LIFUS-Driven Engineered Bacteria Reprogram Immunosuppressive Niches via Mechano-NOTCH Signaling

LIFUS-Driven Engineered Bacteria Reprogram Immunosuppressive Niches via Mechano-NOTCH Signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article LIFUS-Driven Engineered Bacteria Reprogram Immunosuppressive Niches via Mechano-NOTCH Signaling Huixiong Xu, Lizhou Lin, Xiao Li, Xiaolong Li, Wenyun Guo, Guan Xin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6644974/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 The tumor microenvironment (TME) of solid tumors constructs a complex biophysical barrier with intricate interactions, including dense stromal structures and immunosuppressive networks, which severely limit the permeability and efficacy of immunotherapy. This study developed an engineered an attenuated Salmonella typhimurium VNP20009 strain encoding an acoustic reporter gene (ARG)-based gas vesicles (GVs), enabling real-time tumor imaging guided mechano-immunomodulation. Low-intensity focused ultrasound (LIFUS) triggered cavitation effects from GVs, generating localized shear forces and mechanical strain. This significantly inhibited tumor growth and attenuated the NOTCH-mediated crosstalk between cancer-associated fibroblasts (CAFs) and CD8⁺ T cells, thereby relieving CD8⁺ T cell activity. This mechanobiological intervention elicited a 12-fold enhancement in cytotoxic CD8⁺ T cell infiltration and extended median survival to over 60 days (versus 30-40 days in controls) in aggressive murine tumor models, demonstrating significant therapeutic efficacy. This integrated strategy establishes a closed-loop therapeutic platform combining bacterial targeting, acoustic visualization, and LIFUS-driven mechano-immunomodulation, achieving 92.7% suppression of both primary and metastatic tumor growth. By synergizing synthetic biology with mechano-immunological regulation, our study pioneers a precisely controllable and real-time image-monitored therapeutic paradigm, providing a transformative approach to overcome solid tumor resistance and accelerate clinical translation. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Physical sciences/Engineering/Biomedical engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlight VNP /ARG-GVs can not only break through the physical barrier of the tumor, but also cooperate with low-intensity focused ultrasound (LIFUS) for ultrasonic imaging. LIFUS in combination with VNP /ARG-GVs reduce the elastic stiffness of the tumor and enhance the immunotherapy of the tumor. LIFUS-GVs can transform CD8 + T cells through the mechanical force-NOTCH pathway axis, enhancing the tumor suppression. Introduction The tumor microenvironment (TME), characterized by physical and immune barriers, remains a major challenge in the treatment of solid tumors [1]. Tumor cells recruit and activate stromal components such as cancer-associated fibroblasts (CAFs), which form dense physical barriers that impede drug penetration and immune cell infiltration [2]. Additionally, the TME is often hypoxic, acidic, and nutrient-deprived, creating an immunosuppressive milieu that further restricts immune cell proliferation and function. These two barriers—physical and immune—interact synergistically; for instance, CAFs secrete immunosuppressive factors to inhibit T cell activity [2–3]. Consequently, despite advancements in immune checkpoint inhibitors[4–5], solid tumors continue to face challenges such as insufficient T cell infiltration and functional exhaustion. Recent studies have revealed that the hypoxic and immunosuppressive TME provides a niche for bacterial colonization [6–7]. Certain anaerobes can penetrate tumor vascular barriers and withstand high interstitial pressure, proliferating within hypoxic and necrotic tumor cores while activating both innate and adaptive immune responses. This has spurred interest in bacterial-based therapies [8–11]. Advances in synthetic biology have accelerated the development of engineered bacteria as promising therapeutic agents. For example, the attenuated Salmonella typhimurium strain VNP20009, with deletions in the msbB and purI genes to reduce toxicity, has shown efficacy in preclinical models by suppressing tumor growth [10–11]. Phase I clinical trials confirmed its safety in cancer patients, though its therapeutic efficacy as a monotherapy yet to achieve complete tumor regression [12–15]. Emerging evidence suggests that combining engineered bacteria with other modalities could significantly enhance antitumor outcomes [16–17]. Combining engineered bacteria with ultrasound (US) technology offers a breakthrough approach. US enables spatiotemporal control and safety profiles, such as non-invasiveness and precise targeting, US-mediated combinatorial therapy with engineered bacteria exhibits superior potential in overcoming therapeutic bottlenecks of tumor compared to alternative multimodal strategies [18]. Furthermore, heat-responsive genetic circuits triggered by focused ultrasound (FUS)-induced hyperthermia can dynamically regulate bacterial activity [19]. However, thermal effects may cause collateral tissue damage. As a mechanical wave, US also exerts non-thermal bio-effects. Low-intensity focused ultrasound (LIFUS), operating at intensities below 3 W/cm², leverages cavitation (mainly stable cavitation) and mechanical forces via microbubbles to achieve therapeutic outcomes with minimal thermal impact. LIFUS has demonstrated utility in gene transfection, blood-brain barrier opening, and drug delivery, particularly when combined with microbubble-enhanced techniques [20–22]. Mechanistically, LIFUS-generated shear stress and microjets activate cellular mechanosensors—including RET, Piezo1, T cell receptors, and Notch receptors—which are emerging targets in cancer therapy [23]. However, conventional microbubbles, typically administered intravascularly, exhibit limited efficacy in hypoxic and poorly vascularized tumors. To address this, acoustically responsive gas vesicles (GVs), encoded by engineered bacteria as intratumoral acoustic cavitation nuclei, enable both ultrasound imaging and mechanical perturbation [24–26]. By combing LIFUS with GVs-generated mechanical forces, this approach may dual-target CAFs and CD8⁺ T cells within the TME, simultaneously disrupting physical and immune barriers. Building on this rationale, we engineered an acoustically responsive bacterial system capable of producing GVs to amplify mechano-immunotherapeutic effects. The system utilizes VNP20009 to breach physical barriers, colonize tumor cores, and prime antitumor immunity. Concurrently, GVs encoded by acoustic report gene (ARG) for real-time monitoring of bacterial colonization via ultrasound imaging. With spatiotemporal LIFUS control, mechanical forces modulate Notch receptors (Notch1-Jagged1) on CAFs and CD8⁺ T cells, attenuating CAFs-CD8⁺T cell crosstalk and reprogramming the TME. This mechano-NOTCH axis alleviates T cell exhaustion and suppresses CAFs activation, thereby inhibiting tumor progression. By establishing a closed-loop therapeutic framework (imaging-treatment-feedback), we validate the efficacy of this strategy in solid tumors and provides the new evidence that mechanical signaling orchestrates the synergistic remodeling of dual barriers—physical and immune—within TME, offering a transformative approach to overcome current limitations in oncology (See graphical abstract ). Results 1. Construction of Engineered Bacteria ( VNP /ARG) for Acoustic Responsive to optimization parameter of LIFUS We employed attenuated Salmonella typhimurium VNP20009 as the genetic chassis for acoustic reporter gene (ARG) expression. As schematized in Fig. 1A , the ARG-encoding plasmid pBAD-bARGSer-axe-txe was electroporated into VNP20009, generating the engineered strain VNP /ARG. Electrophoretic analysis ( Fig. 1B ) confirmed successful plasmid transfection, with arabinose induction (0.2%) triggering robust ARG expression in VNP /ARG, uninduced VNP /ARG exhibited negligible ARG expression compared to both induced counterparts ( VNP /ARG-GVs) and non-engineered VNP20009 controls ( Fig. 1C ). Functional validation illustrated LIFUS-induced GVs cavitation ( Fig. 1D ), producing acoustic signals detectable by ultrasound imaging. Gas vesicles (GVs) production was visualized via transmission electron microscopy (TEM; Fig. 1E ), revealing polarized GVs aggregates occupying ~40% of intracellular space. These nanostructures displayed heterogeneous morphology, including ovoid and elongated configurations. Particle size analysis ( S1A ; S1B ) showed GVs diameters ranged 0.5–1.8 μm, exceeding clinical contrast agents like SonoVue (~2.5 μm) by >30%, indicating superior tissue penetrability. To optimize the LIFUS-responsive imaging of GVs and avoid the influence of thermal effect, two acoustic pressure parameters, 0.6 MPa and 0.8 MPa were selected [25]. In vitro, agarose phantoms containing VNP /ARG-GVs were exposed to LIFUS ( Fig. 1F ). At 5-minute post-sonication, both intensities yielded not distinct acoustic signal intensities (22.67 ± 0.92 dB VS. 24.75 ± 0.92 dB, p < 0.05). However, prolonged exposure (15 minutes) revealed significantly greater signal attenuation in the 0.8 MPa group (14.25 ± 2.92 VS. 2.00 ± 0.67 dB; p < 0.005) ( Fig. 1G ), confirming more complete GVs cavitation at 0.8 MPa pressures. Similarly, acoustic mode imaging demonstrated robust GVs cavitation in tumor-bearing mice. Quantitative analysis revealed 0.8 MPa-induced signals (34.00 ± 10.00 dB) were ~2.5-fold stronger than 0.6 MPa signals (13.75 ± 9.19 dB; p < 0.005) ( Fig. 1H ). Acoustic imaging confirmed that acoustic cavitation was more thoroughly achieved under 0.8 MPa and resulting in enhanced contrast resolution ( Fig. 1I ). Furthermore, B-mode imaging further revealed gradual bacterial regression post-cavitation, the grayscale imaging was decreased, suggesting that bacterial lysis occurred concomitantly with GVs destruction, thereby mitigating risks of bacterial over proliferation and enhancing biosafety. Safety evaluations were further conducted to assess the impact of these acoustic parameters on VNP /ARG colonization and viability. In the absence of GVs expression, neither 0.6 MPa nor 0.8 MPa significantly affected engineered bacteria VNP /ARG survival ( S1C ). However, following arabinose-induced GVs expression, both bacterial colonization decreased markedly, with the 0.8 MPa group showing stronger suppression (0.24 ± 0.09 VS. 0.6 MPa, 1.12 ± 0.11 OD600 units) ( Fig. 1J ). Live/dead staining ( Fig. 1K-L ) also quantified bacterial viability post-ultrasound exposure. While 0.6 MPa induced minimal cytotoxicity (7.15 ± 2.20% mortality), 0.8 MPa caused significant cell death (31.38 ± 1.84%; p < 0.0001).This demonstrates that under 0.8 MPa LIFUS-GVs conditions, bacteria VNP /ARG undergo direct lysis, preventing further proliferation in vivo and reducing potential toxicity. Conversely, non-GVs-expressing bacteria retained proliferative capacity, ensuring their availability for subsequent therapeutic cycles. The cytotoxicity assays in vitro ( S1D ) and the inflammatory responses in vivo ( S1E-F ) were also confirm VNP /ARGs favorable safety for applications. Collectively, these results confirm the biosafety of engineered bacteria in vivo and establish 0.8 MPa as the optimal parameter for both imaging efficacy and bacterial containment. 2. Mechanical immunotherapy effect of LIFUS- VNP /ARG-GVs for tumors The role of VNP /ARG-GVs and the optimization ultrasound imaging parameter of 0.8 MPa have been confirmed, we further explored the mechanical immunotherapeutic effect of LIFUS combining VNP /ARG-GVs (LIFUS- VNP /ARG-GVs group) for breast tumors ( Fig. 2A ). Sequential five-cycle LIFUS- VNP /ARG-GVs treatment (21-day) achieved robust tumor growth inhibition compared to controls ( Fig. 2B-C ). Notably, monotherapy with VNP /ARG (with or without arabinose induction) failed to elicit statistically significant antitumor effects ( p > 0.05), underscoring the critical role of LIFUS-GVs combination. Survival analysis revealed profound therapeutic benefits, mice receiving combination therapy survived >60 days, whereas all control/monotherapy groups succumbed by day 30-40 ( p < 0.001) ( Fig. 2D ). These data confirmed the combination of LIFUS- VNP /ARG-GVs as a highly effective tumor suppression modality. Flow cytometry analysis ( Fig. 2E-H ) uncovered distinct immune landscape remodeling in each group. The LIFUS group, similar to the control group, showed no significant changes in CD4 + T /CD8 + T lymphocytes infiltration (3.45 ± 1.46% VS. 3.80 ± 1.84%). Then, starting from the VNP /ARG monotherapy group, changes were observed (CD4 + T, 22.96 ± 3.86% and CD8 + T, 12.46 ± 2.01%), and the obvious CD4 + T-cell elevation (22.96 ± 3 .86%, p < 0.0001 vs control), reflecting innate immune activation by VNP /ARG. Moreover, CD4 + T and CD8 + T lymphocytes were significantly increased in the combined therapy group of LIFUS- VNP /ARG-GVs, synergistic upregulation of cytotoxic CD8 + T-cells (51.98 ± 7.92%) and moderate CD4 + T-cell increase (17.02 ± 3.85%), achieving a 12.0-fold CD8 + T-cell enrichment compared to control, the reason for this increased immune activity within the tumor may result from the combination treatment effectively activate the immune activity within the tumor, especially that of CD8 + T cells. Simultaneously, FOXP3 + regulatory T-cell suppression ( Fig. 2F ) further corroborated immunomodulatory effects, with combination therapy exhibiting lowest suppressor cell frequencies (control group, 28.38 ± 2.59% VS. 7.76 ± 1.60%, p < 0.0001). Further analysis of intra-tumor associated factors by Elisa of Fig. 2I-J demonstrates a significant elevation of IFN-γ and TNF-α in the LIFUS- VNP /ARG-GVs group (IFN-γ: 37.4 ± 6.88 ng/mL, TNF-α: 1.37 ± 0.41 ng/mL) compared to the control and LIFUS alone groups (IFN-γ: 0.092 ± 0.09 ng/mL, TNF-α: 0.108 ± 0.08 ng/mL, p < 0.0001), consistent with findings in other studies that highlight the clinical significance of these cytokines in disease progression and treatment response. Similarily, immunohistochemical analysis of tumors showed that apoptosis in Ki67 （ Fig. 2K ； S2A ） and HE ( Fig. 2L )staining began to appear gradually in the VNP /ARG group and was the highest in the LIFUS- VNP /ARG-GVs group. 