, the tumor microenvironment is remodeled, resulting in spatial variation within the tumor ( 26 ). The resulting spatial distribution of tumor and stromal cells leads to the progression of different physiological subregions in tumors and differences in the resistance of tumor areas to treatment. The SI in the MR images of the tumors was positively correlated with Ki-67 staining and negatively correlated with TUNEL staining, which indicated that MRI can potentially be used to monitor tumor growth inhibition induced by radiotherapy and to help evaluate the sensitivity of tumor cells to the radiation dose. Many aspects of vascular biology are affected by radiation and vary according to the dose/grade, time, and model studied ( 27 ). Radiotherapy may preferentially prune poorly covered/functional vessels to redistribute blood vessels, but this vascular change may not systematically translate into long-term effects on the tumor ( 28 , 29 ). Compared with a single dose of high-intensity irradiation, our 2 Gy × 14 and 8 Gy × 3 doses did not cause severe irreversible damage to tumor vessels. After 2 Gy × 14 treatment, the VMI was increased because of the effective stimulation, which may have been related to the retention and improvement of vascular system formation in the first few days after radiotherapy ( 30 ). After 8 Gy × 3 exposure, the MVD did not decrease significantly, possibly because of the death of vascular endothelial cells to varying degrees, which would have been quickly attenuated by the formation of new blood vessels, emphasizing the contribution of the fractionation radiotherapy scheme to tumor vessel remodeling. Hypoxia is a consequence of a high tumor cell proliferation rate and an abnormal structure of the tumor vasculature ( 31 ), and HIF-1α is the main regulator of hypoxic transcription. There was no significant change in the expression of HIF-1α after 2 Gy and 8 Gy exposure. This outcome may have been due to the reoxygenation of tumor parenchyma cells that survived radiotherapy, an outcome that largely depends on the radiation scheme (32). A complex dose–response relationship between radiation-injured tumor vessels, tumor cell reoxygenation and reaggregation is suggested. During PAI monitoring of tumors after radiotherapy, we found that the contents of HbT and HbR in tumor vessels decreased and SO 2 increased in high-dose group, suggesting that the morphology and oxygenation level of tumor vessels was affected to varying degrees, which may depend on the size of tumor vessels ( 33 ). The difference in the response of tumor vessels to different doses of radiation reflects the spatial heterogeneity of the tumor, especially the changes in blood vessel density and oxygenation level and the redistribution of blood flow. Anum and others called the physiological tumor habitat, and differences in imaging and histological findings are evident among different vascular and cellular tumor habitats ( 34 ). In general, the PAI parameters confirmed vascular degenerative changes after radiotherapy and were able to detect subtle changes in tumor oxygenation levels with high sensitivity, suggesting that the degree of vascular degeneration is related to radiotherapy dose, radiation-dose fractionation, regional tumor heterogeneity and other factors. As an immunomodulator, radiation can directly stimulate immune cells to produce cytokines and chemokines that affect the local immune response, and this immune response varies on the basis of the radiotherapy program and tumor model ( 35 – 37 ). The activation status of T and NK cells is suggestive of antitumor immunity; immune cells can kill target cells via various mechanisms following activation. Radiation can enhance the cytokine secretion and cytotoxic activity of NK cells ( 38 ). Compared with the 2 Gy × 14 treatment, 8 Gy × 3 better promoted NK-cell recruitment to the tumor microenvironment, which may have been due to an increase in the radiation dose that was conducive to the release of tumor exosomes, promoting NK-cell polarization ( 39 , 40 ). According to our results, compared to the low dose, 8 Gy × 3 can better promote the aggregation of CD4 + T cells and CD8 + T cells in the tumor. The CD4 + T-cell population contains effector T cells and Treg cells, which play roles in immune stimulation and immunosuppression, respectively ( 41 ), and the Treg cell subpopulation was not significantly changed after irradiation, but overall, high-dose radiotherapy seemed to disrupt the balance of the CD4 + T-cell populations. In addition, the mechanism by which high-dose radiation affects CD8 + T cells involves the promoted infiltration of immune cells into tumors through vascular endothelial cells or through the local production of chemical inducers ( 42 ). TAMs undergo two types of polarization, and TAMs undergoing M2-like polarization show inhibited production of immune cytokines ( 43 ). However, different doses of radiotherapy can lead to conflicting effects on TAM polarization; no significant change in TAMs was observed in this study, and therefore, further study and evaluation of the relative contribution of radiotherapy to TAM polarization types are needed. PD-L1 has been proven to be a key immune checkpoint molecule expressed on the surface of immune cells and highly expressed on the surface of cancer cells. PD-L1 enables tumor cells to escape the host immune response by inducing T-cell depletion and inhibiting effector T-cell function ( 44 ). We found that 8 Gy × 3 radiotherapy triggered more PD-L1 expression than 2 Gy × 14, which suggested that high-dose fraction radiotherapy may induce a greater local inflammatory response in tumor tissue and enhance tumor-specific T-cell infiltration in the tumor microenvironment ( 45 ). Notably, the negative regulation of immune cells by PD-L1/PD-1 may be an important host-mediated mechanism of tumor-acquired radiation resistance, and the expression of PD-L1 induced by radiotherapy indicates an opportunity for the subsequent application of PD-L1/PD-1 axis inhibitors. Combination therapy enhances the antitumor immunity of the host and improves the curative effect of the respective treatments ( 45 , 46 ). In our study, radiotherapy combined with an anti-PD-L1 antibody regimen effectively controlled tumor progression. In terms of survival rate, the radiotherapy dose regimen of 8 Gy × 3 was slightly better than that of 2 Gy × 14 for immunoregulation. Some studies have suggested that the dose-grading regimen of immune checkpoint drugs with 8 Gy × 3 radiation exposure should be the standard grading scheme for an immunotherapy combination ( 47 – 49 ). However, a consensus on the best radiation doses and fractionation plan has not been reached. Our study preliminarily explored the effects of differences in radiation doses and fractionation plans in a combination treatment of breast cancer. The best plan for combined radiotherapy and immunotherapy needs to be assessed considering many variables, such as the effect of tumor subtype, immune response of the tumor and immunotherapy type. In conclusion, different doses of radiotherapy have different effects on tumor vascular remodeling and tumor immune microenvironment activation, and radiotherapy can promote the therapeutic effect of immune checkpoint inhibitors. MR/PA bimodal imaging can be used as an imaging marker to evaluate tumor vessels after radiotherapy and better guide the combined immunotherapy strategy of tumors. Declarations Author contributions YJ, LC contributed to the research and design. XN contributed to the research experiment and thesis writing. WD, ZQ and JH contributed to photoacoustic imaging. GJ, LQ contributed to the formulation of radiotherapy and immunotherapy programs. BS, LY contributed to data statistics and image processing. Funding This study was supported by the National Natural Science Foundation of China (grant no. 82060313,82160340), the Outstanding Youth Science Foundation of Yunnan Basic Research Project (202201AW070002), and the Yunnan Health Training Project of High Level Talents (D-2018009, H-2017005). Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Declaration Ethics approval and consent to participate The Experimental Animal Ethics Committee of Kunming Medical University approved this study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Radiology, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital/Center. No. 519 Kunzhou Road, Xishan District, Kunming 650118, Yunnan, P.R. China 2 School of Optoelectric Engineering, Chongqing University of Posts and Telecommunications, Chongqing 400065, China 3 Department of Radiation Oncology, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital/Center. No. 519 Kunzhou Road, Xishan District, Kunming 650118, Yunnan, P.R. China 4 Department of Medical Engineering, University of South Florida, Tampa 33620, USA References Beck JD, Birtel M, Haefner E, Keil IS, Reidenbach D, Salomon N, et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-2222488","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":148749479,"identity":"b341686a-5eec-42c6-ad57-344f9fea0f19","order_by":0,"name":"Nan Xu","email":"","orcid":"","institution":"The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital/Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Xu","suffix":""},{"id":148749481,"identity":"2c1a4081-4201-4e0a-903f-13f1006e5656","order_by":1,"name":"Dan Wu","email":"","orcid":"","institution":"Chongqing 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15:14:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2222488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2222488/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28765508,"identity":"be38139d-a458-42b3-b55c-a23681e1adfc","added_by":"auto","created_at":"2022-11-07 19:10:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":188596,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of our PAI system for in vivo imaging of tumor-bearing mice.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/a674cdc258719de289aff069.png"},{"id":28766224,"identity":"4e588ad5-4b97-4db0-93c4-ce61cbe847dd","added_by":"auto","created_at":"2022-11-07 19:18:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178979,"visible":true,"origin":"","legend":"\u003cp\u003eTumor response to different doses of radiation therapy. \u003cstrong\u003e(A)\u003c/strong\u003e On the 12th day after tumor transplantation, tumor-bearing mice (TBM) received different doses of radiotherapy (fractionated radiotherapy). \u003cstrong\u003e(B) \u003c/strong\u003eTumor volume growth curve. Compared with the control group, *** p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/b4002b71c9b943c8a9073500.png"},{"id":28765509,"identity":"8cb4d261-b1a8-4b87-821d-7f63c7a41aaa","added_by":"auto","created_at":"2022-11-07 19:10:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1364536,"visible":true,"origin":"","legend":"\u003cp\u003eMR images before and after radiation in the therapy groups. \u003cstrong\u003e(A) \u003c/strong\u003eMR images of the control group, low-dose group and high-dose groups before and after radiotherapy. The contralateral side of the tumor shows the Mn-Au contrast agent. \u003cstrong\u003e(B) \u003c/strong\u003eChanges in signal intensity in the central and peripheral areas of the tumor after radiotherapy. Compared with the control group, * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/685c2bfcfde6f7ab1f8bef6b.png"},{"id":28766227,"identity":"88ce8a78-4a88-4518-9afc-4c5003ff057d","added_by":"auto","created_at":"2022-11-07 19:18:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":532028,"visible":true,"origin":"","legend":"\u003cp\u003ePhotoacoustic parameter image. \u003cstrong\u003e(A)\u003c/strong\u003e PA images of the control group, low-dose group and high-dose group. \u003cstrong\u003e(B)\u003c/strong\u003e Signal intensity change of HbT, HbR, and H\u003csub\u003e2\u003c/sub\u003eO after radiotherapy and the signal differences between the peripheral and central regions of the tumor. Compared with the control group, * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/c91843fada88442714531226.png"},{"id":28765511,"identity":"e76875c0-f234-4cb7-b99e-40396c083e6c","added_by":"auto","created_at":"2022-11-07 19:10:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":992116,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of radiotherapy on tumor progression. \u003cstrong\u003e(A)\u003c/strong\u003e Double-marker immunofluorescence of Ki-67 (red) and TUNEL (green); the blue area represents the nucleus; Double-marker immunofluorescence of CD31 (red) and α-SMA (green); Double-marker immunofluorescence of PD-L1 (red) and HIF-1α (green); Scale bar, 50 µm. \u003cstrong\u003e(B)\u003c/strong\u003eThe percentages of Ki-67+ and TUNEL+cells in the total number of tumor cells per unit area were measured, and cell proliferation and apoptosis were observed; quantitativeevaluation of tumor microvessel density (MVD) and vascular maturity (VMI) after radiotherapy; evaluation of tumor hypoxia and PD-L1 expression after radiotherapy. Compared with the control group, ** p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/46addf10caffba135febefda.png"},{"id":28767139,"identity":"96aa803c-de14-4dde-b9c5-c1d2f53fe483","added_by":"auto","created_at":"2022-11-07 19:26:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":121752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Cleaved caspase-3 expression in tumors from different groups. \u003cstrong\u003e(B)\u003c/strong\u003e Histogram after quantitative analysis performed with ImageJ software. Compared with the control group, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/5daeb916cb5a07ae8add986e.png"},{"id":28765514,"identity":"0672e68f-fad4-4132-a0b1-e23ec2e7349d","added_by":"auto","created_at":"2022-11-07 19:10:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1076715,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometry of immune cells in tumors after radiotherapy. Flow cytometry quantitative data analysis revealed the number of tumor CD4\u003csup\u003e+\u003c/sup\u003e T cells \u003cstrong\u003e(A)\u003c/strong\u003e, CD8\u003csup\u003e+\u003c/sup\u003e T cells \u003cstrong\u003e(B),\u003c/strong\u003e Treg cells \u003cstrong\u003e(C)\u003c/strong\u003e, TAMs \u003cstrong\u003e(D)\u003c/strong\u003e and NK cells \u003cstrong\u003e(E)\u003c/strong\u003e after radiotherapy. Compared with the control group, **p \u0026lt; 0.01, *** p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/d99d8b4ecc25bfc1fe41b474.png"},{"id":28767140,"identity":"849c8777-91d0-4d06-9a8b-aa9978760865","added_by":"auto","created_at":"2022-11-07 19:26:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":286702,"visible":true,"origin":"","legend":"\u003cp\u003eTumor response to different doses of radiation therapy combined with immunotherapy. \u003cstrong\u003e(A)\u003c/strong\u003e On the 12th day after tumor transplantation, tumor-bearing mice (TBM) received different doses of radiotherapy and immunotherapy. \u003cstrong\u003e(B) \u003c/strong\u003eSurvival of mice with different tumor treatment regimens.