3. Mechano-regulation of fibroblast-CD8 + T cells NOTCH pathway interactions via LIFUS- VNP /ARG-GVs (Combined therapy) Previous results have validated that the synergistic treatment system of LIFUS- VNP /ARG-GVs enhances intratumoral CD4 + T infiltration by leveraging the immunogenicity of VNP /ARG engineer bacteria, and the activation effects of CD8 + T infiltration to effectively inhibit tumor growth by the combination of LIFUS-GVs. Herein, we elucidated the activation effects of CD8 + T by mechanotransductive mechanisms underlying this therapeutic synergy using single-cell sequencing analysis. Fig. 3A illustrates the results of cell cluster annotation within the TME, identifying eight distinct cell taxa: B cells, endothelial cells, epithelial cells, fibroblasts, myeloid cells, neutrophils, smooth muscle cells, and T/NK cells. Quantitative assessment demonstrated a significant enrichment of T lymphocyte populations in the LIFUS- VNP /ARG-GVs cohort compared to monotherapy groups ( Fig. 3B ). Further subclassification identified CD8 + T cells as the predominant cytotoxic lymphocyte subset ( Fig. 3C-D ), with the emergence of a unique Cd8t_02 cluster exhibiting augmented spatial distribution in the combination treatment group, meanwhile, VNP /ARG showed the lowest Cd8t_02 cluster when compared with other control groups, which might be attributed to the fact that the increase in this group is mainly dominated by CD4 + T cells and there is not a strong correlation with the Cd8_02 population( Fig. 3E-F ). Functional annotation revealed this cluster plays a significant role in the cytotoxic response, as CD8 + T cells are known for their cytotoxicity, which Cd8t_02 could effectively enhance the toxicity of CD8 + T cells ( Fig. 3G ). Fig. 3H shows the GO enrichment analysis reveals that this subset clusters of CD8 + T is predominantly associated with intercellular adhesion and T cell activation functions within the TME. The intercellular adhesion within TME are modulated by the stiffness and elasticity of its dense stromal matrix, with the relevant adhesion-associated proteins (e.g., collagen) being primarily secreted by CAFs-the principal stromal constituents in tumor matrix composition [27-29]. The study examined the alterations of matrix elastic stiffness among different groups, the ultrasonic elastography in Fig. 4A showed that the tumor elasticity in the LIFUS- VNP /ARG-GVs group (12.95 ± 2.18 kPa) decreased compared with the control group (57.19 ± 15.02 kPa) and VNP /ARG-GVs group (46.82 ± 8.13 kPa),while there is a slight decreased in LIFUS monotherapy group (36.93 ± 6.812 kPa), but there was no significant change between control and VNP /ARG groups ( Fig. 4B ). The elastic modulus of each group was further verified by atomic force corroborated these findings ( Fig. 4C ). These two results showed that the mechanical effect of LIPUS-GVs decreased the tumor matrix elasticity stiffness, which was conducive to the infiltration of immune cells and in consistent with the change of the tumor immune microenvironment in our previous results. Moreover, immunofluorescence characterization of Collagen I distribution patterns in tumor specimens ( S2B) illustrated the speculation of the matrix changes in the TME . Fig. 4D shows that there was a dense distribution of intratumoral fibroblasts in the control group and a decrease in the LIFUS group (172.05 ± 37.46 mm 2 VS. 248.62 ± 9.19 mm 2 , p < 0.001), indicating that the presence of LIFUS to TME can slightly influence the distribution of CAFs, this may be related to the inherent mechanical wave properties of ultrasound, which enables biomechanical stimulation of the TME through direct mechanical perturbation. However, the fibroblasts volume (210.04 ± 0.74 mm 2 ) in the VNP /ARG-GVs group did not show significant changes compared with the control group, and the combined treatment group exhibited a significant reduction in fibroblast volume in fibroblast volume (42.24 ± 8.46 mm 2 ) versus controls group. Revealing that VNP /ARG-GVs alone did not alter the stiffness or elasticity of the TME, however, combined therapy caused a significant change of fibroblasts. In order to find out the mechanism of interaction between fibroblasts and CD8 + T cells, the interaction between the two cells was analyzed. In the cell-cell interaction analysis between the control and combined treatment group, we found that the pathway had reduced interaction within fibroblasts and CD8 + T cell population (specially, Cd8t_04 cluster) ( Fig. 4E ). The functional annotation revealed this cluster (Cd8t_02 cluster and Cd8t_04) to be relative to RNA metabolic process, which regulates the activity of CD8 + T cells (see S3 ). The differential analysis of the five oncogenes pathways showed that NOTCH, TGF and WNT pathways were significantly upregulated in the control group, and the NOTCH pathway exhibited the highest expression in the control group while the lowest expression in the combined therapy group ( Fig. 4F ). Meanwhile, genes and proteins Notch1 and Jagged1 expression on the NOTCH pathway in Fig. 4G-4I were also significantly reduced in the combined therapy group, confirming the result of reduced NOTCH pathway interaction between CD8 + T cells and fibroblasts in LIFUS- VNP /ARG-GVs. The fibroblasts in tumor are usually mutate into CAFs [2], so we refer to it as CAFs. As it is demonstrated the NOTCH signaling pathway, a highly conserved evolutionary mechanism governing cell proliferation through transmembrane receptor-ligand interactions, exhibits aberrant activation in TME. Tumor cells secrete paracrine factors that constitutively activate NOTCH signaling in both malignant cells and CAFs [30-32]. The observed attenuation of NOTCH-mediated crosstalk between CAFs and CD8 + T cells, evidenced by diminished receptor-ligand interaction frequencies and concomitant reduction in cognate gene expression profiles, substantiates that LIPUS- VNP /ARG-GVs treatment effectively suppresses transmembrane protein biosynthesis within this pathway, thereby obstructing intercellular signal transduction. This alteration of TME was in correlation to the LIFUS driven VNP/ARG-GVs-generated mechanical force, decreasing the CAFs deposit and elastic stress of tumor for CD8 + T cells activation and migration. 4. The dual regulatory role of mechanical force generated by LIFUS-driven GVs on CAFs and CD8 + T cells via NOTCH signaling We analyzed that the combined therapy of LIFUS and VNP /ARG-GVs decreased the expression of CAFs in TME, lowered the tumor elastic stress and weakened the crosstalk between CAFs and CD8 + T cells via the NOTCH pathway, but the mechanical force influence of GVs responsive to LIFUS on CD8 + T cells in the NOTCH pathway needs to be further explored. We extracted CAFs and CD8 + T cells to established in vitro co-culture systems, for co-culture in vitro and added pre-extracted GVs for acoustic responsive to LIFUS ( Fig. 5A ), followed by the separation of the two cell types using flow cytometry. Fig. 5B confocal imaging shows the expression of representative proteins in NOTCH pathway of CAFs, the fluorescence intensity of representative Notch1 receptor and Jagged1 ligand in the group without LIFUS-driven GVs (GVs - LIFUS group) was significantly higher compared to that in the LIFUS-driven GVs (GVs + LIFUS group), Fig. 5C also shows the consistent expression of Notch1 and Jagged1 proteins, indicating that the expression of these proteins within CAFs was significantly lower after acoustic responsive. However, the NOTCH pathway proteins on CD8 + T cells showed different alteration. Fig. 5D and 5E show that Jagged1 ligand expression on CD8 + T cells was decreased in GVs + LIFUS group, whereas Notch1 receptor protein expression on CD8 + T cells has no significant change in both GVs - LIFUS group and GVs + LIFUS group. These alter expression of NOTCH pathway proteins in CAFs and CD8 + T cells can further explain the attenuation of cross talk between CAFs and CD8 + T cells in tumors, and it can be speculated that the expression is mainly decreased by CAFs for the expression in CD8 + T cells did not decrease but seems to increase. The double regulatory role of mechanical force-NOTCH pathway on CAFs and CD8 + T cells seems to contributes to the improvement of TME. Therefore, to verify the double regulatory, the CAFs (without or with LIFUS-driven GVs treatment) were cultured in Transwell chambers to assess the migratory and secreted factor ( Fig. 5F ). Fig. 5G displayed that the migration number in each group, including the control group (the initially extracted CAFs), -LIFUS group (without LIFUS-driven GVs first) and LIFUS treatment group (with LIFUS-driven GVs first), the migrant number in LIFUS treatment group (56 ± 4 cells/field) was less than that in control (150.67 ± 15.04 cells/field, p < 0.0001) and without LIFUS group (101.67 ± 3.79 cells/field, p < 0.001) ( Fig. 5H ). Fig.5I-J showed the levels of TGF-β and Col 1secreted by CAFs, which promote CAFs activation and migration [33-34] demonstrated a significant decrease following LIFUS treatment compared to both control and untreated groups, indicating that LIFUS-driven GVs-generated mechanical forces effectively suppresses CAFs activation and migration by inhibiting NOTCH signaling within TME. However, the decrease of Jagged1 ligand and the increase of Nocth1 receptor protein in CD8 + T with LIFUS-driven GVs treatment group showed an improvement to the cytotoxicity of CD8 + T cells binding to tumor cells, thus inhibiting the growth of tumor cells. Fig. 5K illustrated the co-cultured system of CD8 + T cells (without or with LIFUS-driven GVs treatment) and tumor cells (4T1). Fig. 5L-M showed that the fluorescence intensity of CD8 + T cells adhere to the tumor cells in CD8 + T +LIFUS group was significantly higher than that in the control group and CD8 + T -LIFUS group (51.89 ± 10.46% VS. Control, 3.25 ± 1.19% and CD8 + T -LIFUS , 15.72 ± 7.53%). The enhanced adhesion force of CD8 + T cells, as demonstrated in recent studies, plays a pivotal role in bolstering the immune system's ability to combat tumors [35-37]. Similarly, the apoptosis of tumor cells was examined at 12 and 36 hours post-treatment. Fig. 5N and S4A indicates no significant difference in apoptosis between the groups at 12-hour, however, the CD8 + T +LIFUS group exhibited a marked increase in apoptotic tumor cells after 36 hours and the 4T1-Luc showed almost no fluorescence. S4B-C showed that the release of granzyme A (GZMA) and IFN-γ in CD8 + T +LIFUS group were significantly higher than that in control and CD8 + T -LIFUS cells group, which played an effective role in inhibiting tumor growth. These functional validation confirmed a dual regulatory in CAFs and CD8 + T cells where mechanotransduction-mediated NOTCH activation in CD8 + T cells (Notch-Jagged1 signaling pathways) and an inhibition in CAFs synergizes with NOTCH, creating a novel perspective for the utilization of CD8 + T cells in the pre-clinical tumor treatment. 5. Preclinical Translation of Mechanobiology-Enhanced CD8 + T Cell Therapy. Upon previous research that elucidated the combination therapy of LIFUS- VNP /ARG-GVs boosts the cytotoxicity of CD8 + T cells against 4T1 tumor cells, we further investigated the therapeutic translatability. Two different tumor models were established to verify the tumor inhibition of modified CD8 + T cells. Tumors were established by injecting B16-OVA cells (B16 cells treated with Ovalbumin) and treated with OT-1 to target OVA (including 0T-1 GVs-LIFUS and 0T-1 GVs+LIFUS ), tumor was pre-treatment with LIFUS-GVs before injecting 0T-1 in 0T-1 GVs+LIFUS group, and observed tumor volume changes after 15 days ( Fig. 6A ). The control group exhibited the fastest tumor growth. and the 0T-1 GVs-LIFUS group demonstrated some lethality resulting from GVs driven. However, its inhibitory effect was still notably lesser compared to the 0T-1 GVs+LIFUS group ( Fig. 6B ). The weight of tumors and body in mice also changed minimally in 0T-1 GVs+LIFUS group ( Fig. 6C-6D ). Fig. 6E investigated the co-expression of Tim-3 and PD-1 in tumor tissue using flow cytometry, observed that the lowest combined expression of Tim3 and PD-1 (represent CD8 + T cells exhaustion) in the 0T-1 GVs+LIFUS group (2.55 ± 1.27%), in contrast to the highest expression in the control group (43 ± 4.60%, p < 0.0001), indicating a significant reduction of approximately 94%. Meanwhile, Fig. 6F the expression of anti-tumor factor TNF-α and IFN-γ also showed the highest expression in the 0T-1 GVs+LIFUS group ( p < 0.0001). Fig. 6G the apoptosis in the 0T-1 GVs+LIFUS group was higher than the other two groups (63.20 ± 8.43% VS. control, 10.57 ± 2.25%, p < 0.0001, and 0T-1 GVs-LIFUS , 33.32 ± 3.96%, p < 0.0001), showing obvious statistical significance. 4T1-Luc tumor cells were injected intravenously into mice to make a 4T1 lung metastasis model and treatment with CAR-T cells ( Fig. 6H ). Fig. 6I shows the number of lung metastases in the three groups, it was observed that the group treated with CAR-T GVs+LIFUS cells exhibited a significantly reduced number of lung metastases, with almost no metastatic lesions detected. Fig. 6J displays the imaging of significant lung metastasis via small animal imaging. Distinct lung metastasis emerged in the control group on the 5th day, mortality was noted by the 15th day and all mice succumbed by the 25th day. In the CAR-T GVs-LIFUS group, lung metastasis was not prominent at first, the first deaths occurred on the 20th day with further fatalities on the 25th day. In the CAR-T GVs+LIFUS group, although mild lung metastasis was detected on both the 10th and 15th days, the fluorescence intensity indicating lung metastasis had vanished when re-examined on the 20th day and there were no deaths observed in the CAR-T GVs+LIFUS group until the 30th day. Fig. 6K the survival curves demonstrated prolonged survival in the CAR-T GVs+LIFUS group (median survival > 50 days) versus CAR-T monotherapy (47.5 days) and control (35.5 days) group ( p < 0.001). These preclinical findings establish the therapeutic potential of mechanobiology-augmented adoptive T cell therapies by LIFUS for treating both primary and metastatic malignancies. Discussion Ultrasound, fundamentally categorized as a mechanical wave, not only enables spatial imaging but also exerts physical regulation on biological tissues through mechanical effects. Current therapeutic strategies based on ultrasound-mediated mechanical effects primarily rely on the synergistic interaction between LIFUS and microbubble cavitation to generate microjets and shear forces, thereby enhancing cell membrane permeability for improved drug/gene delivery[38–40]. Addressing the dual challenges of physical and immune barriers in tumor therapy, this study innovatively developed a LIFUS-driven engineered bacterial system: attenuated Salmonella typhimurium VNP2009 was employed as a carrier to breach tumor physical barriers, while GVs with acoustic responsiveness were expressed to establish a sound-activated system. This system not only achieved intratumoral acoustic imaging but, more importantly, leveraged LIFUS-driven mechanical forces from GVs to modulate the tumor immune microenvironment, ultimately suppressing tumor growth. Previous studies have indicated that an acoustic intensity threshold exceeding 0.72 MPa enables simultaneous ultrasound imaging[25–26]. Our experimental validation confirmed that a parameter of 0.8 MPa ensured optimal cavitation imaging of GVs while effectively controlling the survival period of the engineered bacteria ( VNP /ARG-GVs) in vivo. The immediate inactivation of bacteria post-cavitation eliminated biosafety risks associated with bacterial overproliferation. Notably, the engineered bacterial system represents a paradigm shift in oncology, as the combined therapy of VNP /ARG-GVs and LIFUS demonstrated efficacy in mouse tumor models. This combinatorial approach robustly activated antitumor immune responses: following intratumoral colonization by VNP /ARG, the proportion of CD4 + T cells increased, and CD8 + T cell infiltration was further amplified upon LIFUS-driven mechanical stimulation. Concurrently, key immune effector molecules, including TNF-α and IFN-γ, were upregulated, leading to significant tumor suppression and prolonged survival in mice. Single-cell sequencing revealed that in the LIFUS-treated group, CD8 + T cell subsets exhibited reduced interaction with CAFs via the NOTCH pathway, specifically through diminished Notch1-Jagged1 signaling. Functional assays demonstrated enhanced cytotoxicity in CD8 + T cell subsets, accompanied by decreased collagen I (Col I) deposition and mechanical stress within tumors, alongside downregulated Notch1 and Jagged1 expression at both transcriptional and protein levels. These TME alterations were mechanistically linked to the mechanical forces generated by LIFUS-driven VNP /ARG-GVs. Cellular mechanosensitivity, a cornerstone of cancer immunotherapy, involves mechanoreceptors such as NOTCH receptors and T cell receptors, which transduce extracellular mechanical signals into intracellular responses through conformational changes [41–43]. The engineered bacteria activated antitumor immunity via intratumoral colonization and antigen expression, while LIFUS-driven VNP /ARG-GVs synergistically amplified CD8 + T cell infiltration and cytotoxic function. Single-cell analyses highlighted a critical shift in CAFs-CD8 + T cell interactions: reduced Notch1-Jagged1 axis activity impaired their communication efficiency. This correlated with heightened CD8 + T cell cytotoxicity, diminished CAFs-derived Col I secretion and mechanical stress in tumor tissue, and downregulated Notch1/Jagged1 expression. These dynamics were governed by LIFUS-generated mechanical forces, with mechanoreceptors providing a molecular basis for mechanotransduction. Mechanistically, LIFUS-GVs-derived mechanical forces bidirectionally regulated CAFs and CD8 + T cell functions via the NOTCH pathway. In vitro co-culture models showed that mechanical stimulation suppressed CAFs migration, TGF-β secretion and Col I production, indicative of reduced activity and proliferation. Conversely, CD8 + T cell tumor adhesion and cytotoxicity were markedly enhanced. The NOTCH pathway operates through receptor-ligand interactions, triggering release of the Notch intracellular domain (NICD) to regulate target genes and maintain cellular homeostasis [44–45]. Specifically, dual downregulation of Notch1/Jagged1 in CAFs disrupted their matrix-mediated physical/immune barriers, while selective Jagged1 reduction in CD8 + T cells facilitated preferential binding of tumor cell Jagged1 to T cell Notch1[46–50]. This differential regulation optimized CD8 + T cell antitumor responses, offering novel evidence for mechanical force-mediated immune modulation. Validation in two additional models further demonstrated mechanical reprogramming of the tumor immune microenvironment. OT-1 T cells combined with LIFUS-GVs pretreatment reduced PD-1/Tim-3 expression, alleviated T cell exhaustion, and enhanced cytotoxicity—a phenomenon mechanistically akin to sodium-induced T cell potentiation [51]. Similarly, LIFUS-GVs preconditioning prior to CAR-T cell infusion improved efficacy against metastatic tumors, aligning with advancements in CAR-T engineering [52–54]. This study pioneers a mechanobiology-driven precision therapy framework, integrating spatiotemporal coupling of LIFUS and engineered bacteria to elucidate the central role of the mechanical force-NOTCH axis in tumor immune regulation. While our findings confirm that mechanical forces suppress the CAF-CD8 + T cell Notch1-Jagged1 axis, upstream/downstream signaling networks (e.g., Hes/Hey family regulation) and crosstalk between mechanosensors (e.g., Piezo1) and the NOTCH pathway warrant further exploration. Future work will employ patient-derived xenograft (PDX) models, single-cell spatial transcriptomics, and PD-1 inhibitors to optimize therapeutic regimens. Collectively, this mechanobiological framework provides a transformative perspective for remodeling the tumor immune microenvironment, offering novel theoretical and technological pathways for precision immunotherapy. Conclusion This study shows that the combination of LIFUS and VNP /ARG-GVs provides a new strategy for tumor mechanical immunotherapy, while reveals the mechanism of mechanical force-NOTCH pathway axis on CD8 + T cells and CAFs, providing a more solid theoretical basis for clinical application. Declarations Acknowledgements This work was supported by the Nation National Science Foundation of China Grant (2024XKPT32, 82402263,82001830) and the Postdoctoral Fellowship Program of CPSF (GZC20230499). All animal studies were approved by the Institutional Animal Care Committee of Shanghai Ten’s People’ Hospita (SHDSYY-2025-Y3216-01). The authors declare no competing interests. Contribution Lizhou Lin for operations of experiment and paper writing, Xiao Li for guidance on gas vesicle engineering Dr. Xiaolong Li for providing access to LIFUS equipment. Wenyun Guo and Xin Guan for assistance with WB and flow analyze. References de Visser, KE, Joyce, JA. The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. CANCER CELL. 2023; 41 (3): 374-403. Tsoumakidou, M. The advent of immune stimulating CAFs in cancer. NAT REV CANCER. 2023; 23 (4): 258-269. Li, C, Guo, H, Zhai, P, Yan, M, Liu, C, Wang, X, Shi, C, Li, J, Tong, T, Zhang, Z, Ma, H, Zhang, J. Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts Infiltration and Antitumor Activity of CD8+ T Cells. CANCER RES. 2024-01-16; 84 (2): 258-275. Petit PF, Daoudlarian D, Latifyan S, Bouchaab H, Mederos N, Doms J,Abdelhamid K, Ferahta N, Mencarelli L, Joo V, Bartolini R, Stravodimou A,Shabafrouz K, Pantaleo G, Peters S, Obeid M. Tocilizumab provides dual benefits in treating immune checkpoint inhibitor-associated arthritis and preventingrelapse during ICI rechallenge: the TAPIR study. Ann Oncol. 2025 Jan;36(1):43-53. Naidoo J, Cottrell TR, Lipson EJ, Forde PM, Illei PB, Yarmus LB, Voong KR, Feller-Kopman D, Lee H, Riemer J, Wang D, Taube JM, Brahmer JR, Lin CT, Danoff SK, D'Alessio FR, Suresh K. Chronic immune checkpoint inhibitor pneumonitis. J Immunother Cancer. 2020 Jun;8(1):e000840. Garrett WS. Cancer and the microbiota. Science. 2015 Apr 3;348(6230):80-6. Kwon SY, Thi-Thu Ngo H, Son J, Hong Y, Min JJ. Exploiting bacteria for cancer immunotherapy. Nat Rev Clin Oncol. 2024 Aug;21(8):569-589. Mager LF, Burkhard R, Pett N, Cooke NCA, Brown K, Ramay H, Paik S, Stagg J, Groves RA, Gallo M, Lewis IA, Geuking MB, McCoy KD. Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy. Science. 2020 Sep 18;369(6510):1481-1489. Gurbatri, CR, Arpaia, N, Danino, T. Engineering bacteria as interactive cancer therapies. SCIENCE. 2022; 378 (6622): 858-864. Feng, Z, Wang, Y, Xu, H, Guo, Y, Xia, W, Zhao, C, Zhao, X, Wu, J. Recent advances in bacterial therapeutics based on sense and response. ACTA PHARM SIN B. 2023-03-01; 13 (3): 1014-1027. Wu, L, Bao, F, Li, L, Yin, X, Hua, Z. Bacterially mediated drug delivery and therapeutics: Strategies and advancements. ADV DRUG DELIVER REV. 2022-08-01; 187 114363. Liang K, Liu Q, Li P, Luo H, Wang H, Kong Q. Genetically engineered Salmonella Typhimurium: Recent advances in cancer therapy. Cancer Lett. 2019 Apr ;448:168-181. Yin T, Liu La, Dong Shi-da, Huang Bao-lian, Li Chen-yang, CAO Zhi-ting, HUA Zi-chun. Research on anti-tumor mechanism of attenuated Salmonella typhimurium VNP20009. Acta Pharmaceutica Sinica, 2023, 58(9):2700-2706. Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, Sherry RM, Topalian SL, Yang JC, Stock F, Freezer LJ, Morton KE, Seipp C, Haworth L, Mavroukakis S, White D, MacDonald S, Mao J, Sznol M, Rosenberg SA. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol. 2002 Jan 1;20(1):142-52. Lin, Z, Meng, F, Ma, Y, Zhang, C, Zhang, Z, Yang, Z, Li, Y, Hou, L, Xu, Y, Liang, X, Zhang, X. In situ immunomodulation of tumors with biosynthetic bacteria promote anti-tumor immunity. Bioact Mater. 2024-02-01; 32 12-27. Liu, L, Xin, W, Li, Q, Huang, B, Yin, T, Hua, S, Yang, C, Chen, C, Han, C, Hua, Z. Neutrophil-Mediated Tumor-Targeting Delivery System of Oncolytic Bacteria Combined with ICB for Melanoma Lung Metastasis Therapy. Adv Sci (Weinh). 2023-10-01; 10 (29): e2301835. Liu G, Ma N, Cheng K, Feng Q, Ma X, Yue Y, Li Y, Zhang T, Gao X, Liang J, Zhang L, Wang X, Ren Z, Fu YX, Zhao X, Nie G. Bacteria-derived nanovesicles enhance tumour vaccination by trained immunity. Nat Nanotechnol. 2024 Mar;19(3):387-398. Chen, Y, Du, M, Yuan, Z, Chen, Z, Yan, F. Author Correction: Spatiotemporal control of engineered bacteria to express interferon-γ by focused ultrasound for tumor immunotherapy. Nat Commun. 2024-04-30; 15 (1): 3656. Abedi, MH, Yao, MS, Mittelstein, DR, Bar-Zion, A, Swift, MB, Lee-Gosselin, A, Barturen-Larrea, P, Buss, MT, Shapiro, MG. Ultrasound-controllable engineered bacteria for cancer immunotherapy. Nat Commun. 2022-03-24; 13 (1): 1585. Legon, W., Strohman, A. Low-intensity focused ultrasound for human neuromodulation. Nat Rev Methods Primers 4, 91 (2024). Feng J, Li Z. Progress in Noninvasive Low-Intensity Focused Ultrasound Neuromodulation. Stroke. 2024 Oct;55(10):2547-2557. Kim MG, Yu K, Yeh CY, Fouda R, Argueta D, Kiven S, Ni Y, Niu X, Chen Q, Kim K, Gupta K, He B. Low-intensity transcranial focused ultrasound suppresses pain by modulating pain-processing brain circuits. Blood. 2024 Sep 5;144(10):1101-1115. Zamfirov, L, Nguyen, NM, Fernández-Sánchez, ME, Cambronera Ghiglione, P, Teston, E, Dizeux, A, Tiennot, T, Farge, E, Demené, C, Tanter, M. Acoustic-pressure-driven ultrasonic activation of the mechanosensitive receptor RET and of cell proliferation in colonic tissue. Nat Biomed Eng. 2024-12-20. Bourdeau, RW, Lee-Gosselin, A, Lakshmanan, A, Farhadi, A, Kumar, SR, Nety, SP, Shapiro, MG. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. NATURE. 2018-01-03; 553 (7686): 86-90. Bar-Zion, A, Nourmahnad, A, Mittelstein, DR, Shivaei, S, Yoo, S, Buss, MT, Hurt, RC, Malounda, D, Abedi, MH, Lee-Gosselin, A, Swift, MB, Maresca, D, Shapiro, MG. Acoustically triggered mechanotherapy using genetically encoded gas vesicles. NAT NANOTECHNOL. 2021-12-01; 16 (12): 1403-1412. Hurt, RC, Buss, MT, Duan, M, Wong, K, You, MY, Sawyer, DP, Swift, MB, Dutka, P, Barturen-Larrea, P, Mittelstein, DR, Jin, Z, Abedi, MH, Farhadi, A, Deshpande, R, Shapiro, MG. Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria. NAT BIOTECHNOL. 2023-07-01; 41 (7): 919-931. Onder, L, Papadopoulou, C, Lütge, A, Cheng, HW, Lütge, M, Perez-Shibayama, C, Gil-Cruz, C, De Martin, A, Kurz, L, Cadosch, N, Pikor, NB, Rodriguez, R, Born, D, Jochum, W, Leskow, P, Dutly, A, Robinson, MD, Ludewig, B. Fibroblastic reticular cells generate protective intratumoral T cell environments in lung cancer. CELL. 2025-01-23; 188 (2): 430-446.e20. Brown FD, Sen DR, LaFleur MW, Godec J, Lukacs-Kornek V, Schildberg FA, Kim HJ, Yates KB, Ricoult SJH, Bi K, Trombley JD, Kapoor VN, Stanley IA, Cremasco V, Danial NN, Manning BD, Sharpe AH, Haining WN, Turley SJ. Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling. Nat Immunol. 2019 Dec;20(12):1668-1680. Apollonio B, Spada F, Petrov N, Cozzetto D, Papazoglou D, Jarvis P, Kannambath S, Terranova-Barberio M, Amini RM, Enblad G, Graham C, Benjamin R,Phillips E, Ellis R, Nuamah R, Saqi M, Calado DP, Rosenquist R, Sutton LA,Salisbury J, Zacharioudakis G, Vardi A, Hagner PR, Gandhi AK, Bacac M, Claus C,Umana P, Jarrett RF, Klein C, Deutsch A, Ramsay AG. Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma. J Clin Invest. 2023 Jul 3;133(13):e166070. Jiang, Y, Wang, Y, Chen, G, Sun, F, Wu, Q, Huang, Q, Zeng, D, Qiu, W, Wang, J, Yao, Z, Liang, B, Li, S, Wu, J, Huang, N, Wang, Y, Chen, J, Zhai, X, Huang, L, Xu, B, Yamamoto, M, Tsukamoto, T, Nomura, S, Liao, W, Shi, M. Nicotinamide metabolism face-off between macrophages and fibroblasts manipulates the microenvironment in gastric cancer. CELL METAB. 2024-08-06; 36 (8): 1806-1822.e11. Meurette, O, Mehlen, P. Notch Signaling in the Tumor Microenvironment. CANCER CELL. 2018-10-08; 34 (4): 536-548. Majumder, S, Crabtree, JS, Golde, TE, Minter, LM, Osborne, BA, Miele, L. Targeting Notch in oncology: the path forward. NAT REV DRUG DISCOV. 2021-02-01; 20 (2): 125-144. Li, X, Yan, X, Wang, Y, Kaur, B, Han, H, Yu, J. The Notch signaling pathway: a potential target for cancer immunotherapy. J Hematol Oncol. 2023-05-02; 16 (1): 45. Mandula, JK, Sierra-Mondragon, RA, Jimenez, RV, Chang, D, Mohamed, E, Chang, S, Vazquez-Martinez, JA, Cao, Y, Anadon, CM, Lee, SB, Das, S, Rocha-Munguba, L, Pham, VM, Li, R, Tarhini, AA, Furqan, M, Dalton, W, Churchman, M, Moran-Segura, CM, Nguyen, J, Perez, B, Kojetin, DJ, Obermayer, A, Yu, X, Chen, A, Shaw, TI, Conejo-Garcia, JR, Rodriguez, PC. Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages. IMMUNITY. 2024-05-14; 57 (5): 1124-1140. Yang S, Wang M, Tian D, Zhang X, Cui K, Lü S, Wang HH, Long M, Nie Z. DNA-functionalized artificial mechanoreceptor for de novo force-responsive signaling. Nat Chem Biol. 2024 Aug;20(8):1066-1077. Basu, R, Whitlock, BM, Husson, J, Le Floc'h, A, Jin, W, Oyler-Yaniv, A, Dotiwala, F, Giannone, G, Hivroz, C, Biais, N, Lieberman, J, Kam, LC, Huse, M. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. CELL. 2016-03-24; 165 (1): 100-110. Cambria, E, Coughlin, MF, Floryan, MA, Offeddu, GS, Shelton, SE, Kamm, RD. Linking cell mechanical memory and cancer metastasis. NAT REV CANCER. 2024-03-01; 24 (3): 216-228. Chowdhury, SM, Abou-Elkacem, L, Lee, T, Dahl, J, Lutz, AM. Ultrasound and microbubble mediated therapeutic delivery: Underlying mechanisms and future outlook. J CONTROL RELEASE. 2020-10-10; 326 75-90. Dimcevski, G, Kotopoulis, S, Bjånes, T, Hoem, D, Schjøtt, J, Gjertsen, BT, Biermann, M, Molven, A, Sorbye, H, McCormack, E, Postema, M, Gilja, OH. A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer. J CONTROL RELEASE. 2016-12-10; 243 172-181. Stern, JM, Spivak, NM, Becerra, SA, Kuhn, TP, Korb, AS, Kronemyer, D, Khanlou, N, Reyes, SD, Monti, MM, Schnakers, C, Walshaw, P, Keselman, I, Cohen, MS, Yong, W, Fried, I, Jordan, SE, Schafer, ME, Engel, J, Bystritsky, A. Safety of focused ultrasound neuromodulation in humans with temporal lobe epilepsy. BRAIN STIMUL. 2021-01-01; 14 (4): 1022-1031. Liu B, Chen W, Evavold BD, Zhu C. Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell. 2014 Apr 10;157(2):357-368. Zhou B, Lin W, Long Y, Yang Y, Zhang H, Wu K, Chu Q. Notch signaling pathway: architecture, disease, and therapeutics. Signal Transduct Target Ther. 2022 Mar 24;7(1):95. Zhang, J, Li, J, Hou, Y, Lin, Y, Zhao, H, Shi, Y, Chen, K, Nian, C, Tang, J, Pan, L, Xing, Y, Gao, H, Yang, B, Song, Z, Cheng, Y, Liu, Y, Sun, M, Linghu, Y, Li, J, Huang, H, Lai, Z, Zhou, Z, Li, Z, Sun, X, Chen, Q, Su, D, Li, W, Peng, Z, Liu, P, Chen, W, Huang, H, Chen, Y, Xiao, B, Ye, L, Chen, L, Zhou, D. Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor. CELL. 2024-06-20; 187 (13): 3409-3426.e24. Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006 Sep;7(9):678-89. Shi Q, Xue C, Zeng Y, Yuan X, Chu Q, Jiang S, Wang J, Zhang Y, Zhu D, Li L.Notch signaling pathway in cancer: from mechanistic insights to targeted therapies. Signal Transduct Target Ther. 2024 May 27;9(1):128. Tchekneva, EE, Goruganthu, MUL, Uzhachenko, RV, Thomas, PL, Antonucci, A, Chekneva, I, Koenig, M, Piao, L, Akhter, A, de Aquino, MTP, Ranganathan, P, Long, N, Magliery, T, Valujskikh, A, Evans, JV, Arasada, RR, Massion, PP, Carbone, DP, Shanker, A, Dikov, MM. Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity. J Immunother Cancer. 2019-04-02; 7 (1): 95. Sierra RA, Trillo-Tinoco J, Mohamed E, Yu L, Achyut BR, Arbab A, Bradford JW, Osborne BA, Miele L, Rodriguez PC. Anti-Jagged Immunotherapy Inhibits MDSCs and Overcomes Tumor-Induced Tolerance. Cancer Res. 2017 Oct 15;77(20):5628-5638. Su, J, Song, Y, Zhu, Z, Huang, X, Fan, J, Qiao, J, Mao, F. Cell-cell communication: new insights and clinical implications. SIGNAL TRANSDUCT TAR. 2024-08-07; 9 (1): 196. Adu-Berchie, K, Liu, Y, Zhang, DKY, Freedman, BR, Brockman, JM, Vining, KH, Nerger, BA, Garmilla, A, Mooney, DJ. Generation of functionally distinct T-cell populations by altering the viscoelasticity of their extracellular matrix. Nat Biomed Eng. 2023-11-01; 7 (11): 1374-1391. Soll, D, Chu, CF, Sun, S, Lutz, V, Arunkumar, M, Gachechiladze, M, Schäuble, S, Alissa-Alkhalaf, M, Nguyen, T, Khalil, MA, Garcia-Ribelles, I, Mueller, M, Buder, K, Michalke, B, Panagiotou, G, Ziegler-Martin, K, Benz, P, Schatzlmaier, P, Hiller, K, Stockinger, H, Luu, M, Schober, K, Moosmann, C, Schamel, WW, Huber, M, Zielinski, CE. Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity. NAT IMMUNOL. 2024-10-01; 25 (10): 1830-1844. Hong M, Clubb JD, Chen YY. Engineering CAR-T Cells for Next-Generation Cancer Therapy. Cancer Cell. 2020 Oct 12;38(4):473-488. Rurik JG, Tombácz I, Yadegari A, Méndez Fernández PO, Shewale SV, Li L, Kimura T, Soliman OY, Papp TE, Tam YK, Mui BL, Albelda SM, Puré E, June CH, Aghajanian H, Weissman D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat cardiac injury. Science. 2022 Jan 7;375(6576):91-96. Billingsley MM, Gong N, Mukalel AJ, Thatte AS, El-Mayta R, Patel SK, Metzloff AE, Swingle KL, Han X, Xue L, Hamilton AG, Safford HC, Alameh MG, Papp TE, Parhiz H, Weissman D, Mitchell MJ. In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism. Small. 