\u003cstrong\u003e (C) \u003c/strong\u003eTumor volume growth curve. Compared with the control group, ** p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/8d070a336c901afb72f19563.png"},{"id":28951835,"identity":"bd32eb85-c9e4-4c81-899d-0e6956ede1ea","added_by":"auto","created_at":"2022-11-11 14:14:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3350477,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2222488/v1/4bd484bd-ae0f-4613-a3c7-2150dccbe9c0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A MR/PA Dual-Modality Imaging for quantitative evaluation of tumor vascular and immune microenvironment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer immunotherapy is based on the immunogenicity of tumors, which is achieved through the induction of active or passive immunity for tumor suppression and clearance (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, low immune response rates and drug resistance remain problems in immunotherapy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). During tumor progression, the interactions between the tumor microenvironment and tumor cells mediate tumor immune tolerance, which affects the clinical effect of immunotherapy. The state of the tumor vasculature plays an important role in immunotherapy. For effective immunotherapy, sufficient T cells must sufficiently infiltrate tumor tissue; however, abnormal tumor vessels reduce immune cell infiltration through a variety of mechanisms, thereby limiting immunotherapy effectiveness. Moderate antitumor angiogenic therapy can correct the immature and chaotic blood vessels in the tumor, rendering tumor angiogenesis more dynamically balanced, and this improvement of vascular structure combined with immunotherapy drugs can lead to obvious therapeutic effects (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, antiangiogenic drugs can lead to many complications and cannot effectively stimulate tumor immunogenicity, which limits their applications in the comprehensive treatment of tumors (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRadiotherapy not only can kill tumor cells but can also directly promote immune-mediated tumor rejection by enhancing the total effect of tumor antigen processing and cross presentation, making tumor cells sensitive to immune recognition and attack, and increasing the immunosuppressive state of the tumor microenvironment, which benefits the recruitment, activation and action of T cells (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In addition, tumor vessels are properly pruned but not completely destroyed by radiotherapy (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), the distribution of blood vessels is more uniform, the interstitial pressure in the tumor tissue is decreased, and the oxygen content in the tumor tissue and the distribution of combined therapy drugs are maximized (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Therefore, accurate evaluation of tumor vascular repair and microenvironmental changes is the key to the success of tumor combined therapy (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, imaging techniques such as dynamic contrast-enhanced magnetic resonance imaging (MRI), computed tomography (CT) perfusion imaging, sonography and nuclear medicine are often used to evaluate tumor vessels (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Adequate detection of complex vascular changes is difficult with a single imaging method, although MRI and ultrasound are the measurements most reported in the clinical literature, and their reliability and stability are worthy of further observation. Photoacoustic imaging (PAI) is an emerging imaging technique that combines optics with ultrasound and can provide tissue structure and functional information. PAI has higher resolution than traditional ultrasonic imaging, and its image resolution can reach the order of microns, which can achieve high-resolution tissue imaging (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Second, PAI uses different tissues and components in the body to reflect tissue structure and functional information by different light absorption of different wavelengths (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In addition, PAI can achieve noninvasive physiological and pathological imaging at the molecular level by combining molecular imaging probe technology and contrast-enhanced imaging technology (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Finally, through MRI to provide tumor structure information, perfusion information and tumor vascular information, hypoxia status and other functional information provided by PAI, we can accurately identify the changes in tumor blood vessels and the corresponding microenvironment (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Hence, we conducted this study to evaluate whether MR/PA can potentially be used to assess tumor vascular remodeling after radiotherapy and to observe changes in the immune-related tumor microenvironment at the molecular biological level with the aim of optimizing combination immunotherapy.\u003c/p\u003e"},{"header":"Methods And Materials","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eCell Line and Animals\u003c/h2\u003e\n \u003cp\u003eMouse 4T1 breast cancer cells were provided by the Chinese Academy of Sciences Stem Cell Bank and cultured in RPMI-1640 containing 10% fetal bovine serum and penicillin\u0026ndash;streptomycin. Female BALB/c mice (5\u0026ndash;6 weeks old, weighing approximately 18\u0026ndash;20 g) were purchased from the Department of Experimental Animals, Kunming Medical University. The Experimental Animal Ethics Committee of Kunming Medical University approved this study, and all experimental protocols were conducted in accordance with the committee\u0026rsquo;s guidelines. Approximately 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in 200 \u0026micro;L of phosphate-buffered saline (PBS) were inoculated subcutaneously in the right lower limb of the mice. Twelve days after implantation, these 4T1 mouse breast cancer models (V\u003csub\u003etumor\u003c/sub\u003e\u0026ge;200 mm\u003csup\u003e3\u003c/sup\u003e) were used for treatment experiments. The tumor volume calculation formula was V\u0026thinsp;=\u0026thinsp;0.5\u0026times;a\u0026times;b\u003csup\u003e2\u003c/sup\u003e, where a is the vertical long diameter measured by calipers and b is the vertical short diameter.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eTumor Treatment\u003c/h2\u003e\n \u003cp\u003eThe tumor-bearing mice (TBM) were randomly allocated to six groups: the low-dose group, high-dose group, low-dose\u0026thinsp;+\u0026thinsp;PD-L1 group, high-dose\u0026thinsp;+\u0026thinsp;PD-L1 group, anti-PD-L1 group and control group. Animals received 2 Gy/14 Fx (low-dose group) or 8 Gy/3 Fx (high-dose group) radiation for two consecutive weeks. The combination treatment groups were given an anti-PD-L1 antibody (clone 10F.9G2, isotype rat IgG2b, Bio-Xcell) by intraperitoneal injection (200 \u0026micro;g/3 Fx, on days 12, 18, and 25), and the control group was injected with the same amount of normal saline and evaluated in a survival analysis. The TBM were exposed to X-ray radiation from an RS2000 Biological X-ray Biological Irradiator with an accelerating voltage of 225 kV and a dose rate of 1.8 Gy/min (Georgia, USA). To prevent radiation exposure of normal tissue, the mice were immobilized in the irradiator, the area away from the tumor was covered by a lead screen, and the other parts of the body were covered with lead screens to minimize the radiation to normal tissues. Tumor volume and body weight were measured every 2 days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eMR/PA Bimodal Imaging and Analysis\u003c/h2\u003e\n \u003cp\u003eThe mice from the radiotherapy group