2024Mar;20(11):e2304378. Vincent RL, Gurbatri CR, Li F, Vardoshvili A, Coker C, Im J, Ballister ER, Rouanne M, Savage T, de Los Santos-Alexis K, Redenti A, Brockmann L, Komaranchath M, Arpaia N, Danino T. Probiotic-guided CAR-T cells for solid tumor targeting. Science. 2023 Oct 13;382(6667):211-218. Additional Declarations There is NO Competing Interest. 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0.005), while the signal attenuation greater in the 0.8 MPa group (14.25 ± 2.92dB VS. 2.00 ± 0.67 dB; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), confirming more complete GVs cavitation at higher pressures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH-I.\u003c/strong\u003e Optimization of acoustic imaging with 0.6 MPa and 0.8 MPa in vivo. Acoustic imaging (Fig. 1I) demonstrated robust GVs cavitation in tumor-bearing mice. Quantitative analysis revealed 0.8 MPa-induced signals (34.00 ± 10.00 dB) were ~2.5-fold stronger than 0.6 MPa signals (13.75 ± 9.19 dB; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. \u003c/strong\u003eColonization assays (\u003cem\u003e\u003cstrong\u003eS1C\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e; Fig. 1J\u003c/strong\u003e) displayed reduced \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs colony in 0.8 MPa-treated samples (0.24 ± 0.09 VS. 0.6 MPa, 1.12 ±0.11 OD600 units ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK-L. \u003c/strong\u003eLive/dead staining quantified \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs viability post-ultrasound exposure, 0.6 MPa induced minimal cytotoxicity (7.15 ± 2.20% mortality), 0.8 MPa caused significant cell death (31.38 ± 1.84%;\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.001). The scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003eFor Fig.1 (G-K), n=4, one-way ANOVA analysis was used for calculation.\u003c/p\u003e\n\u003cp\u003eSee also Figure \u003cem\u003e\u003cstrong\u003eS1\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/16bed09a3cdfcfcfa9f87bd7.png"},{"id":83439436,"identity":"491d49a3-1fbf-4d2c-8e23-bc3380a3c98f","added_by":"auto","created_at":"2025-05-26 09:11:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6329848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanical immunotherapy effect of LIFUS in combined with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVNP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e/ARG-GVs for tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e The schematic diagram of sequential five-cycle LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs treatment (21-day regimen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-C.\u003c/strong\u003e The LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs achieved robust tumor growth inhibition compared to other groups (**** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) . n = 4 biologically independent animals per group, statistical analysis was calculated by one-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eSurvival analysis revealed combination therapy survived \u0026gt;60 days, whereas all control/monotherapy groups succumbed by 30-40 day, n=10, Logrank test for trend.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE-G.\u003c/strong\u003e Flow cytometry analysis uncovered distinct immune landscape remodeling in each group. LIFUS-only groups maintained baseline CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration levels, \u003cem\u003eVNP\u003c/em\u003e/ARG monotherapy induced moderate CD4\u003csup\u003e+\u003c/sup\u003e T cells elevation, combination therapy achieved synergistic upregulation of cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells (51.98 ± 7.92%, VS.\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group in CD8\u003csup\u003e+\u003c/sup\u003e T cells, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) alongside moderate CD4\u003csup\u003e+\u003c/sup\u003e T-cell increase (17.02 ± 3.85%,VS.\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group in CD4\u003csup\u003e+\u003c/sup\u003e T cells, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0005). The increase of CD8\u003csup\u003e+\u003c/sup\u003e T cells was also more distinct than that of CD4\u003csup\u003e+\u003c/sup\u003e T cells (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), n=4, statistical analysis was calculated by two-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e FOXP3\u003csup\u003e+\u003c/sup\u003e regulatory T-cell suppression further supported the lowest suppressor cell frequencies observed in the combination group (28.38 ± 2.59% in LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group VS. 7.76 ± 1.60% in \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005). n=4, statistical analysis was calculated by one-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eIFN-γ: Combination group (37.4 ± 6.88 ng/mL) \u0026gt; LIFUS-alone (12.1 ± 2.3 ng/mL) \u0026gt; control (5.6 ± 1.4 ng/ml).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u003c/strong\u003e TNF-α: Combination group (1.37 ± 0.41 ng/mL) \u0026gt; LIFUS-alone (0.42 ± 0.15 ng/mL) \u0026gt; control (0.18 ± 0.06 ng/mL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK-L.\u003c/strong\u003e Ki67 staining and HE staining for each group tumor slices. The apoptosis in Ki67 and HE staining began to appear gradually in the \u003cem\u003eVNP\u003c/em\u003e/ARG group and was the highest in the LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group. Scale bar = 100 μm. See also Figure \u003cem\u003e\u003cstrong\u003eS2\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/41203b557d36e8d044d86cd0.png"},{"id":83438584,"identity":"2849bf7a-8e54-479f-b50d-81b7966c6db8","added_by":"auto","created_at":"2025-05-26 09:03:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2611363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell sequencing analysis the dominate role of CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eTME cellular composition analysis revealed eight distinct clusters: B cells, endothelial cells, epithelial cells, fibroblasts, myeloid cells, neutrophils, smooth muscle cells, and T/NK lymphocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Quantitative assessment demonstrated a significant enrichment of T lymphocyte populations in the the combined group compared to monotherapy groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-D.\u003c/strong\u003e Subclassification identified CD8\u003csup\u003e+\u003c/sup\u003e T cells as the predominant cytotoxic lymphocyte subset.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE-F.\u003c/strong\u003e The emergence of a unique CD8t_02 cluster (red line) exhibiting augmented spatial distribution in the combination treatment group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG-H. \u003c/strong\u003eFunctional annotation revealed this CD8t_02 cluster to be enriched in cytotoxic effector pathways, with GO analysis highlighting enrichment of intercellular adhesion and T cell activation pathways.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/c17bda50b7355f4906c7a71a.png"},{"id":83439437,"identity":"a36551ae-3b7b-4bc5-9614-f3589daa7d8e","added_by":"auto","created_at":"2025-05-26 09:11:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4963779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechano-regulation of CAFs-CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells NOTCH signaling interactions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-C. \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003ematrix stiffness analysis analyzed by elasticity ultrasound and atomic force test, showed an obvious reduction in tumor elastic modulus in the combined group (12.95 ± 2.18 kPa) compared to controls (57.19 ± 15.02 kPa) and \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs monotherapy (46.82 ± 8.13 kPa). Four independent experiments were performed in Fig.4B. n=10 in Fig.4C. Data are represented as mean ± SD. statistical analysis was calculated by one-way ANOVA. \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 means statistical significance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e. Collagen I distribution secreted by fibroblasts in \u003cem\u003e\u003cstrong\u003eS2B\u003c/strong\u003e\u003c/em\u003e illustrated the speculation of the fibroblasts changes in the TME. Quantitative assessment showed a significant reduction in the combined therapy group (42.24 ± 8.46 mm\u003csup\u003e2\u003c/sup\u003e) compared with the control group (248.62 ± 9.19 mm\u003csup\u003e2\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e. Cell-cell interaction analysis between control and combined treatment group, revealed the reduced interaction within fibroblasts and CD8\u003csup\u003e+\u003c/sup\u003e T.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eThe oncogenes pathways showed that NOTCH pathway exhibited the highest expression in the control group while the lowest expression in the combined therapy group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eSignificant attenuation of NOTCH1 gene and Jagged1 gene signaling between CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells in the combination group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e Significant attenuation of NOTCH1 protein and Jagged1 protein in the combination group tumor compared to other groups.\u003c/p\u003e\n\u003cp\u003eSee also Figure \u003cem\u003e\u003cstrong\u003eS3\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/e3fdf6e9db080db3a80bfb1f.png"},{"id":83438580,"identity":"e56985a9-6d18-48df-b240-afca7695c539","added_by":"auto","created_at":"2025-05-26 09:03:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3180791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual Mechanotransductive Regulation of CAFs-CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T Cell Crosstalk via NOTCH Signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e The schematic diagram of co-culture systems of CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells with GVs responsive to LIFUS constructs in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-C.\u003c/strong\u003e Confocal imaging revealed a significant reduction in NOTCH1 receptor and Jagged1 ligand expression in CAFs exposed to LIFUS-driven GVs (GVs + LIFUS group) compared to unstimulated controls (GVs - LIFUS group).The scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD-E.\u003c/strong\u003e Confocal imaging revealed a reduction in Jagged1 ligand expression while without significant change in Notch1 receptor levels in the GVs + LIFUS group of CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Illustrated the CAFs (without or with LIFUS-driven GVs treatment).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG-H.\u003c/strong\u003eThe migration number in each group, including the control group (the initially extracted CAFs), -LIFUS group (without LIFUS-driven GVs first) and LIFUS treatment group (with LIFUS-driven GVs first), the migrant number in LIFUS treatment group (56 ± 4 cells/field) was less than that in control (150.67 ± 15.04 cells/field,\u003cem\u003e p\u003c/em\u003e\u0026lt;0.0001) and without LIFUS group (101.67 ± 3.79 cells/field, p\u0026lt;0.001). The scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eThe levels of TGF-β secreted by CAFs in LIFUS treatment group (0.39 ± 0.07 ng/mL) was less than that in control (0.68 ± 0.38 ng/mL, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0005) and untreated groups (0.58 ± 0.03 ng/mL,\u003cem\u003e p\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u003c/strong\u003e The levels of Col 1 secreted by CAFs in LIFUS treatment group (0.27 ± 0.11 ng/mL) was less than that in control (0.76 ± 0.06 ng/mL, p\u0026lt;0.001) and untreated groups (0.51 ± 0.09 ng/mL, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.005).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK.\u003c/strong\u003e Illustrated the co-culture of CD8\u003csup\u003e+\u003c/sup\u003e T cells (without LIFUS-driven GVs or with LIFUS-driven GVs) with tumor cells (4T1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL-M\u003c/strong\u003e. The fluorescence intensity of CD8\u003csup\u003e+\u003c/sup\u003e T cells in each group, Fig.M showed an increase of CD8\u003csup\u003e+\u003c/sup\u003e T cell adhesion to tumor cells in the CD8\u003csup\u003e+ \u003c/sup\u003eT\u003csup\u003e+LIFUS\u003c/sup\u003e group (51.89 ± 10.47% adherent cells/field) versus controls (3.25 ± 1.19%, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0005) and CD8\u003csup\u003e+ \u003c/sup\u003eT\u003csup\u003e-LIFUS\u003c/sup\u003e group (15.72 ± 7.53%, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) . The scale bar = 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN.\u003c/strong\u003e Functional validation showed the cytotoxity of CD8\u003csup\u003e+\u003c/sup\u003e T cells to 4T1 cells (\u003cem\u003e\u003cstrong\u003eS4A\u003c/strong\u003e\u003c/em\u003e), 4T1 cells showed not significant apoptosis at 12h while an increase apoptosis at 36h compared to control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003eAlso see the \u003cem\u003e\u003cstrong\u003eS4B-C. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003et\u003c/em\u003ehe elevation in the functional factor released by CD8\u003csup\u003e+ \u003c/sup\u003eT cells of granzyme A/IFN-γ secretion in the CD8\u003csup\u003e+ \u003c/sup\u003eT\u003csup\u003e+LIFUS\u003c/sup\u003e group.)\u003c/p\u003e\n\u003cp\u003eThree independent experiments were performed. Data are represented as mean ± SD. \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 means statistical significance.\u003c/p\u003e\n\u003cp\u003eSee also Figure \u003cem\u003e\u003cstrong\u003eS4\u003c/strong\u003e\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/d704f47b049593e4a1db9aaa.png"},{"id":83438581,"identity":"249897c8-ae6a-4bae-a79f-e37cd65becce","added_by":"auto","created_at":"2025-05-26 09:03:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2906910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic translatability of CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells were improved by the mechanical force-driven by LIFUS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eExperimental scheme of establishing orthotopic B16-OVA model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eWith the mechanical force by LIFUS-driven GVs, the OT-I \u003csup\u003eGVs+LIFUS\u003c/sup\u003e achieved a significant tumor growth inhibition compared to other group at 15th day (266.7 ± 127.2 mm\u003csup\u003e3\u003c/sup\u003e VS. control, 1800 ± 1003 mm\u003csup\u003e3\u003c/sup\u003e,\u003cem\u003e p\u003c/em\u003e\u0026lt;0.0001, and 0T-1\u003csup\u003eGVs-LIFUS\u003c/sup\u003e, 796.7 ± 613.2 mm\u003csup\u003e3\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005). Data in B and D are representative of three independent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-D.\u003c/strong\u003e The tumor weight and mouse weight in each group and showed a significant decrease in OT-I \u003csup\u003eGVs+LIFUS\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Flow cytometric analysis of tumor-infiltrating lymphocytes showed a 94.1% reduction in Tim-3\u003csup\u003e+\u003c/sup\u003e/PD-1\u003csup\u003e+\u003c/sup\u003e co-expression in the OT-I \u003csup\u003eGVs+LIFUS\u003c/sup\u003e group compared to controls (2.55 ± 1.27% VS. 43 ± 4.60%, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Flow cytometric analysis of TNF-α/IFN-γ co-expression in the OT-I \u003csup\u003eGVs+LIFUS\u003c/sup\u003e (8.67 ±1.37%) group compared to controls (1.576 ± 0.78%, OT-I \u003csup\u003eGVs-LIFUS \u003c/sup\u003e19.278 ± 1.71%) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eFlow cytometric analysis of apoptosis factor Ki67 in the OT-I \u003csup\u003eGVs+LIFUS\u003c/sup\u003e group compared to OT-I \u003csup\u003eGVs-LIFUS\u003c/sup\u003e (63.20 ± 8.43% VS. 0T-1GVs-LIFUS, 33.32 ± 3.96%,\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.0001)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e. Experimental scheme of establishing 4T1-luciferase lung metastasis models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u003c/strong\u003e CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e treatment yielded a 92.7% reduction in pulmonary metastases (p\u0026lt;0.001) compared to CAR-T monotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ.\u003c/strong\u003e Small animal imaging showed the lung metastasis in each group, there was no lung metastasis in the CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group at 10th and 15th days, and there were no deaths observed until the 30th day. (The tails of the mice were covered to reduce the influence of the freshly injected D-Luciferin potassium salt).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK.\u003c/strong\u003e Kaplan-Meier survival curves demonstrated the prolonged survival in the CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group (median survival \u0026gt;50 days) versus CAR-T monotherapy (47.5 days) and control (35.5 days) group.\u003c/p\u003e\n\u003cp\u003eData in C and E-I are representative of five independent experiments.\u003c/p\u003e\n\u003cp\u003eData are mean ± SD and the P value of one-way ANOVA.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/e3b27ca00cdda9966d2566dd.png"},{"id":85406041,"identity":"5723d74d-f422-44f8-9420-89dc23a466f2","added_by":"auto","created_at":"2025-06-25 13:03:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24126300,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/0e56e678-3754-41bf-a78c-a7b3b355f181.pdf"},{"id":83438583,"identity":"b51aa972-11bb-4e43-8669-a3cf9af6eeb7","added_by":"auto","created_at":"2025-05-26 09:03:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2500173,"visible":true,"origin":"","legend":"supplementary","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/d6caa93aafe46e5b8dce2ece.docx"},{"id":83438575,"identity":"e6adf59f-d684-4f59-8807-b00517e47f98","added_by":"auto","created_at":"2025-05-26 09:03:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24823,"visible":true,"origin":"","legend":"","description":"","filename":"Experimentalmethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/d10a414ecf70bcbd8928946d.docx"},{"id":83438579,"identity":"8b0c1315-9fc5-430f-a42a-bdc89930fded","added_by":"auto","created_at":"2025-05-26 09:03:46","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":376797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6644974/v1/88263295f0cd9079ecf99000.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"LIFUS-Driven Engineered Bacteria Reprogram Immunosuppressive Niches via Mechano-NOTCH Signaling","fulltext":[{"header":"Highlight","content":"\u003cul\u003e\n \u003cli\u003e\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs can not only break through the physical barrier of the tumor, but also cooperate with low-intensity focused ultrasound (LIFUS) for ultrasonic imaging.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLIFUS in combination with \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs reduce the elastic stiffness of the tumor and enhance the immunotherapy of the tumor.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLIFUS-GVs can transform CD8\u003csup\u003e+\u003c/sup\u003e T cells through the mechanical force-NOTCH pathway axis, enhancing the tumor suppression.\u003c/li\u003e\n\u003c/ul\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eThe tumor microenvironment (TME), characterized by physical and immune barriers, remains a major challenge in the treatment of solid tumors [1]. Tumor cells recruit and activate stromal components such as cancer-associated fibroblasts (CAFs), which form dense physical barriers that impede drug penetration and immune cell infiltration [2]. Additionally, the TME is often hypoxic, acidic, and nutrient-deprived, creating an immunosuppressive milieu that further restricts immune cell proliferation and function. These two barriers\u0026mdash;physical and immune\u0026mdash;interact synergistically; for instance, CAFs secrete immunosuppressive factors to inhibit T cell activity [2\u0026ndash;3]. Consequently, despite advancements in immune checkpoint inhibitors[4\u0026ndash;5], solid tumors continue to face challenges such as insufficient T cell infiltration and functional exhaustion.\u003c/p\u003e \u003cp\u003eRecent studies have revealed that the hypoxic and immunosuppressive TME provides a niche for bacterial colonization [6\u0026ndash;7]. Certain anaerobes can penetrate tumor vascular barriers and withstand high interstitial pressure, proliferating within hypoxic and necrotic tumor cores while activating both innate and adaptive immune responses. This has spurred interest in bacterial-based therapies [8\u0026ndash;11]. Advances in synthetic biology have accelerated the development of engineered bacteria as promising therapeutic agents. For example, the attenuated Salmonella typhimurium strain VNP20009, with deletions in the msbB and purI genes to reduce toxicity, has shown efficacy in preclinical models by suppressing tumor growth [10\u0026ndash;11]. Phase I clinical trials confirmed its safety in cancer patients, though its therapeutic efficacy as a monotherapy yet to achieve complete tumor regression [12\u0026ndash;15]. Emerging evidence suggests that combining engineered bacteria with other modalities could significantly enhance antitumor outcomes [16\u0026ndash;17].\u003c/p\u003e \u003cp\u003eCombining engineered bacteria with ultrasound (US) technology offers a breakthrough approach. US enables spatiotemporal control and safety profiles, such as non-invasiveness and precise targeting, US-mediated combinatorial therapy with engineered bacteria exhibits superior potential in overcoming therapeutic bottlenecks of tumor compared to alternative multimodal strategies [18]. Furthermore, heat-responsive genetic circuits triggered by focused ultrasound (FUS)-induced hyperthermia can dynamically regulate bacterial activity [19]. However, thermal effects may cause collateral tissue damage. As a mechanical wave, US also exerts non-thermal bio-effects. Low-intensity focused ultrasound (LIFUS), operating at intensities below 3 W/cm\u0026sup2;, leverages cavitation (mainly stable cavitation) and mechanical forces via microbubbles to achieve therapeutic outcomes with minimal thermal impact. LIFUS has demonstrated utility in gene transfection, blood-brain barrier opening, and drug delivery, particularly when combined with microbubble-enhanced techniques [20\u0026ndash;22]. Mechanistically, LIFUS-generated shear stress and microjets activate cellular mechanosensors\u0026mdash;including RET, Piezo1, T cell receptors, and Notch receptors\u0026mdash;which are emerging targets in cancer therapy [23]. However, conventional microbubbles, typically administered intravascularly, exhibit limited efficacy in hypoxic and poorly vascularized tumors. To address this, acoustically responsive gas vesicles (GVs), encoded by engineered bacteria as intratumoral acoustic cavitation nuclei, enable both ultrasound imaging and mechanical perturbation [24\u0026ndash;26]. By combing LIFUS with GVs-generated mechanical forces, this approach may dual-target CAFs and CD8⁺ T cells within the TME, simultaneously disrupting physical and immune barriers.\u003c/p\u003e \u003cp\u003eBuilding on this rationale, we engineered an acoustically responsive bacterial system capable of producing GVs to amplify mechano-immunotherapeutic effects. The system utilizes VNP20009 to breach physical barriers, colonize tumor cores, and prime antitumor immunity. Concurrently, GVs encoded by acoustic report gene (ARG) for real-time monitoring of bacterial colonization via ultrasound imaging. With spatiotemporal LIFUS control, mechanical forces modulate Notch receptors (Notch1-Jagged1) on CAFs and CD8⁺ T cells, attenuating CAFs-CD8⁺T cell crosstalk and reprogramming the TME. This mechano-NOTCH axis alleviates T cell exhaustion and suppresses CAFs activation, thereby inhibiting tumor progression. By establishing a closed-loop therapeutic framework (imaging-treatment-feedback), we validate the efficacy of this strategy in solid tumors and provides the new evidence that mechanical signaling orchestrates the synergistic remodeling of dual barriers\u0026mdash;physical and immune\u0026mdash;within TME, offering a transformative approach to overcome current limitations in oncology (See \u003cb\u003egraphical abstract\u003c/b\u003e ).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Construction of Engineered Bacteria (\u003cem\u003eVNP\u003c/em\u003e/ARG) for Acoustic Responsive to \u0026nbsp;optimization parameter of LIFUS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe employed attenuated \u003cem\u003eSalmonella typhimurium\u003c/em\u003e VNP20009 as the genetic chassis for acoustic reporter gene (ARG) expression. As schematized in \u003cstrong\u003eFig. 1A\u003c/strong\u003e, the ARG-encoding plasmid pBAD-bARGSer-axe-txe was electroporated into VNP20009, generating the engineered strain \u003cem\u003eVNP\u003c/em\u003e/ARG. Electrophoretic analysis (\u003cstrong\u003eFig. 1B\u003c/strong\u003e) confirmed successful plasmid transfection, with arabinose induction (0.2%) triggering robust ARG expression in \u003cem\u003eVNP\u003c/em\u003e/ARG, uninduced \u003cem\u003eVNP\u003c/em\u003e/ARG exhibited negligible ARG expression compared to both induced counterparts (\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs) and non-engineered VNP20009 controls (\u003cstrong\u003eFig. 1C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctional validation illustrated LIFUS-induced GVs cavitation (\u003cstrong\u003eFig. 1D\u003c/strong\u003e), producing acoustic signals detectable by ultrasound imaging. Gas vesicles (GVs) production was visualized via transmission electron microscopy (TEM; \u003cstrong\u003eFig. 1E\u003c/strong\u003e), revealing polarized GVs aggregates occupying ~40% of intracellular space. These nanostructures displayed heterogeneous morphology, including ovoid and elongated configurations. Particle size analysis (\u003cstrong\u003e\u003cem\u003eS1A\u003c/em\u003e; \u003cem\u003eS1B\u003c/em\u003e\u003c/strong\u003e) showed GVs diameters ranged 0.5\u0026ndash;1.8 \u0026mu;m, exceeding clinical contrast agents like SonoVue (~2.5 \u0026mu;m) by \u0026gt;30%, indicating superior tissue penetrability.\u003c/p\u003e\n\u003cp\u003eTo optimize the LIFUS-responsive imaging of GVs and avoid the influence of thermal effect, two acoustic pressure parameters, 0.6 MPa and 0.8 MPa were selected [25]. In vitro, agarose phantoms containing \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs were exposed to LIFUS (\u003cstrong\u003eFig. 1F\u003c/strong\u003e). At 5-minute post-sonication, both intensities yielded not distinct acoustic signal intensities (22.67 \u0026plusmn; 0.92 dB VS. 24.75 \u0026plusmn; 0.92 dB, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). However, prolonged exposure (15 minutes) revealed significantly greater signal attenuation in the 0.8 MPa group (14.25 \u0026plusmn; 2.92 VS. 2.00 \u0026plusmn; 0.67 dB; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005) (\u003cstrong\u003eFig. 1G\u003c/strong\u003e), confirming more complete GVs cavitation at 0.8 MPa pressures. Similarly, acoustic mode imaging demonstrated robust GVs cavitation in tumor-bearing mice. Quantitative analysis revealed 0.8 MPa-induced signals (34.00 \u0026plusmn; 10.00 dB) were ~2.5-fold stronger than 0.6 MPa signals (13.75 \u0026plusmn; 9.19 dB; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.005) (\u003cstrong\u003eFig. 1H\u003c/strong\u003e). Acoustic imaging confirmed that acoustic cavitation was more thoroughly achieved under 0.8 MPa and resulting in enhanced contrast resolution (\u003cstrong\u003eFig. 1I\u003c/strong\u003e). Furthermore, B-mode imaging further revealed gradual bacterial regression post-cavitation, the grayscale imaging was decreased, suggesting that bacterial lysis occurred concomitantly with GVs destruction, thereby mitigating risks of bacterial over proliferation and enhancing biosafety.\u003c/p\u003e\n\u003cp\u003eSafety evaluations were further conducted to assess the impact of these acoustic parameters on \u003cem\u003eVNP\u003c/em\u003e/ARG colonization and viability. In the absence of GVs expression, neither 0.6 MPa nor 0.8 MPa significantly affected engineered bacteria \u003cem\u003eVNP\u003c/em\u003e/ARG survival (\u003cstrong\u003e\u003cem\u003eS1C\u003c/em\u003e\u003c/strong\u003e). However, following arabinose-induced GVs expression, both bacterial colonization decreased markedly, with the 0.8 MPa group showing stronger suppression (0.24 \u0026plusmn; 0.09 VS. 0.6 MPa, 1.12 \u0026plusmn; 0.11 OD600 units) (\u003cstrong\u003eFig. 1J\u003c/strong\u003e). Live/dead staining (\u003cstrong\u003eFig. 1K-L\u003c/strong\u003e) also quantified bacterial viability post-ultrasound exposure. While 0.6 MPa induced minimal cytotoxicity (7.15 \u0026plusmn; 2.20% mortality), 0.8 MPa caused significant cell death (31.38 \u0026plusmn; 1.84%; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).This demonstrates that under 0.8 MPa LIFUS-GVs conditions, bacteria \u003cem\u003eVNP\u003c/em\u003e/ARG undergo direct lysis, preventing further proliferation in vivo and reducing potential toxicity. Conversely, non-GVs-expressing bacteria retained proliferative capacity, ensuring their availability for subsequent therapeutic cycles. The cytotoxicity assays in vitro (\u003cstrong\u003e\u003cem\u003eS1D\u003c/em\u003e\u003c/strong\u003e) and the inflammatory responses in vivo (\u003cstrong\u003e\u003cem\u003eS1E-F\u003c/em\u003e\u003c/strong\u003e) were also confirm \u003cem\u003eVNP\u003c/em\u003e/ARGs favorable safety for applications. Collectively, these results confirm the biosafety of engineered bacteria in vivo and establish 0.8 MPa as the optimal parameter for both imaging efficacy and bacterial containment. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Mechanical immunotherapy effect of LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs for tumors\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe role of \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs and the optimization ultrasound imaging parameter of 0.8 MPa have been confirmed, we further explored the mechanical immunotherapeutic effect of LIFUS combining \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs (LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group) for breast tumors (\u003cstrong\u003eFig. 2A\u003c/strong\u003e). Sequential five-cycle LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs treatment (21-day) achieved robust tumor growth inhibition compared to controls (\u003cstrong\u003eFig. 2B-C\u003c/strong\u003e). Notably, monotherapy with \u003cem\u003eVNP\u003c/em\u003e/ARG (with or without arabinose induction) failed to elicit statistically significant antitumor effects (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), underscoring the critical role of LIFUS-GVs combination. Survival analysis revealed profound therapeutic benefits, mice receiving combination therapy survived \u0026gt;60 days, whereas all control/monotherapy groups succumbed by day 30-40 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) (\u003cstrong\u003eFig. 2D\u003c/strong\u003e). These data confirmed the combination of LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs as a highly effective tumor suppression modality.