and the control group were scanned on days 12 and 26 with a 3.0 T MRI scanner (Ingenia, Philips Medical Systems, Netherlands). The mice were injected intraperitoneally with 200 \u0026micro;L of Mn-Au contrast agent (APN-AU-40, 50 mmol/L MnCI\u003csub\u003e2\u003c/sub\u003e, and 7x10\u003csup\u003e10\u003c/sup\u003e N/mL) 24 hours before imaging. During imaging, the mice in the prone position were anesthetized with 10% chloral hydrate administered intraperitoneally, and eight-channel phased array coils were used for scanning the animals. The tumor was placed in the center of the coil, and the Mn-Au contrast agent was placed in the opposite lower limb of the tumor for image signal intensity (SI) comparison. TSE-T1WI was performed with the following parameters: a repetition time (TR)/echo time (TE) of 510 ms/27 ms, an average signal number (NSA) of 8, a slice thickness/slice spacing of 1.5 mm/0.15 mm, a slice number equal to 14, a flip angle of 90\u0026deg;, a scanning field of view (FOV) equal to 5 cm \u0026times; 5 cm, a matrix of 200 \u0026times; 198, and a total imaging time of 588 s. The image data were analyzed using software on multifunction workstations (Syngo.via, version VB10B, Siemens, Erlangen, Germany). The regions of interest (ROIs) were drawn manually on the tumor (including the periphery and center of the tumor) and the contrast agent on the image of one slice, and each region ROI consisted of approximately the same size (approximately 0.5 mm\u003csup\u003e2\u003c/sup\u003e) and shape. The SI produced by the Mn-Au contrast medium was obtained. The mean signal of the contrast agent was standardized in each slice. The enhanced Mn\u003csup\u003e2+\u003c/sup\u003e intensity in the peripheral and central tumor ROIs was calculated by the following formula: SI\u0026thinsp;=\u0026thinsp;SMn (Con)-SMn (IP \u003csub\u003e(per/c)\u003c/sub\u003e)/SMn (air), in which SMn (IP\u003csub\u003eper\u003c/sub\u003e), SMn (IP\u003csub\u003ec\u003c/sub\u003e) and SMn (air) represented the SI of the peripheral and central Mn\u003csup\u003e2+\u003c/sup\u003e of the tumor and the SI of the peripheral air, respectively.\u003c/p\u003e\n \u003cp\u003eThe raw PAI data in this study were obtained with an in vivo laboratory-built PAI image acquisition system (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A laser (Surelite OPO, Continuum USA) emitted pulses with a width of 6\u0026ndash;7 ns and a wavelength of 690\u0026ndash;960 nm at a repetition rate of 20 Hz to excite the target and thus produce PA signals. The energy of the pulsed light was controlled to extend 10 mJ/cm\u003csup\u003e2\u003c/sup\u003e below the surface of the animal\u0026rsquo;s skin, which was well beneath the safety standard (20 mJ/cm\u003csup\u003e2\u003c/sup\u003e). A 128 element half-ring ultrasonic array probe (with a center frequency of 5 MHz and a bandwidth of 75%, Japan Probe Co., Ltd.) was used to receive the PA signal. The received PA signal was amplified with a preamplifier (gain of 54 dB and frequency range from 460 kHz to 9 MHz) made in house, and the signal was collected through data acquisition cards (NI 5105). Ten-time averaging of the signal can minimize laser energy instability, and thus, the image quality is improved, making the calculation of the tumor area more accurate. During the acquisition process, real-time imaging was realized with a LabVIEW panel. The PAI tumor images were reconstructed from the PA signals using a delay and sum reconstruction algorithm.\u003c/p\u003e\n \u003cp\u003eThe PAI tumor images were reconstructed from photoacoustic signals using a delay and sum reconstruction algorithm. According to the algorithm, we identified changes in the cerebral deoxyhemoglobin (HbR), oxyhemoglobin (HbO) and oxygen saturation (SO\u003csub\u003e2\u003c/sub\u003e), levels in tumor regions at two different wavelengths (760 nm and 840 nm). The absorption coefficient of HbR is higher than that of HbO at 760 nm, while the absorption coefficient of HbO is higher than that of HbR at 840 nm. The HbO, HbR and SO\u003csub\u003e2\u003c/sub\u003e changes were calculated pixel by pixel via Eqs. 1 and 2, and the pixels were chosen according to the calculated total hemoglobin (HbT) concentration (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({C}_{HbT}={C}_{HbO}+{C}_{HbR}\\)\u003c/span\u003e\u003c/span\u003e), which was greater than 0.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003eTumor Immunofluorescence\u003c/h2\u003e\n \u003cp\u003eAll mice were sacrificed by spinal dislocation. Tumor tissues were fixed with 4% paraformaldehyde and cut into 4-\u0026micro;m-thick sections after dehydration and embedment. In brief, specimens were incubated with anti-CD31 (1:500, Abcam, ab182981), anti-\u0026alpha;-SMA (1:200, Servicebio, GB13044), anti-ki67 (1:200, Servicebio, GB111141), anti-HIF-1\u0026alpha; (1:200, Cell Signaling Technology, 36169T), and anti-PD-L1 (1:200, Servicebio, GB11339A) antibodies for staining. The slices were imaged with a NIKON ECLIPSE C1 fluorescence microscope and scanned and analyzed with a PANNORAMIC panoramic slice scanner and Image-Pro Plus 6.0 analysis software. Tumor slices were divided into three equal areas, and the microvessel density (MVD) of each area was detected by CD31 staining of perivascular cells. The area was quantified on the basis of the total number of microvessels per unit area; similarly, the coverage of the perivascular cells stained for \u0026alpha;-SMA in each area was calculated. The vascular maturity index (VMI) refers to the percentage of blood vessels stained with the anti-\u0026alpha;-SMA antibody compared to the total number of blood vessels stained with CD31 (\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e). An apoptosis kit (Servicebio, G1501) was used to perform TUNEL assays and thus evaluate the number of apoptotic tumor cells. Tumor cell proliferation was quantitatively analyzed based on the percentage of the number of positive cells stained for Ki-67 in each tumor area compared to the total number of cells. The ratio of HIF-1\u0026alpha;-positive cells to total cells per unit area was calculated to analyze tumor hypoxia, and the proportion of PD-L1-positive cells to total cells was analyzed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003eFlow Cytometry\u003c/h2\u003e\n \u003cp\u003eTo obtain a single-cell suspension, the tumor tissue was mechanically cut into small pieces and subjected to cell filtration. The number of single cells in suspension was counted, and the cells were stained with specific antibodies against CD4 (BioLegend, clone GK1.5, 100408), CD8a (BioLegend, clone 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7, 100707), CD16 (BioLegend, clone S17014E, 158007), CD3 (BioLegend, clone 17A2, 100273), CD163 (BioLegend, clone S15049I, 155308), FOXP3 (BioLegend, clone 150D, 320011), CD25 (BioLegend, clone PC61, 102015), CD68 (eBioscience, clone FA-11, 25-0681-80), CD80 (eBioscience, clone 16-10A1, 11-0801-81), and CD56 (Santa Cruz, clone 123C3, SC-7326). The expression levels of CD4, CD8, tumor-associated macrophages (TAMs), regulatory T (Treg) cells, and natural killer (NK) cells were detected with an acoustic focus flow cytometer (Invitrogen Attune NxT, AFC2), and the data were analyzed using FlowJo software (Tree Star Inc.).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e\n \u003cp\u003eThe apoptosis of tumor cells in the tumor microenvironment was detected by Western blotting. As previously described(\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e), the tumor tissue was mechanically lysed with RIPA cleavage buffer (Solarbio, Beijing, China). After 30 min of incubation on ice, the total protein was extracted from the cells, centrifuged for 15 min at 4\u0026deg;C and 12000 rpm, and quantified with a BCA protein assay kit (Biosharp, Hefei, Anhui, China). The protein in the supernatant was dissolved in 5x electrophoresis sample buffer at a ratio of 1:4, heated to a high temperature and stored at -20 ℃ until use. The isolated proteins were separated by polyacrylamide gel electrophoresis (Biosharp, Hefei, Anhui, China), transferred to a polyvinylidene fluoride membrane (0.22 \u0026micro;m, Merck KGaA, Darmstadt, Germany), soaked in TBST (Solarbio, Beijing, China) blocking solution containing 5% w/v skim milk (Biofroxx, Germany) for 2 hours, and then incubated for 0.5 hours with a primary antibody; horseradish peroxidase-bound secondary antibodies (1:10000, Cell Signaling Technology, #7074) were added to the membrane and incubated for 1 hour at room temperature. Cleaved caspase-3 was visualized with an enhanced chemiluminescence enhancement kit (Biosharp, Hefei, Anhui, China), and the protein expression level was detected with ImageJ software (GE Healthcare Bio-Sciences, Uppsala, Sweden). The image was decolorized into an 8-bit black-and-white image, and its gray value represented the grayscale state of the image. The depth and area of the bands in the image comprehensively represented the amount of protein. The following antibody was used: rabbit anti-cleaved caspase-3 (1:1000, Cell Signaling Technology, 9664).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eAll data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD). Independent-samples t test, one-way ANOVA or nonparametric tests were performed to assess the differences between different groups. The correlation between MR/PA parameters and histological results was analyzed on the basis of Pearson correlation coefficients. SPSS 25.0 software (SPSS Company, Chicago, Illinois, USA) was used for the statistical analysis, and the survival data were analyzed by the Kaplan\u0026ndash;Meier test; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTumor Volume\u003c/h2\u003e \u003cp\u003eThe changes in tumor volume were continuously observed after implantation of the breast cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). No significant difference in tumor volume was observed among the three groups before radiotherapy. On the 10th day, the tumor volume was 205.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69 mm\u003csup\u003e3\u003c/sup\u003e in the control group, 199.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.18 mm\u003csup\u003e3\u003c/sup\u003e in the low-dose group and 203.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 mm\u003csup\u003e3\u003c/sup\u003e in the high-dose group. After fractionation radiotherapy, the percentage of tumor volume increased on the 26th day: in the control group, it increased by 83.60%, in the low-dose group, it increased by 77.59%, and in the high-dose group, it increased by 71.21%. Compared with tumor progression in the control group, radiotherapy significantly inhibited tumor progression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The tumor growth in the high-dose group was lower than that in the low-dose group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMR/PA Imaging\u003c/h2\u003e \u003cp\u003eMR images were obtained from each group of mice before and after radiotherapy. The images taken before radiotherapy showed no significant difference in signal intensity among the control group, low-dose group and high-dose group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The radiotherapy group image showed a low enhancement area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), which was significantly different than that taken before radiotherapy. This finding suggested local necrosis inside the tumor. Although the images of the control group showed localized hypointense areas inside the tumor compared with those in the images taken before radiotherapy, the difference in tumor signal change was not significant. Further analysis showed that compared with that in the control group, the signal enhancement of the tumor center was decreased in the radiotherapy groups, and the SI decreased more significantly in the high-dose group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The SI in the peripheral region of the tumor was also decreased after radiotherapy (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the PAI experiments, 5 representative images were continuously obtained for quantitative analysis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. The levels of HbT and HbR in the high-dose group and low-dose group were significantly lower than those in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The levels of HbO\u003csub\u003e2\u003c/sub\u003e and SO\u003csub\u003e2\u003c/sub\u003e in the high-dose group and low-dose group was higher than that in the control group, and there was a significant difference in SO\u003csub\u003e2\u003c/sub\u003e between the high dose group and the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further analysis showed that HbT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and HbR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) levels in different regions of the tumor in the radiotherapy groups were different from those in the control group. In the high-dose and low-dose groups, the HbT levels in the central area of the tumor were lower than those in the peripheral region in the high-dose (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the HbR levels in the central area of the tumor were lower than those in the peripheral region (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no regional difference in HbT and HbR levels in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Assay of the Tumors\u003c/h2\u003e \u003cp\u003eCompared with that in the control group, the number of Ki67-positive proliferative cells decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the number of TUNEL-positive apoptotic cells increased in both the low-dose and high-dose groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Furthermore, in the high-dose group, the number of Ki-67-positive cells was decreased most significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the number of TUNEL-positive cells increased most significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the high-dose group, Ki-67 was positively correlated with SI(per) (r\u0026thinsp;=\u0026thinsp;0.879, p\u0026thinsp;=\u0026thinsp;0.021) and SI(c) (r\u0026thinsp;=\u0026thinsp;0.938, p\u0026thinsp;=\u0026thinsp;0.006), but TUNEL staining was negatively correlated with SI(c) (r=-0.867, p\u0026thinsp;=\u0026thinsp;0.025). In the low-dose group, Ki-67 was positively correlated with SI(per) (r\u0026thinsp;=\u0026thinsp;0.826, p\u0026thinsp;=\u0026thinsp;0.043) and SI(c) (r\u0026thinsp;=\u0026thinsp;0.907, p\u0026thinsp;=\u0026thinsp;0.012).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTumor sections were stained with anti-CD31 and anti-α SMA antibodies to detect tumor vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared with that in the control group, the number of blood vessels decreased; in the low-dose group, it decreased to 48.93% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and in the high-dose group, it decreased to 14.67%. In addition, compared with the control group, radiotherapy increased the VMI; in the low-dose group, it increased to 55.13% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and in the high-dose group, it increased to 29.60% (p༞0.05).