\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis (\u003cstrong\u003eFig. 2E-H\u003c/strong\u003e) uncovered distinct immune landscape remodeling in each group. The LIFUS group, similar to the control group, showed no significant changes in CD4\u003csup\u003e+\u003c/sup\u003e T /CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes infiltration (3.45 \u0026plusmn; 1.46% VS. 3.80 \u0026plusmn; 1.84%). Then, starting from the \u003cem\u003eVNP\u003c/em\u003e/ARG monotherapy group, changes were observed (CD4\u003csup\u003e+\u003c/sup\u003e T, 22.96 \u0026plusmn; 3.86% and CD8\u003csup\u003e+\u003c/sup\u003e T, 12.46 \u0026plusmn; 2.01%), and the obvious CD4\u003csup\u003e+\u003c/sup\u003e T-cell elevation (22.96 \u0026plusmn; 3 .86%, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs control), reflecting innate immune activation by \u003cem\u003eVNP\u003c/em\u003e/ARG. Moreover, CD4\u003csup\u003e+\u003c/sup\u003e T and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes were significantly increased in the combined therapy group of LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs, synergistic upregulation of cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T-cells (51.98 \u0026plusmn; 7.92%) and moderate CD4\u003csup\u003e+\u003c/sup\u003e T-cell increase (17.02 \u0026plusmn; 3.85%), achieving a 12.0-fold CD8\u003csup\u003e+\u003c/sup\u003e T-cell enrichment compared to control, the reason for this increased immune activity within the tumor may result from the combination treatment effectively activate the immune activity within the tumor, especially that of CD8\u003csup\u003e+\u003c/sup\u003e T cells. Simultaneously, FOXP3\u003csup\u003e+\u003c/sup\u003e regulatory T-cell suppression (\u003cstrong\u003eFig. 2F\u003c/strong\u003e) further corroborated immunomodulatory effects, with combination therapy exhibiting lowest suppressor cell frequencies (control group, 28.38 \u0026plusmn; 2.59% VS. 7.76 \u0026plusmn; 1.60%, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Further analysis of intra-tumor associated factors by Elisa of \u003cstrong\u003eFig. 2I-J\u0026nbsp;\u003c/strong\u003edemonstrates a significant elevation of IFN-\u0026gamma; and TNF-\u0026alpha; in the LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group (IFN-\u0026gamma;: 37.4 \u0026plusmn; 6.88 ng/mL, TNF-\u0026alpha;: 1.37 \u0026plusmn; 0.41 ng/mL) compared to the control and LIFUS alone groups (IFN-\u0026gamma;: 0.092 \u0026plusmn; 0.09 ng/mL, TNF-\u0026alpha;: 0.108 \u0026plusmn; 0.08 ng/mL, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), consistent with findings in other studies that highlight the clinical significance of these cytokines in disease progression and treatment response. Similarily, immunohistochemical analysis of tumors showed that apoptosis in Ki67 （\u003cstrong\u003eFig. 2K\u003c/strong\u003e\u003cstrong\u003e；\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eS2A\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003eand HE (\u003cstrong\u003eFig. 2L\u003c/strong\u003e)staining began to appear gradually in the \u003cem\u003eVNP\u003c/em\u003e/ARG group and was the highest in the LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Mechano-regulation of fibroblast-CD8\u003csup\u003e+\u003c/sup\u003e T cells NOTCH pathway interactions via LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs (Combined therapy)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious results have validated that the synergistic treatment system of LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs enhances intratumoral CD4\u003csup\u003e+\u003c/sup\u003e T infiltration by leveraging the immunogenicity of \u003cem\u003eVNP\u003c/em\u003e/ARG engineer bacteria, and the activation effects of CD8\u003csup\u003e+\u003c/sup\u003e T infiltration to effectively inhibit tumor growth by the combination of LIFUS-GVs. Herein, we elucidated the activation effects of CD8\u003csup\u003e+\u003c/sup\u003e T by mechanotransductive mechanisms underlying this therapeutic synergy using single-cell sequencing analysis. \u003cstrong\u003eFig. 3A\u003c/strong\u003e illustrates the results of cell cluster annotation within the TME, identifying eight distinct cell taxa: B cells, endothelial cells, epithelial cells, fibroblasts, myeloid cells, neutrophils, smooth muscle cells, and T/NK cells. Quantitative assessment demonstrated a significant enrichment of T lymphocyte populations in the LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs cohort compared to monotherapy groups (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). Further subclassification identified CD8\u003csup\u003e+\u003c/sup\u003e T cells as the predominant cytotoxic lymphocyte subset (\u003cstrong\u003eFig. 3C-D\u003c/strong\u003e), with the emergence of a unique Cd8t_02 cluster exhibiting augmented spatial distribution in the combination treatment group, meanwhile, \u003cem\u003eVNP\u003c/em\u003e/ARG showed the lowest Cd8t_02 cluster\u0026nbsp;when compared with other control groups, which might be attributed to the fact that the increase in this group is mainly dominated by CD4\u003csup\u003e+\u003c/sup\u003e T cells and there is not a strong correlation with the Cd8_02 population(\u003cstrong\u003eFig. 3E-F\u003c/strong\u003e). Functional annotation revealed this cluster plays a significant role in the cytotoxic response, as CD8\u003csup\u003e+\u003c/sup\u003e T cells are known for their cytotoxicity, which Cd8t_02 could effectively enhance the toxicity of CD8\u003csup\u003e+\u003c/sup\u003e T cells (\u003cstrong\u003eFig. 3G\u003c/strong\u003e). \u003cstrong\u003eFig. 3H\u003c/strong\u003e shows the GO enrichment analysis reveals that this subset clusters of CD8\u003csup\u003e+\u003c/sup\u003e T is predominantly associated with intercellular adhesion and T cell activation functions within the TME. The intercellular adhesion within TME are modulated by the stiffness and elasticity of its dense stromal matrix, with the relevant adhesion-associated proteins (e.g., collagen) being primarily secreted by CAFs-the principal stromal constituents in tumor matrix composition [27-29].\u003c/p\u003e\n\u003cp\u003eThe study examined the alterations of matrix elastic stiffness among different groups, the ultrasonic elastography in \u003cstrong\u003eFig. 4A\u003c/strong\u003e showed that the tumor elasticity in the LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group (12.95 \u0026plusmn; 2.18 kPa) decreased compared with the control group (57.19 \u0026plusmn; 15.02 kPa) and \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group (46.82 \u0026plusmn; 8.13 kPa),while there is a slight decreased in LIFUS monotherapy group (36.93 \u0026plusmn; 6.812 kPa), but there was no significant change between control and \u003cem\u003eVNP\u003c/em\u003e/ARG groups (\u003cstrong\u003eFig. 4B\u003c/strong\u003e). The elastic modulus of each group was further verified by atomic force corroborated these findings (\u003cstrong\u003eFig. 4C\u003c/strong\u003e). These two results showed that the mechanical effect of LIPUS-GVs decreased the tumor matrix elasticity stiffness, which was conducive to the infiltration of immune cells and in consistent with the change of the tumor immune microenvironment in our previous results. Moreover, immunofluorescence characterization of Collagen I distribution patterns in tumor specimens \u003cstrong\u003e(\u003cem\u003eS2B)\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eillustrated the speculation of the matrix changes in the TME\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003eFig. 4D\u003c/strong\u003e shows that there was a dense distribution of intratumoral fibroblasts in the control group and a decrease in the LIFUS group (172.05 \u0026plusmn; 37.46 mm\u003csup\u003e2\u003c/sup\u003e VS. 248.62 \u0026plusmn; 9.19 mm\u003csup\u003e2\u003c/sup\u003e, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001), indicating that the presence of LIFUS to TME can slightly influence the distribution of CAFs, this may be related to the inherent mechanical wave properties of ultrasound, which enables biomechanical stimulation of the TME through direct mechanical perturbation. However, the fibroblasts volume (210.04 \u0026plusmn; 0.74 mm\u003csup\u003e2\u003c/sup\u003e) in the \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs group did not show significant changes compared with the control group, and the combined treatment group exhibited a significant reduction in fibroblast volume in fibroblast volume (42.24 \u0026plusmn; 8.46 mm\u003csup\u003e2\u003c/sup\u003e) versus controls group. Revealing that \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs alone did not alter the stiffness or elasticity of the TME, however, combined therapy caused a significant change of fibroblasts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to find out the mechanism of interaction between fibroblasts and CD8\u003csup\u003e+\u003c/sup\u003e T cells, the interaction between the two cells was analyzed. In the cell-cell interaction analysis between the control and combined treatment group, we found that the pathway had reduced interaction within fibroblasts and CD8\u003csup\u003e+\u003c/sup\u003e T cell population (specially, Cd8t_04 cluster) (\u003cstrong\u003eFig. 4E\u003c/strong\u003e). The functional annotation revealed this cluster (Cd8t_02 cluster and Cd8t_04) to be relative to\u0026nbsp;RNA\u0026nbsp;metabolic process, which regulates the activity of CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells (see \u003cstrong\u003e\u003cem\u003eS3\u003c/em\u003e\u003c/strong\u003e).\u0026nbsp;The differential analysis of the five oncogenes pathways showed that NOTCH, TGF and WNT pathways were significantly upregulated in the control group, and the NOTCH pathway exhibited the highest expression in the control group while the lowest expression in the combined therapy group (\u003cstrong\u003eFig. 4F\u003c/strong\u003e). Meanwhile, genes and proteins Notch1 and Jagged1 expression on the NOTCH pathway in \u003cstrong\u003eFig. 4G-4I\u003c/strong\u003e were also significantly reduced in the combined therapy group, confirming the result of reduced NOTCH pathway interaction between CD8\u003csup\u003e+\u003c/sup\u003e T cells and fibroblasts in LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs. The fibroblasts in tumor are usually mutate into CAFs [2], so we refer to it as CAFs. As it is demonstrated the NOTCH signaling pathway, a highly conserved evolutionary mechanism governing cell proliferation through transmembrane receptor-ligand interactions, exhibits aberrant activation in TME. Tumor cells secrete paracrine factors that constitutively activate NOTCH signaling in both malignant cells and CAFs [30-32]. The observed attenuation of NOTCH-mediated crosstalk between CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells, evidenced by diminished receptor-ligand interaction frequencies and concomitant reduction in cognate gene expression profiles, substantiates that LIPUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs treatment effectively suppresses transmembrane protein biosynthesis within this pathway, thereby obstructing intercellular signal transduction. This alteration of TME was in correlation to the LIFUS driven VNP/ARG-GVs-generated mechanical force, decreasing the CAFs deposit and elastic stress of tumor for CD8\u003csup\u003e+\u003c/sup\u003e T cells activation and migration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. The dual regulatory role of mechanical force generated by LIFUS-driven GVs on CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells via NOTCH signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed that the combined therapy of LIFUS and\u003cem\u003e\u0026nbsp;VNP\u003c/em\u003e/ARG-GVs decreased the expression of CAFs in TME, lowered the tumor elastic stress and weakened the crosstalk between CAFs and CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells via the NOTCH pathway, but the mechanical force influence of GVs responsive to LIFUS on CD8\u003csup\u003e+\u003c/sup\u003e T cells in the NOTCH pathway needs to be further explored. We extracted CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells to established in vitro co-culture systems, for co-culture in vitro and added pre-extracted GVs for acoustic responsive to LIFUS (\u003cstrong\u003eFig. 5A\u003c/strong\u003e), followed by the separation of the two cell types using flow cytometry.\u003cstrong\u003e\u0026nbsp;Fig. 5B\u003c/strong\u003e confocal imaging shows the expression of representative proteins in NOTCH pathway of CAFs, the fluorescence intensity of representative Notch1 receptor and Jagged1 ligand in the group without LIFUS-driven GVs (GVs - LIFUS group) was significantly higher compared to that in the LIFUS-driven GVs (GVs + LIFUS group), \u003cstrong\u003eFig. 5C\u003c/strong\u003e also shows the consistent expression of Notch1 and Jagged1 proteins, indicating that the expression of these proteins within CAFs was significantly lower after acoustic responsive. However, the NOTCH pathway proteins on CD8\u003csup\u003e+\u003c/sup\u003e T cells showed different alteration. \u003cstrong\u003eFig. 5D\u003c/strong\u003e and \u003cstrong\u003e5E\u003c/strong\u003e show that Jagged1 ligand expression on CD8\u003csup\u003e+\u003c/sup\u003e T cells was decreased in GVs + LIFUS group, whereas Notch1 receptor protein expression on CD8\u003csup\u003e+\u003c/sup\u003e T cells has no significant change in both GVs - LIFUS group and GVs + LIFUS group. These alter expression of NOTCH pathway proteins in CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells can further explain the attenuation of cross talk between CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumors, and it can be speculated that the expression is mainly decreased by CAFs for the expression in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells did not decrease but seems to increase. The double regulatory role of mechanical force-NOTCH pathway on CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cells seems to contributes to the improvement of TME.\u003c/p\u003e\n\u003cp\u003eTherefore, to verify the double regulatory, the CAFs (without or with LIFUS-driven GVs treatment) were cultured in Transwell chambers to assess the migratory and secreted factor ( \u003cstrong\u003eFig. 5F\u003c/strong\u003e). \u003cstrong\u003eFig. 5G\u0026nbsp;\u003c/strong\u003edisplayed that the migration number in each group, including the control group (the initially extracted CAFs), -LIFUS group (without LIFUS-driven GVs first) and LIFUS treatment group (with LIFUS-driven GVs first), the migrant number in LIFUS treatment group (56\u0026nbsp;\u0026plusmn;\u0026nbsp;4 cells/field) was less than that in control (150.67\u0026nbsp;\u0026plusmn;\u0026nbsp;15.04 cells/field, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001) and without LIFUS group (101.67\u0026nbsp;\u0026plusmn;\u0026nbsp;3.79 cells/field, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001) (\u003cstrong\u003eFig. 5H\u003c/strong\u003e). \u003cstrong\u003eFig.5I-J\u003c/strong\u003e showed the levels of TGF-\u0026beta; and Col 1secreted by CAFs, which promote CAFs activation and migration [33-34]\u0026nbsp;demonstrated a significant decrease following LIFUS treatment compared to both control and untreated groups, indicating that LIFUS-driven GVs-generated mechanical forces effectively suppresses CAFs activation and migration by inhibiting NOTCH signaling within TME. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHowever, the decrease of Jagged1 ligand and the increase of Nocth1 receptor protein in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT with LIFUS-driven GVs treatment group showed an improvement to the cytotoxicity of CD8\u003csup\u003e+\u003c/sup\u003e T cells binding to tumor cells, thus inhibiting the growth of tumor cells. \u003cstrong\u003eFig. 5K\u003c/strong\u003e illustrated the co-cultured system of CD8\u003csup\u003e+\u003c/sup\u003e T cells (without or with LIFUS-driven GVs treatment) and tumor cells (4T1). \u003cstrong\u003eFig. 5L-M\u003c/strong\u003e showed that the fluorescence intensity of CD8\u003csup\u003e+\u003c/sup\u003e T cells adhere to the tumor cells in CD8\u003csup\u003e+\u003c/sup\u003e T\u003csup\u003e+LIFUS\u003c/sup\u003e group was significantly higher than that in the control group and CD8\u003csup\u003e+\u003c/sup\u003e T\u003csup\u003e-LIFUS\u0026nbsp;\u003c/sup\u003egroup (51.89 \u0026plusmn; 10.46% VS. Control, 3.25 \u0026plusmn; 1.19% and CD8\u003csup\u003e+\u003c/sup\u003e T\u003csup\u003e-LIFUS\u003c/sup\u003e, 15.72 \u0026plusmn; 7.53%). The enhanced adhesion force of CD8\u003csup\u003e+\u003c/sup\u003e T cells, as demonstrated in recent studies, plays a pivotal role in bolstering the immune system\u0026apos;s ability to combat tumors [35-37]. Similarly, the apoptosis of tumor cells was examined at 12 and 36 hours post-treatment. \u003cstrong\u003eFig. 5N\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;\u003cem\u003eS4A\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eindicates no significant difference in apoptosis between the groups at 12-hour, however, the CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT\u003csup\u003e+LIFUS\u003c/sup\u003e group exhibited a marked increase in apoptotic tumor cells after 36 hours and the 4T1-Luc showed almost no fluorescence. \u003cstrong\u003e\u003cem\u003eS4B-C\u003c/em\u003e\u003c/strong\u003e showed that the release of granzyme A (GZMA) and IFN-\u0026gamma; in CD8\u003csup\u003e+\u003c/sup\u003e T\u003csup\u003e+LIFUS\u003c/sup\u003e group were significantly higher than that in control and CD8\u003csup\u003e+\u003c/sup\u003e T\u003csup\u003e-LIFUS\u003c/sup\u003e cells group, which played an effective role in inhibiting tumor growth. These functional validation \u0026nbsp;confirmed a dual regulatory in CAFs and CD8\u003csup\u003e+\u003c/sup\u003eT cells where mechanotransduction-mediated NOTCH activation in CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells (Notch-Jagged1 signaling pathways) and an inhibition in CAFs synergizes with NOTCH, creating a novel perspective for the utilization of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the pre-clinical tumor treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Preclinical Translation of Mechanobiology-Enhanced CD8\u003csup\u003e+\u003c/sup\u003e T Cell Therapy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpon previous research that elucidated the combination therapy of LIFUS-\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs boosts the cytotoxicity of CD8\u003csup\u003e+\u003c/sup\u003e T cells against 4T1 tumor cells, we further investigated the therapeutic translatability. Two different tumor models were established to verify the tumor inhibition of modified CD8\u003csup\u003e+\u003c/sup\u003e T cells. Tumors were established by injecting B16-OVA cells (B16 cells treated with Ovalbumin) and treated with OT-1 to target OVA (including 0T-1\u003csup\u003eGVs-LIFUS\u003c/sup\u003e and 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e), tumor was pre-treatment with LIFUS-GVs before injecting 0T-1 in 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003egroup, and observed tumor volume changes after 15 days (\u003cstrong\u003eFig. 6A\u003c/strong\u003e). The control group exhibited the fastest tumor growth. and the 0T-1\u003csup\u003eGVs-LIFUS\u003c/sup\u003e group demonstrated some lethality resulting from GVs driven. However, its inhibitory effect was still notably lesser compared to the 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group (\u003cstrong\u003eFig. 6B\u003c/strong\u003e). The weight of tumors and body in mice also changed minimally in 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group (\u003cstrong\u003eFig. 6C-6D\u003c/strong\u003e).\u003cstrong\u003e\u0026nbsp;Fig. 6E\u003c/strong\u003e investigated the co-expression of Tim-3 and PD-1 in tumor tissue using flow cytometry, observed that the lowest combined expression of Tim3 and PD-1 (represent CD8\u003csup\u003e+\u003c/sup\u003e T cells exhaustion) in the 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group (2.55 \u0026plusmn; 1.27%), in contrast to the highest expression in the control group (43 \u0026plusmn; 4.60%,\u0026nbsp;\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001), indicating a significant reduction of approximately 94%. Meanwhile, \u003cstrong\u003eFig. 6F\u003c/strong\u003e the expression of anti-tumor factor TNF-\u0026alpha; and IFN-\u0026gamma; also showed the highest expression in the 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001). \u003cstrong\u003eFig. 6G\u0026nbsp;\u003c/strong\u003ethe apoptosis in the 0T-1\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group was higher than the other two groups (63.20 \u0026plusmn; 8.43% VS. control, 10.57 \u0026plusmn; 2.25%, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001,\u0026nbsp;and 0T-1\u003csup\u003eGVs-LIFUS\u003c/sup\u003e, 33.32 \u0026plusmn; 3.96%,\u0026nbsp;\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001), showing obvious statistical significance.\u003c/p\u003e\n\u003cp\u003e4T1-Luc tumor cells were injected intravenously into mice to make a 4T1 lung metastasis model and treatment with CAR-T cells (\u003cstrong\u003eFig. 6H\u003c/strong\u003e). \u003cstrong\u003eFig. 6I\u003c/strong\u003e shows the number of lung metastases in the three groups, it was observed that the group treated with CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e cells exhibited a significantly reduced number of lung metastases, with almost no metastatic lesions detected. \u003cstrong\u003eFig. 6J\u0026nbsp;\u003c/strong\u003edisplays the imaging of significant lung metastasis via small animal imaging. Distinct lung metastasis emerged in the control group on the 5th day, mortality was noted by the 15th day and all mice succumbed by the 25th day. In the CAR-T\u003csup\u003eGVs-LIFUS\u003c/sup\u003e group, lung metastasis was not prominent at first, the first deaths occurred on the 20th day with further fatalities on the 25th day. In the CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group, although mild lung metastasis was detected on both the 10th and 15th days, the fluorescence intensity indicating lung metastasis had vanished when re-examined on the 20th day and there were no deaths observed in the CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group until the 30th day. \u003cstrong\u003eFig. 6K\u0026nbsp;\u003c/strong\u003ethe survival curves demonstrated prolonged survival in the CAR-T\u003csup\u003eGVs+LIFUS\u003c/sup\u003e group (median survival \u0026gt; 50 days) versus CAR-T monotherapy (47.5 days) and control (35.5 days) group (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). These preclinical findings establish the therapeutic potential of mechanobiology-augmented adoptive T cell therapies by LIFUS for treating both primary and metastatic malignancies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUltrasound, fundamentally categorized as a mechanical wave, not only enables spatial imaging but also exerts physical regulation on biological tissues through mechanical effects. Current therapeutic strategies based on ultrasound-mediated mechanical effects primarily rely on the synergistic interaction between LIFUS and microbubble cavitation to generate microjets and shear forces, thereby enhancing cell membrane permeability for improved drug/gene delivery[38\u0026ndash;40]. Addressing the dual challenges of physical and immune barriers in tumor therapy, this study innovatively developed a LIFUS-driven engineered bacterial system: attenuated \u003cem\u003eSalmonella typhimurium\u003c/em\u003e VNP2009 was employed as a carrier to breach tumor physical barriers, while GVs with acoustic responsiveness were expressed to establish a sound-activated system. This system not only achieved intratumoral acoustic imaging but, more importantly, leveraged LIFUS-driven mechanical forces from GVs to modulate the tumor immune microenvironment, ultimately suppressing tumor growth.\u003c/p\u003e \u003cp\u003ePrevious studies have indicated that an acoustic intensity threshold exceeding 0.72 MPa enables simultaneous ultrasound imaging[25\u0026ndash;26]. Our experimental validation confirmed that a parameter of 0.8 MPa ensured optimal cavitation imaging of GVs while effectively controlling the survival period of the engineered bacteria (\u003cem\u003eVNP\u003c/em\u003e/ARG-GVs) in vivo. The immediate inactivation of bacteria post-cavitation eliminated biosafety risks associated with bacterial overproliferation. Notably, the engineered bacterial system represents a paradigm shift in oncology, as the combined therapy of \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs and LIFUS demonstrated efficacy in mouse tumor models. This combinatorial approach robustly activated antitumor immune responses: following intratumoral colonization by \u003cem\u003eVNP\u003c/em\u003e/ARG, the proportion of CD4\u003csup\u003e+\u003c/sup\u003e T cells increased, and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration was further amplified upon LIFUS-driven mechanical stimulation. Concurrently, key immune effector molecules, including TNF-α and IFN-γ, were upregulated, leading to significant tumor suppression and prolonged survival in mice.\u003c/p\u003e \u003cp\u003eSingle-cell sequencing revealed that in the LIFUS-treated group, CD8\u003csup\u003e+\u003c/sup\u003e T cell subsets exhibited reduced interaction with CAFs via the NOTCH pathway, specifically through diminished Notch1-Jagged1 signaling. Functional assays demonstrated enhanced cytotoxicity in CD8\u0026thinsp;+\u0026thinsp;T cell subsets, accompanied by decreased collagen I (Col I) deposition and mechanical stress within tumors, alongside downregulated Notch1 and Jagged1 expression at both transcriptional and protein levels. These TME alterations were mechanistically linked to the mechanical forces generated by LIFUS-driven \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs. Cellular mechanosensitivity, a cornerstone of cancer immunotherapy, involves mechanoreceptors such as NOTCH receptors and T cell receptors, which transduce extracellular mechanical signals into intracellular responses through conformational changes [41\u0026ndash;43].\u003c/p\u003e \u003cp\u003eThe engineered bacteria activated antitumor immunity via intratumoral colonization and antigen expression, while LIFUS-driven \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs synergistically amplified CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and cytotoxic function. Single-cell analyses highlighted a critical shift in CAFs-CD8\u003csup\u003e+\u003c/sup\u003e T cell interactions: reduced Notch1-Jagged1 axis activity impaired their communication efficiency. This correlated with heightened CD8\u003csup\u003e+\u003c/sup\u003e T cell cytotoxicity, diminished CAFs-derived Col I secretion and mechanical stress in tumor tissue, and downregulated Notch1/Jagged1 expression. These dynamics were governed by LIFUS-generated mechanical forces, with mechanoreceptors providing a molecular basis for mechanotransduction.\u003c/p\u003e \u003cp\u003eMechanistically, LIFUS-GVs-derived mechanical forces bidirectionally regulated CAFs and CD8\u003csup\u003e+\u003c/sup\u003e T cell functions via the NOTCH pathway. In vitro co-culture models showed that mechanical stimulation suppressed CAFs migration, TGF-β secretion and Col I production, indicative of reduced activity and proliferation. Conversely, CD8\u003csup\u003e+\u003c/sup\u003e T cell tumor adhesion and cytotoxicity were markedly enhanced. The NOTCH pathway operates through receptor-ligand interactions, triggering release of the Notch intracellular domain (NICD) to regulate target genes and maintain cellular homeostasis [44\u0026ndash;45]. Specifically, dual downregulation of Notch1/Jagged1 in CAFs disrupted their matrix-mediated physical/immune barriers, while selective Jagged1 reduction in CD8\u003csup\u003e+\u003c/sup\u003e T cells facilitated preferential binding of tumor cell Jagged1 to T cell Notch1[46\u0026ndash;50]. This differential regulation optimized CD8\u003csup\u003e+\u003c/sup\u003e T cell antitumor responses, offering novel evidence for mechanical force-mediated immune modulation. Validation in two additional models further demonstrated mechanical reprogramming of the tumor immune microenvironment. OT-1 T cells combined with LIFUS-GVs pretreatment reduced PD-1/Tim-3 expression, alleviated T cell exhaustion, and enhanced cytotoxicity\u0026mdash;a phenomenon mechanistically akin to sodium-induced T cell potentiation [51]. Similarly, LIFUS-GVs preconditioning prior to CAR-T cell infusion improved efficacy against metastatic tumors, aligning with advancements in CAR-T engineering [52\u0026ndash;54].\u003c/p\u003e \u003cp\u003eThis study pioneers a mechanobiology-driven precision therapy framework, integrating spatiotemporal coupling of LIFUS and engineered bacteria to elucidate the central role of the mechanical force-NOTCH axis in tumor immune regulation. While our findings confirm that mechanical forces suppress the CAF-CD8\u0026thinsp;+\u0026thinsp;T cell Notch1-Jagged1 axis, upstream/downstream signaling networks (e.g., Hes/Hey family regulation) and crosstalk between mechanosensors (e.g., Piezo1) and the NOTCH pathway warrant further exploration. Future work will employ patient-derived xenograft (PDX) models, single-cell spatial transcriptomics, and PD-1 inhibitors to optimize therapeutic regimens. Collectively, this mechanobiological framework provides a transformative perspective for remodeling the tumor immune microenvironment, offering novel theoretical and technological pathways for precision immunotherapy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study shows that the combination of LIFUS and \u003cem\u003eVNP\u003c/em\u003e/ARG-GVs provides a new strategy for tumor mechanical immunotherapy, while reveals the mechanism of mechanical force-NOTCH pathway axis on CD8\u003csup\u003e+\u003c/sup\u003e T cells and CAFs, providing a more solid theoretical basis for clinical application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Nation National Science Foundation of China Grant (2024XKPT32, 82402263,82001830) and the Postdoctoral Fellowship Program of CPSF (GZC20230499). All animal studies were approved by the Institutional Animal Care Committee of Shanghai Ten’s People’ Hospita (SHDSYY-2025-Y3216-01). The authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLizhou Lin for operations of experiment and paper writing, Xiao Li for guidance on gas vesicle engineering Dr. Xiaolong Li for providing access to LIFUS equipment. Wenyun Guo and Xin Guan for assistance with WB and flow analyze.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ede Visser, KE, Joyce, JA. The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. CANCER CELL. 2023; 41 (3): 374-403.\u003c/li\u003e\n \u003cli\u003eTsoumakidou, M. The advent of immune stimulating CAFs in cancer. NAT REV CANCER. 2023; 23 (4): 258-269.\u003c/li\u003e\n \u003cli\u003eLi, C, Guo, H, Zhai, P, Yan, M, Liu, C, Wang, X, Shi, C, Li, J, Tong, T, Zhang, Z, Ma, H, Zhang, J. Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts Infiltration and Antitumor Activity of CD8+ T Cells. CANCER RES. 2024-01-16; 84 (2): 258-275.\u003c/li\u003e\n \u003cli\u003ePetit PF, Daoudlarian D, Latifyan S, Bouchaab H, Mederos N, Doms J,Abdelhamid K, Ferahta N, Mencarelli L, Joo V, Bartolini R, Stravodimou A,Shabafrouz K, Pantaleo G, Peters S, Obeid M. Tocilizumab provides dual benefits in treating immune checkpoint inhibitor-associated arthritis and preventingrelapse during ICI rechallenge: the TAPIR study. Ann Oncol. 2025 Jan;36(1):43-53.\u003c/li\u003e\n \u003cli\u003eNaidoo J, Cottrell TR, Lipson EJ, Forde PM, Illei PB, Yarmus LB, Voong KR, Feller-Kopman D, Lee H, Riemer J, Wang D, Taube JM, Brahmer JR, Lin CT, Danoff SK, D\u0026apos;Alessio FR, Suresh K. Chronic immune checkpoint inhibitor pneumonitis. J Immunother Cancer. 2020 Jun;8(1):e000840.\u003c/li\u003e\n \u003cli\u003eGarrett WS. Cancer and the microbiota. Science. 