\u003c/p\u003e \u003cp\u003eTumors were stained with anti-PD-L1 and anti-HIF-1α antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), and the effects of radiation on tumor immunity and hypoxia were evaluated. Compared with that in the control group, radiotherapy increased the expression of PD-L1 in the tumor, which increased by 25.69% in the low-dose group and 30.8% in the high-dose group, and the expression of PD-L1 in the high-dose group was significantly higher than that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with the control group, radiotherapy slightly reduced the content of HIF-1α in the tumor tissue (p༞0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExpression of Cleaved Caspase-3 in the Tumor Tissue\u003c/h2\u003e \u003cp\u003eCompared with the control group, the expression of cleaved caspase-3 in the high-dose group and low-dose group was significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Radiotherapy on Immune Cells in Tumors\u003c/h2\u003e \u003cp\u003eTo determine the changes in the number of tumor immune cells after radiotherapy, tumor-associated immune cells were analyzed by flow cytometry. Compared with the control group, the high-dose CD4\u003csup\u003e+\u003c/sup\u003e group showed an increase of 73.96% (p\u0026thinsp;=\u0026thinsp;0.006), CD8\u003csup\u003e+\u003c/sup\u003e cells showed an increase of 74.91% (p\u0026thinsp;=\u0026thinsp;0.015), and NK cells showed an increase of 29.25% (p\u0026thinsp;=\u0026thinsp;0.001); the low-dose CD4\u003csup\u003e+\u003c/sup\u003e group showed an increase of 67.46%, CD8\u003csup\u003e+\u003c/sup\u003e cells showed an increase of 3.89%, and NK cells showed an increase of 15.98% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B, E). However, there was no significant difference in the levels of CD4\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e+\u003c/sup\u003e, and NK cells between the low-dose group and the control group. In addition, no significant changes in TAMs (p\u0026thinsp;=\u0026thinsp;0.643) or Treg cells (p\u0026thinsp;=\u0026thinsp;0.932) were observed in the radiotherapy groups compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic Effects of the Combination Therapy on the Tumors\u003c/h2\u003e \u003cp\u003eTo evaluate the efficacy of combination therapy, the mice were treated with a single anti-PD-L1 antibody, high-dose irradiation\u0026thinsp;+\u0026thinsp;the anti-PD-L1 antibody (8 Gy/3 Fx, 200 \u0026micro;g/3 Fx on days 12, 18, and 25), or low-dose irradiation (2 Gy/14 Fx)\u0026thinsp;+\u0026thinsp;the anti-PD-L1 antibody (200 \u0026micro;g/3 Fx on days 12, 18, and 25) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). During the course of treatment, the tumor growth in the three treatment groups was significantly lower than that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Compared with anti-PD-L1 therapy alone, low-dose irradiation\u0026thinsp;+\u0026thinsp;anti-PD-L1 antibody and high-dose irradiation\u0026thinsp;+\u0026thinsp;anti-PD-L1 antibody treatment significantly slowed tumor progression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Compared with the control group regimen, the dose regimen of combined therapy showed a more significant inhibitory effect on tumor growth (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and a tendency to increase the survival rate (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Furthermore, compared with anti-PD-L1 antibody therapy alone, the survival of the low-dose irradiation\u0026thinsp;+\u0026thinsp;anti-PD-L1 antibody therapy group was significantly increased (p\u0026thinsp;=\u0026thinsp;0.035), and this increase in the survival rate was the most obvious in the high-dose irradiation\u0026thinsp;+\u0026thinsp;anti-PD-L1 antibody treatment group (p\u0026thinsp;=\u0026thinsp;0. 002).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, radiotherapy induced tumor vascular remodeling by changing the vascular density and oxygenation level and increased the infiltration of immune cells and the expression of PD-L1 in the tumors, thus promoting the inhibition of tumor immune environment activation. Compared with histopathological biopsy, noninvasive quantitative imaging is valuable in providing anatomical and vascular information on the tumor response to radiotherapy. To the best of our knowledge, this is the first study to use MR/PA bimodal imaging to observe the response of tumor vessels and the microenvironment to different radiotherapy regimens in tumor-bearing mice.\u003c/p\u003e \u003cp\u003eTumor growth is affected in many ways during radiotherapy, and the differences in a radiotherapy fractionation scheme are important factors in controlling tumor progression and metastasis. In this study, we found that tumor growth was continuously inhibited by radiotherapy, and the effect of high-dose fractionated radiotherapy was more obvious than that of low-dose fractionated radiotherapy. Of course, this effect may have been related to our radiotherapy program. The inhibitory effect of low-dose fractionation radiotherapy on tumor cell growth was short-lived and weak, while interval high-dose fractionation radiotherapy can cause a wide range of DNA double-strand breaks, destroy the ability of cellular self-repair, and thus accelerate tumor cell apoptosis. Therefore, we should not only ensure the inhibitory effect of high-dose radiotherapy on tumor growth but also consider the interval of treatment. Ki-67 expression, the best marker of cell proliferation, and apoptosis marker expression were assessed to compare the effects of dose differences on tumor growth. The apoptosis index significantly increased, indicating that high-dose irradiation caused tumor cell death and effectively prevented tumor cell proliferation. In addition to analyzing the growth status of tumors, we also used manganese-enhanced MRI to monitor the changes in apoptosis of tumor cells after radiotherapy. Manganese ions are actively transported to biologically active cells through calcium channels, so T1-weighted positive signals of magnetic resonance imaging were used to reflect the survival status of cells, especially to observe the response of tumor cells to different doses of radiotherapy (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Compared with that of low-dose radiotherapy, the killing effect of high-dose fraction radiation on tumor cells was more direct and persistent. After high-dose radiotherapy, the degree of signal enhancement in the tumor center was decreased significantly, and the peripheral area of the tumor was more sensitive to high-dose radiotherapy and showed little enhancement, suggesting that the tumor cells in the peripheral region underwent apoptosis at a significant rate. More importantly, through MRI, we found that the radiosensitivity in different regions of the tumor differed. This finding may have been a result of phenotypic and genetically diverse tumor cell populations, and the proliferation rate and therapeutic sensitivity of these cells may differ (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In addition, when tumor cells recruit stromal cell groups, such as immune cells and endothelial cells, the tumor microenvironment is remodeled, resulting in spatial variation within the tumor (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The resulting spatial distribution of tumor and stromal cells leads to the progression of different physiological subregions in tumors and differences in the resistance of tumor areas to treatment. The SI in the MR images of the tumors was positively correlated with Ki-67 staining and negatively correlated with TUNEL staining, which indicated that MRI can potentially be used to monitor tumor growth inhibition induced by radiotherapy and to help evaluate the sensitivity of tumor cells to the radiation dose.\u003c/p\u003e \u003cp\u003eMany aspects of vascular biology are affected by radiation and vary according to the dose/grade, time, and model studied (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Radiotherapy may preferentially prune poorly covered/functional vessels to redistribute blood vessels, but this vascular change may not systematically translate into long-term effects on the tumor (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Compared with a single dose of high-intensity irradiation, our 2 Gy \u0026times; 14 and 8 Gy \u0026times; 3 doses did not cause severe irreversible damage to tumor vessels. After 2 Gy \u0026times; 14 treatment, the VMI was increased because of the effective stimulation, which may have been related to the retention and improvement of vascular system formation in the first few days after radiotherapy (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). After 8 Gy \u0026times; 3 exposure, the MVD did not decrease significantly, possibly because of the death of vascular endothelial cells to varying degrees, which would have been quickly attenuated by the formation of new blood vessels, emphasizing the contribution of the fractionation radiotherapy scheme to tumor vessel remodeling.\u003c/p\u003e \u003cp\u003eHypoxia is a consequence of a high tumor cell proliferation rate and an abnormal structure of the tumor vasculature (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and HIF-1α is the main regulator of hypoxic transcription. There was no significant change in the expression of HIF-1α after 2 Gy and 8 Gy exposure. This outcome may have been due to the reoxygenation of tumor parenchyma cells that survived radiotherapy, an outcome that largely depends on the radiation scheme (32). A complex dose\u0026ndash;response relationship between radiation-injured tumor vessels, tumor cell reoxygenation and reaggregation is suggested. During PAI monitoring of tumors after radiotherapy, we found that the contents of HbT and HbR in tumor vessels decreased and SO\u003csub\u003e2\u003c/sub\u003e increased in high-dose group, suggesting that the morphology and oxygenation level of tumor vessels was affected to varying degrees, which may depend on the size of tumor vessels (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The difference in the response of tumor vessels to different doses of radiation reflects the spatial heterogeneity of the tumor, especially the changes in blood vessel density and oxygenation level and the redistribution of blood flow. Anum and others called the physiological tumor habitat, and differences in imaging and histological findings are evident among different vascular and cellular tumor habitats (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In general, the PAI parameters confirmed vascular degenerative changes after radiotherapy and were able to detect subtle changes in tumor oxygenation levels with high sensitivity, suggesting that the degree of vascular degeneration is related to radiotherapy dose, radiation-dose fractionation, regional tumor heterogeneity and other factors.\u003c/p\u003e \u003cp\u003eAs an immunomodulator, radiation can directly stimulate immune cells to produce cytokines and chemokines that affect the local immune response, and this immune response varies on the basis of the radiotherapy program and tumor model (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The activation status of T and NK cells is suggestive of antitumor immunity; immune cells can kill target cells via various mechanisms following activation. Radiation can enhance the cytokine secretion and cytotoxic activity of NK cells (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Compared with the 2 Gy \u0026times; 14 treatment, 8 Gy \u0026times; 3 better promoted NK-cell recruitment to the tumor microenvironment, which may have been due to an increase in the radiation dose that was conducive to the release of tumor exosomes, promoting NK-cell polarization (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). According to our results, compared to the low dose, 8 Gy \u0026times; 3 can better promote the aggregation of CD4\u003csup\u003e+\u003c/sup\u003e T cells and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the tumor. The CD4\u003csup\u003e+\u003c/sup\u003e T-cell population contains effector T cells and Treg cells, which play roles in immune stimulation and immunosuppression, respectively (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), and the Treg cell subpopulation was not significantly changed after irradiation, but overall, high-dose radiotherapy seemed to disrupt the balance of the CD4\u003csup\u003e+\u003c/sup\u003e T-cell populations. In addition, the mechanism by which high-dose radiation affects CD8\u003csup\u003e+\u003c/sup\u003e T cells involves the promoted infiltration of immune cells into tumors through vascular endothelial cells or through the local production of chemical inducers (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). TAMs undergo two types of polarization, and TAMs undergoing M2-like polarization show inhibited production of immune cytokines (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). However, different doses of radiotherapy can lead to conflicting effects on TAM polarization; no significant change in TAMs was observed in this study, and therefore, further study and evaluation of the relative contribution of radiotherapy to TAM polarization types are needed.\u003c/p\u003e \u003cp\u003ePD-L1 has been proven to be a key immune checkpoint molecule expressed on the surface of immune cells and highly expressed on the surface of cancer cells. PD-L1 enables tumor cells to escape the host immune response by inducing T-cell depletion and inhibiting effector T-cell function (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). We found that 8 Gy \u0026times; 3 radiotherapy triggered more PD-L1 expression than 2 Gy \u0026times; 14, which suggested that high-dose fraction radiotherapy may induce a greater local inflammatory response in tumor tissue and enhance tumor-specific T-cell infiltration in the tumor microenvironment (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Notably, the negative regulation of immune cells by PD-L1/PD-1 may be an important host-mediated mechanism of tumor-acquired radiation resistance, and the expression of PD-L1 induced by radiotherapy indicates an opportunity for the subsequent application of PD-L1/PD-1 axis inhibitors. Combination therapy enhances the antitumor immunity of the host and improves the curative effect of the respective treatments (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). In our study, radiotherapy combined with an anti-PD-L1 antibody regimen effectively controlled tumor progression. In terms of survival rate, the radiotherapy dose regimen of 8 Gy \u0026times; 3 was slightly better than that of 2 Gy \u0026times; 14 for immunoregulation. Some studies have suggested that the dose-grading regimen of immune checkpoint drugs with 8 Gy \u0026times; 3 radiation exposure should be the standard grading scheme for an immunotherapy combination (\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). However, a consensus on the best radiation doses and fractionation plan has not been reached. Our study preliminarily explored the effects of differences in radiation doses and fractionation plans in a combination treatment of breast cancer. The best plan for combined radiotherapy and immunotherapy needs to be assessed considering many variables, such as the effect of tumor subtype, immune response of the tumor and immunotherapy type.