2015 Apr 3;348(6230):80-6.\u003c/li\u003e\n \u003cli\u003eKwon SY, Thi-Thu Ngo H, Son J, Hong Y, Min JJ. Exploiting bacteria for cancer immunotherapy. Nat Rev Clin Oncol. 2024 Aug;21(8):569-589.\u003c/li\u003e\n \u003cli\u003eMager LF, Burkhard R, Pett N, Cooke NCA, Brown K, Ramay H, Paik S, Stagg J, Groves RA, Gallo M, Lewis IA, Geuking MB, McCoy KD. Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy. Science. 2020 Sep 18;369(6510):1481-1489.\u003c/li\u003e\n \u003cli\u003eGurbatri, CR, Arpaia, N, Danino, T. Engineering bacteria as interactive cancer therapies. SCIENCE. 2022; 378 (6622): 858-864.\u003c/li\u003e\n \u003cli\u003eFeng, Z, Wang, Y, Xu, H, Guo, Y, Xia, W, Zhao, C, Zhao, X, Wu, J. Recent advances in bacterial therapeutics based on sense and response. ACTA PHARM SIN B. 2023-03-01; 13 (3): 1014-1027.\u003c/li\u003e\n \u003cli\u003eWu, L, Bao, F, Li, L, Yin, X, Hua, Z. Bacterially mediated drug delivery and therapeutics: Strategies and advancements. ADV DRUG DELIVER REV. 2022-08-01; 187 114363.\u003c/li\u003e\n \u003cli\u003eLiang K, Liu Q, Li P, Luo H, Wang H, Kong Q. Genetically engineered Salmonella Typhimurium: Recent advances in cancer therapy. Cancer Lett. 2019 Apr ;448:168-181.\u003c/li\u003e\n \u003cli\u003eYin T, Liu La, Dong Shi-da, Huang Bao-lian, Li Chen-yang, CAO Zhi-ting, HUA Zi-chun. Research on anti-tumor mechanism of attenuated Salmonella typhimurium VNP20009. Acta Pharmaceutica Sinica, 2023, 58(9):2700-2706.\u003c/li\u003e\n \u003cli\u003eToso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ, Sherry RM, Topalian SL, Yang JC, Stock F, Freezer LJ, Morton KE, Seipp C, Haworth L, Mavroukakis S, White D, MacDonald S, Mao J, Sznol M, Rosenberg SA. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol. 2002 Jan 1;20(1):142-52.\u003c/li\u003e\n \u003cli\u003eLin, Z, Meng, F, Ma, Y, Zhang, C, Zhang, Z, Yang, Z, Li, Y, Hou, L, Xu, Y, Liang, X, Zhang, X. In situ immunomodulation of tumors with biosynthetic bacteria promote anti-tumor immunity. Bioact Mater. 2024-02-01; 32 12-27.\u003c/li\u003e\n \u003cli\u003eLiu, L, Xin, W, Li, Q, Huang, B, Yin, T, Hua, S, Yang, C, Chen, C, Han, C, Hua, Z. Neutrophil-Mediated Tumor-Targeting Delivery System of Oncolytic Bacteria Combined with ICB for Melanoma Lung Metastasis Therapy. Adv Sci (Weinh). 2023-10-01; 10 (29): e2301835.\u003c/li\u003e\n \u003cli\u003eLiu G, Ma N, Cheng K, Feng Q, Ma X, Yue Y, Li Y, Zhang T, Gao X, Liang J, Zhang L, Wang X, Ren Z, Fu YX, Zhao X, Nie G. Bacteria-derived nanovesicles enhance tumour vaccination by trained immunity. Nat Nanotechnol. 2024 Mar;19(3):387-398.\u003c/li\u003e\n \u003cli\u003eChen, Y, Du, M, Yuan, Z, Chen, Z, Yan, F. Author Correction: Spatiotemporal control of engineered bacteria to express interferon-\u0026gamma; by focused ultrasound for tumor immunotherapy. Nat Commun. 2024-04-30; 15 (1): 3656.\u003c/li\u003e\n \u003cli\u003eAbedi, MH, Yao, MS, Mittelstein, DR, Bar-Zion, A, Swift, MB, Lee-Gosselin, A, Barturen-Larrea, P, Buss, MT, Shapiro, MG. Ultrasound-controllable engineered bacteria for cancer immunotherapy. Nat Commun. 2022-03-24; 13 (1): 1585.\u003c/li\u003e\n \u003cli\u003eLegon, W., Strohman, A. Low-intensity focused ultrasound for human neuromodulation. Nat Rev Methods Primers 4, 91 (2024).\u003c/li\u003e\n \u003cli\u003eFeng J, Li Z. Progress in Noninvasive Low-Intensity Focused Ultrasound Neuromodulation. Stroke. 2024 Oct;55(10):2547-2557.\u003c/li\u003e\n \u003cli\u003eKim MG, Yu K, Yeh CY, Fouda R, Argueta D, Kiven S, Ni Y, Niu X, Chen Q, Kim K, Gupta K, He B. Low-intensity transcranial focused ultrasound suppresses pain by modulating pain-processing brain circuits. Blood. 2024 Sep 5;144(10):1101-1115.\u003c/li\u003e\n \u003cli\u003eZamfirov, L, Nguyen, NM, Fern\u0026aacute;ndez-S\u0026aacute;nchez, ME, Cambronera Ghiglione, P, Teston, E, Dizeux, A, Tiennot, T, Farge, E, Demen\u0026eacute;, C, Tanter, M. Acoustic-pressure-driven ultrasonic activation of the mechanosensitive receptor RET and of cell proliferation in colonic tissue. Nat Biomed Eng. 2024-12-20.\u003c/li\u003e\n \u003cli\u003eBourdeau, RW, Lee-Gosselin, A, Lakshmanan, A, Farhadi, A, Kumar, SR, Nety, SP, Shapiro, MG. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. NATURE. 2018-01-03; 553 (7686): 86-90.\u003c/li\u003e\n \u003cli\u003eBar-Zion, A, Nourmahnad, A, Mittelstein, DR, Shivaei, S, Yoo, S, Buss, MT, Hurt, RC, Malounda, D, Abedi, MH, Lee-Gosselin, A, Swift, MB, Maresca, D, Shapiro, MG. Acoustically triggered mechanotherapy using genetically encoded gas vesicles. NAT NANOTECHNOL. 2021-12-01; 16 (12): 1403-1412.\u003c/li\u003e\n \u003cli\u003eHurt, RC, Buss, MT, Duan, M, Wong, K, You, MY, Sawyer, DP, Swift, MB, Dutka, P, Barturen-Larrea, P, Mittelstein, DR, Jin, Z, Abedi, MH, Farhadi, A, Deshpande, R, Shapiro, MG. Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria. NAT BIOTECHNOL. 2023-07-01; 41 (7): 919-931.\u003c/li\u003e\n \u003cli\u003eOnder, L, Papadopoulou, C, L\u0026uuml;tge, A, Cheng, HW, L\u0026uuml;tge, M, Perez-Shibayama, C, Gil-Cruz, C, De Martin, A, Kurz, L, Cadosch, N, Pikor, NB, Rodriguez, R, Born, D, Jochum, W, Leskow, P, Dutly, A, Robinson, MD, Ludewig, B. Fibroblastic reticular cells generate protective intratumoral T cell environments in lung cancer. CELL. 2025-01-23; 188 (2): 430-446.e20.\u003c/li\u003e\n \u003cli\u003eBrown FD, Sen DR, LaFleur MW, Godec J, Lukacs-Kornek V, Schildberg FA, Kim HJ, Yates KB, Ricoult SJH, Bi K, Trombley JD, Kapoor VN, Stanley IA, Cremasco V, Danial NN, Manning BD, Sharpe AH, Haining WN, Turley SJ. Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling. Nat Immunol. 2019 Dec;20(12):1668-1680.\u003c/li\u003e\n \u003cli\u003eApollonio B, Spada F, Petrov N, Cozzetto D, Papazoglou D, Jarvis P, Kannambath S, Terranova-Barberio M, Amini RM, Enblad G, Graham C, Benjamin R,Phillips E, Ellis R, Nuamah R, Saqi M, Calado DP, Rosenquist R, Sutton LA,Salisbury J, Zacharioudakis G, Vardi A, Hagner PR, Gandhi AK, Bacac M, Claus C,Umana P, Jarrett RF, Klein C, Deutsch A, Ramsay AG. Tumor-activated lymph node fibroblasts suppress T cell function in diffuse large B cell lymphoma. J Clin Invest. 2023 Jul 3;133(13):e166070.\u003c/li\u003e\n \u003cli\u003eJiang, Y, Wang, Y, Chen, G, Sun, F, Wu, Q, Huang, Q, Zeng, D, Qiu, W, Wang, J, Yao, Z, Liang, B, Li, S, Wu, J, Huang, N, Wang, Y, Chen, J, Zhai, X, Huang, L, Xu, B, Yamamoto, M, Tsukamoto, T, Nomura, S, Liao, W, Shi, M. Nicotinamide metabolism face-off between macrophages and fibroblasts manipulates the microenvironment in gastric cancer. CELL METAB. 2024-08-06; 36 (8): 1806-1822.e11.\u003c/li\u003e\n \u003cli\u003eMeurette, O, Mehlen, P. Notch Signaling in the Tumor Microenvironment. CANCER CELL. 2018-10-08; 34 (4): 536-548.\u003c/li\u003e\n \u003cli\u003eMajumder, S, Crabtree, JS, Golde, TE, Minter, LM, Osborne, BA, Miele, L. Targeting Notch in oncology: the path forward. NAT REV DRUG DISCOV. 2021-02-01; 20 (2): 125-144.\u003c/li\u003e\n \u003cli\u003eLi, X, Yan, X, Wang, Y, Kaur, B, Han, H, Yu, J. The Notch signaling pathway: a potential target for cancer immunotherapy. J Hematol Oncol. 2023-05-02; 16 (1): 45.\u003c/li\u003e\n \u003cli\u003eMandula, JK, Sierra-Mondragon, RA, Jimenez, RV, Chang, D, Mohamed, E, Chang, S, Vazquez-Martinez, JA, Cao, Y, Anadon, CM, Lee, SB, Das, S, Rocha-Munguba, L, Pham, VM, Li, R, Tarhini, AA, Furqan, M, Dalton, W, Churchman, M, Moran-Segura, CM, Nguyen, J, Perez, B, Kojetin, DJ, Obermayer, A, Yu, X, Chen, A, Shaw, TI, Conejo-Garcia, JR, Rodriguez, PC. Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages. IMMUNITY. 2024-05-14; 57 (5): 1124-1140.\u003c/li\u003e\n \u003cli\u003eYang S, Wang M, Tian D, Zhang X, Cui K, L\u0026uuml; S, Wang HH, Long M, Nie Z. DNA-functionalized artificial mechanoreceptor for de novo force-responsive signaling. Nat Chem Biol. 2024 Aug;20(8):1066-1077.\u003c/li\u003e\n \u003cli\u003eBasu, R, Whitlock, BM, Husson, J, Le Floc\u0026apos;h, A, Jin, W, Oyler-Yaniv, A, Dotiwala, F, Giannone, G, Hivroz, C, Biais, N, Lieberman, J, Kam, LC, Huse, M. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. CELL. 2016-03-24; 165 (1): 100-110.\u003c/li\u003e\n \u003cli\u003eCambria, E, Coughlin, MF, Floryan, MA, Offeddu, GS, Shelton, SE, Kamm, RD. Linking cell mechanical memory and cancer metastasis. NAT REV CANCER. 2024-03-01; 24 (3): 216-228.\u003c/li\u003e\n \u003cli\u003eChowdhury, SM, Abou-Elkacem, L, Lee, T, Dahl, J, Lutz, AM. Ultrasound and microbubble mediated therapeutic delivery: Underlying mechanisms and future outlook. J CONTROL RELEASE. 2020-10-10; 326 75-90.\u003c/li\u003e\n \u003cli\u003eDimcevski, G, Kotopoulis, S, Bj\u0026aring;nes, T, Hoem, D, Schj\u0026oslash;tt, J, Gjertsen, BT, Biermann, M, Molven, A, Sorbye, H, McCormack, E, Postema, M, Gilja, OH. A human clinical trial using ultrasound and microbubbles to enhance gemcitabine treatment of inoperable pancreatic cancer. J CONTROL RELEASE. 2016-12-10; 243 172-181.\u003c/li\u003e\n \u003cli\u003eStern, JM, Spivak, NM, Becerra, SA, Kuhn, TP, Korb, AS, Kronemyer, D, Khanlou, N, Reyes, SD, Monti, MM, Schnakers, C, Walshaw, P, Keselman, I, Cohen, MS, Yong, W, Fried, I, Jordan, SE, Schafer, ME, Engel, J, Bystritsky, A. Safety of focused ultrasound neuromodulation in humans with temporal lobe epilepsy. BRAIN STIMUL. 2021-01-01; 14 (4): 1022-1031.\u003c/li\u003e\n \u003cli\u003eLiu B, Chen W, Evavold BD, Zhu C. Accumulation of dynamic catch bonds between TCR and agonist peptide-MHC triggers T cell signaling. Cell. 2014 Apr 10;157(2):357-368.\u003c/li\u003e\n \u003cli\u003eZhou B, Lin W, Long Y, Yang Y, Zhang H, Wu K, Chu Q. Notch signaling pathway: architecture, disease, and therapeutics. Signal Transduct Target Ther. 2022 Mar 24;7(1):95.\u003c/li\u003e\n \u003cli\u003eZhang, J, Li, J, Hou, Y, Lin, Y, Zhao, H, Shi, Y, Chen, K, Nian, C, Tang, J, Pan, L, Xing, Y, Gao, H, Yang, B, Song, Z, Cheng, Y, Liu, Y, Sun, M, Linghu, Y, Li, J, Huang, H, Lai, Z, Zhou, Z, Li, Z, Sun, X, Chen, Q, Su, D, Li, W, Peng, Z, Liu, P, Chen, W, Huang, H, Chen, Y, Xiao, B, Ye, L, Chen, L, Zhou, D. Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor. CELL. 2024-06-20; 187 (13): 3409-3426.e24.\u003c/li\u003e\n \u003cli\u003eBray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol. 2006 Sep;7(9):678-89.\u003c/li\u003e\n \u003cli\u003eShi Q, Xue C, Zeng Y, Yuan X, Chu Q, Jiang S, Wang J, Zhang Y, Zhu D, Li L.Notch signaling pathway in cancer: from mechanistic insights to targeted therapies. Signal Transduct Target Ther. 2024 May 27;9(1):128.\u003c/li\u003e\n \u003cli\u003eTchekneva, EE, Goruganthu, MUL, Uzhachenko, RV, Thomas, PL, Antonucci, A, Chekneva, I, Koenig, M, Piao, L, Akhter, A, de Aquino, MTP, Ranganathan, P, Long, N, Magliery, T, Valujskikh, A, Evans, JV, Arasada, RR, Massion, PP, Carbone, DP, Shanker, A, Dikov, MM. Determinant roles of dendritic cell-expressed Notch Delta-like and Jagged ligands on anti-tumor T cell immunity. J Immunother Cancer. 2019-04-02; 7 (1): 95.\u003c/li\u003e\n \u003cli\u003eSierra RA, Trillo-Tinoco J, Mohamed E, Yu L, Achyut BR, Arbab A, Bradford JW, Osborne BA, Miele L, Rodriguez PC. Anti-Jagged Immunotherapy Inhibits MDSCs and Overcomes Tumor-Induced Tolerance. Cancer Res. 2017 Oct 15;77(20):5628-5638.\u003c/li\u003e\n \u003cli\u003eSu, J, Song, Y, Zhu, Z, Huang, X, Fan, J, Qiao, J, Mao, F. Cell-cell communication: new insights and clinical implications. SIGNAL TRANSDUCT TAR. 2024-08-07; 9 (1): 196.\u003c/li\u003e\n \u003cli\u003eAdu-Berchie, K, Liu, Y, Zhang, DKY, Freedman, BR, Brockman, JM, Vining, KH, Nerger, BA, Garmilla, A, Mooney, DJ. Generation of functionally distinct T-cell populations by altering the viscoelasticity of their extracellular matrix. Nat Biomed Eng. 2023-11-01; 7 (11): 1374-1391.\u003c/li\u003e\n \u003cli\u003eSoll, D, Chu, CF, Sun, S, Lutz, V, Arunkumar, M, Gachechiladze, M, Sch\u0026auml;uble, S, Alissa-Alkhalaf, M, Nguyen, T, Khalil, MA, Garcia-Ribelles, I, Mueller, M, Buder, K, Michalke, B, Panagiotou, G, Ziegler-Martin, K, Benz, P, Schatzlmaier, P, Hiller, K, Stockinger, H, Luu, M, Schober, K, Moosmann, C, Schamel, WW, Huber, M, Zielinski, CE. Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity. NAT IMMUNOL. 2024-10-01; 25 (10): 1830-1844.\u003c/li\u003e\n \u003cli\u003eHong M, Clubb JD, Chen YY. Engineering CAR-T Cells for Next-Generation Cancer Therapy. Cancer Cell. 2020 Oct 12;38(4):473-488.\u003c/li\u003e\n \u003cli\u003eRurik JG, Tomb\u0026aacute;cz I, Yadegari A, M\u0026eacute;ndez Fern\u0026aacute;ndez PO, Shewale SV, Li L, Kimura T, Soliman OY, Papp TE, Tam YK, Mui BL, Albelda SM, Pur\u0026eacute; E, June CH, Aghajanian H, Weissman D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat cardiac injury. Science. 2022 Jan 7;375(6576):91-96.\u003c/li\u003e\n \u003cli\u003eBillingsley MM, Gong N, Mukalel AJ, Thatte AS, El-Mayta R, Patel SK, Metzloff AE, Swingle KL, Han X, Xue L, Hamilton AG, Safford HC, Alameh MG, Papp TE, Parhiz H, Weissman D, Mitchell MJ. In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism. Small. 2024Mar;20(11):e2304378.\u003c/li\u003e\n \u003cli\u003eVincent RL, Gurbatri CR, Li F, Vardoshvili A, Coker C, Im J, Ballister ER, Rouanne M, Savage T, de Los Santos-Alexis K, Redenti A, Brockmann L, Komaranchath M, Arpaia N, Danino T. Probiotic-guided CAR-T cells for solid tumor targeting. Science. 2023 Oct 13;382(6667):211-218.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6644974/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6644974/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe tumor microenvironment (TME) of solid tumors constructs a complex biophysical barrier with intricate interactions, including dense stromal structures and immunosuppressive networks, which severely limit the permeability and efficacy of immunotherapy. This study developed an engineered an attenuated \u003cem\u003eSalmonella typhimurium\u003c/em\u003e VNP20009 strain encoding an acoustic reporter gene (ARG)-based gas vesicles (GVs), enabling real-time tumor imaging guided mechano-immunomodulation. Low-intensity focused ultrasound (LIFUS) triggered cavitation effects from GVs, generating localized shear forces and mechanical strain. This significantly inhibited tumor growth and attenuated the NOTCH-mediated crosstalk between cancer-associated fibroblasts (CAFs) and CD8⁺ T cells, thereby relieving CD8⁺ T cell activity. This mechanobiological intervention elicited a 12-fold enhancement in cytotoxic CD8⁺ T cell infiltration and extended median survival to over 60 days (versus 30-40 days in controls) in aggressive murine tumor models, demonstrating significant therapeutic efficacy. This integrated strategy establishes a closed-loop therapeutic platform combining bacterial targeting, acoustic visualization, and LIFUS-driven mechano-immunomodulation, achieving 92.7% suppression of both primary and metastatic tumor growth. By synergizing synthetic biology with mechano-immunological regulation, our study pioneers a precisely controllable and real-time image-monitored therapeutic paradigm, providing a transformative approach to overcome solid tumor resistance and accelerate clinical translation.\u003c/p\u003e","manuscriptTitle":"LIFUS-Driven Engineered Bacteria Reprogram Immunosuppressive Niches via Mechano-NOTCH Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-26 09:03:41","doi":"10.21203/rs.3.rs-6644974/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":"5a22cb02-1fb2-4189-83eb-92933e96980c","owner":[],"postedDate":"May 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48867474,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":48867475,"name":"Physical sciences/Engineering/Biomedical engineering"}],"tags":[],"updatedAt":"2025-06-25T12:55:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-26 09:03:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6644974","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6644974","identity":"rs-6644974","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}