\u003c/p\u003e \u003cp\u003eIn conclusion, different doses of radiotherapy have different effects on tumor vascular remodeling and tumor immune microenvironment activation, and radiotherapy can promote the therapeutic effect of immune checkpoint inhibitors. MR/PA bimodal imaging can be used as an imaging marker to evaluate tumor vessels after radiotherapy and better guide the combined immunotherapy strategy of tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYJ, LC contributed to the research and design.\u0026nbsp;XN contributed to the research experiment and thesis writing.\u0026nbsp;WD,\u0026nbsp;ZQ and JH contributed to photoacoustic imaging.\u0026nbsp;GJ,\u0026nbsp;LQ contributed to the formulation of radiotherapy and immunotherapy programs.\u0026nbsp;BS,\u0026nbsp;LY contributed to data statistics and image processing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (grant no. 82060313,82160340),\u0026nbsp;the Outstanding Youth Science Foundation of Yunnan Basic Research Project (202201AW070002),\u0026nbsp;and\u0026nbsp;the Yunnan Health Training Project of High Level Talents (D-2018009, H-2017005).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003eDeclaration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Experimental Animal Ethics Committee of Kunming Medical University approved this study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Radiology, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital/Center. No. 519 Kunzhou Road, Xishan District, Kunming 650118, Yunnan, P.R. China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eSchool of Optoelectric Engineering, Chongqing University of Posts and Telecommunications, Chongqing 400065, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Radiation Oncology, The Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Hospital/Center. No. 519 Kunzhou Road, Xishan District, Kunming 650118, Yunnan, P.R. China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eDepartment of Medical Engineering, University of South Florida, Tampa 33620, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBeck JD, Birtel M, Haefner E, Keil IS, Reidenbach D, Salomon N, et al. Cimt 2019: Report on the 17th Annual Meeting of the Association for Cancer Immunotherapy. \u003cem\u003eHum Vaccin Immunother\u003c/em\u003e (2020) 16(4):808-15. 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Combination Strategies with Pd-1/Pd-L1 Blockade: Current Advances and Future Directions. \u003cem\u003eMol Cancer\u003c/em\u003e (2022) 21(1):28. Epub 2022/01/23. doi: 10.1186/s12943-021-01489-2.\u003c/li\u003e\n\u003cli\u003eDemaria S, Guha C, Schoenfeld J, Morris Z, Monjazeb A, Sikora A, et al. Radiation Dose and Fraction in Immunotherapy: One-Size Regimen Does Not Fit All Settings, So How Does One Choose? \u003cem\u003eJ Immunother Cancer\u003c/em\u003e (2021) 9(4). Epub 2021/04/09. doi: 10.1136/jitc-2020-002038.\u003c/li\u003e\n\u003cli\u003eDewan MZ, Galloway AE, Kawashima N, Dewyngaert JK, Babb JS, Formenti SC, et al. Fractionated but Not Single-Dose Radiotherapy Induces an Immune-Mediated Abscopal Effect When Combined with Anti-Ctla-4 Antibody. \u003cem\u003eClin Cancer Res\u003c/em\u003e (2009) 15(17):5379-88. Epub 2009/08/27. doi: 10.1158/1078-0432.Ccr-09-0265.\u003c/li\u003e\n\u003cli\u003eArina A, Gutiontov SI, Weichselbaum RR. Radiotherapy and Immunotherapy for Cancer: From \u0026quot;Systemic\u0026quot; to \u0026quot;Multisite\u0026quot;. \u003cem\u003eClin Cancer Res\u003c/em\u003e (2020) 26(12):2777-82. Epub 2020/02/13. doi: 10.1158/1078-0432.Ccr-19-2034.\u003c/li\u003e\n\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":"tumor microenvironment, immunotherapy, tumor vasculature, radiotherapy, magnetic resonance imaging, photoacoustic imaging","lastPublishedDoi":"10.21203/rs.3.rs-2222488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2222488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackgroud:\u003c/h2\u003e \u003cp\u003eTumor radiotherapy combined with immunotherapy for solid tumors has been proposed, but tumor vascular structure abnormalities and immune microenvironment often affect the therapeutic effect of tumor, and there are few reports on multimodal imaging markers in the study of tumor therapeutic response. The purpose of this study was to evaluate the dynamic monitoring of tumor blood vessels and microenvironment induced by radiotherapy by magnetic resonance imaging /photoacoustic imaging (MR/PA) imaging, and to explore the therapeutic effect of tumor radiotherapy combined with PD-L1 immunocheckpoint inhibitor.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe tumor-bearing mice (TBM) were randomly allocated to six groups: the low-dose group, high-dose group, low-dose\u0026thinsp;+\u0026thinsp;PD-L1 group, high-dose\u0026thinsp;+\u0026thinsp;PD-L1 group, anti-PD-L1 group and control group. Animals received 2 Gy/14 Fx (low-dose group) or 8 Gy/3 Fx (high-dose group) radiation and the combination treatment groups were given an anti-PD-L1 antibody for two consecutive weeks. MR/PA imaging was used to noninvasively evaluate the response of breast cancer model to different doses of radiotherapy, combined with histopathological techniques to observe the changes of tumor vessels and microenvironment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe inhibitory effect of high-dose radiotherapy on tumors was significantly greater than that of low-dose radiotherapy, with the MR images revealing that the signal intensity (SI) decreased significantly (p༜0.05). Compared with those in the other groups, the tumor vascular density decreased significantly, and the vascular maturity index (VMI) increased significantly in the low-dose group (p༜0.05). The PA images showed that the deoxyhemoglobin (HbR) and total hemoglobin (HbT) levels decreased and the SO\u003csub\u003e2\u003c/sub\u003e level increased after radiation treatment. In addition, the high-dose group had an increased number of tumor-infiltrating lymphocytes (CD4\u003csup\u003e+\u003c/sup\u003e T and CD8\u003csup\u003e+\u003c/sup\u003e T cells) and natural killer (NK) cells and increased PD-L1 expression in the tumors (p༜0.05). The combination of radiotherapy and immunotherapy increased the survival rate of the mice, and a regimen of an 8 Gy dose of radiation combined with immunotherapy inhibited tumor growth and increased the survival rate of the mice to a greater degree than the 2 Gy radiation dose with immunotherapy combination (p\u0026thinsp;=\u0026thinsp;0. 002).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eDifferential fractionation radiotherapy doses exert different effects on tumor vascular remodeling and the immune microenvironment, and MR/PA can be used to evaluate tumor vascular remodeling after radiotherapy, which is of great significance for the clinical applications of radiotherapy combined with immunotherapy.\u003c/p\u003e","manuscriptTitle":"A MR/PA Dual-Modality Imaging for quantitative evaluation of tumor vascular and immune microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-07 19:10:33","doi":"10.21203/rs.3.rs-2222488/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":"5c5b928b-6188-41b1-9440-d1718d72c936","owner":[],"postedDate":"November 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-11T14:14:36+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-07 19:10:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2222488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2222488","identity":"rs-2222488","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}