Spatial heterogeneity represents a pivotal driver of therapeutic resistance and metastatic progression in colorectal cancer (CRC). However, the mechanical regulatory mechanisms governing intratumoral regional specialization remain incompletely understood. This study integrates spatial transcriptomics, single-cell RNA sequencing, pseudotime analysis, biomimetic peristaltic organ-on-a-chip technology, and immunocompetent mouse models to systematically delineate how membrane potential differences, shaped by intratumoral mechanical pressure gradients, spatially modulate mechanoptosis resistance, chemoresistance, invasive phenotypes, and immune evasion via mechanotransduction and the PVR/CD96 immune checkpoint axis.

We demonstrate that CRC tumors exhibit a distinct center-periphery mechanical and electrophysiological dichotomy. The peripheral tumor region, exposed to lower mechanical compression, displays membrane potential depolarization, accompanied by Hippo pathway inactivation, enhanced YAP nuclear translocation, and elevated expression of mechanosensitive genes including PIEZO1, YAP1, and ITGB1, as well as proliferation-related genes. This region manifests a "mechanically active, highly proliferative" phenotype linked to aggressive behavior. In contrast, the tumor core, subjected to high mechanical pressure, undergoes membrane potential hyperpolarization, concurrent with Hippo pathway activation, cytoplasmic sequestration of YAP, and downregulation of mechanotransduction and proliferation genes, yielding a "mechanically suppressed, stress-adapted" phenotype associated with survival under compressive stress.

Depolarization further amplifies YAP transcriptional activity, upregulates ABCG2-dependent chemoresistance, stimulates epithelial-mesenchymal transition (EMT), augments migratory and invasive capacity, and reinforces resistance to mechanoptosis. Conversely, pharmacologically induced hyperpolarization or targeted inhibition of YAP effectively reverses these malignant phenotypes. Mechanistically, tumor cells with heightened mechanosignaling activity exhibit robust expression of the immune checkpoint ligand PVR, which engages the CD96 receptor on CD8+ T cells, thereby triggering T cell exhaustion, suppressing the secretion of IFN-γ and Granzyme B, and blunting anti-tumor cytotoxicity. Depolarization exacerbates this immunosuppressive signaling axis, whereas hyperpolarization or antibody-mediated blockade of the PVR/CD96 interaction restores T cell effector function and reinstates anti-tumor immunity.

In vivo experiments using subcutaneous CRC xenografts confirm that depolarization promotes tumor growth and suppresses apoptosis by inhibiting the Hippo pathway and activating the YAP/PVR signaling cascade. Hyperpolarization acts oppositely: it activates Hippo signaling, inhibits YAP/PVR expression, induces apoptotic cell death, curtails proliferation, and reverses CD8+ T cell exhaustion. Overexpression of YAP or PVR partially offsets the tumor-suppressive effects of hyperpolarization, verifying the functional hierarchy of this electro-mechano-immune regulatory network.

Collectively, these findings establish the membrane potential-mechanotransduction-PVR/CD96 axis as a central regulator of spatial-specific mechanoptosis resistance, chemoresistance, and immune evasion in CRC. This work bridges mechanobiology, electrophysiology, and tumor immunology, revealing a previously unrecognized mechano-electro-immune regulatory module that governs intratumoral heterogeneity. By defining how mechanical forces and membrane potential cooperatively shape regional tumor phenotypes and immune suppression, this study provides a mechanistic foundation for innovative therapeutic strategies targeting membrane potential, mechanosignaling pathways, or the PVR/CD96 checkpoint in combination with conventional chemotherapy and immunotherapy for metastatic CRC.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with spatial heterogeneity serving as a major barrier to effective treatment, chemosensitivity, and immunotherapeutic response. While biochemical and genetic heterogeneity have been extensively studied, the mechanical and electrophysiological heterogeneity within CRC tumors-particularly how mechanical pressure gradients govern regional cell fate, apoptosis resistance, and immune suppression-remains poorly defined. This study systematically investigates the functional roles and molecular mechanisms of cell membrane potential in regulating spatially specific mechanoptosis resistance, metastatic potential, chemoresistance, and CD8+ T cell exhaustion via mechanotransduction signaling and the PVR/CD96 immune checkpoint axis.

Intratumoral mechanical stress exhibits clear regional variation: the central region of solid CRC tumors is exposed to high compressive pressure due to dense extracellular matrix and rapid cell proliferation, while the peripheral invasive front experiences relatively low mechanical compression. We hypothesized that these pressure gradients establish distinct membrane potential states across tumor regions, which in turn drive mechanotransduction-dependent transcriptional programs and immune checkpoint expression to shape malignant phenotypes. To test this hypothesis, we integrated spatial transcriptomics, single-cell RNA sequencing, pseudotime trajectory analysis, biomimetic peristaltic organ-on-a-chip models, and immunocompetent mouse xenograft studies to map regional electro-mechanical signaling and its downstream immune regulatory networks.

Spatial transcriptomic analysis of human CRC tissues revealed three major cell compartments: epithelial cells, myeloid immune cells, and perivascular stromal cells. Mechanosensitive genes including PIEZO1, YAP1, TAZ, and ITGB1 were significantly enriched in the peripheral tumor region, accompanied by high expression of proliferation markers (PCNA, CCND1, CDK4, MYC). In contrast, the central tumor region showed low mechanical activity and reduced proliferative gene expression. GSVA-based mechanical activity scoring confirmed a robust "peripherally high, centrally low" spatial pattern, consistent with immunofluorescence validation of PIEZO1, YAP nuclear localization, and Ki-67. Pseudotime trajectory analysis further demonstrated that peripheral epithelial cells occupy the late differentiation state with elevated mechanosignaling, while central epithelial cells remain in an early, stress-adaptive differentiation state enriched for extracellular matrix organization and mechanical adaptation pathways.

Single-cell RNA sequencing analysis identified multiple CD4+ and CD8+ T cell subpopulations within CRC tissues, including naive, cytotoxic, and exhausted CD8+ T cells. Cell-cell interaction analysis using CellphoneDB revealed significantly enhanced crosstalk between mechanically high epithelial cells and T cells, with marked enrichment of the PVR-CD96 receptor-ligand pair. Spatial transcriptomics confirmed that PVR (the ligand) is predominantly expressed in the mechanically active peripheral tumor region, colocalizing with CD96+ CD8+ T cells at the invasive front. These observations established a spatial link between mechanosignaling activity and the PVR/CD96 immunosuppressive axis.

To recapitulate physiological intestinal peristalsis and intratumoral pressure gradients, we developed a custom multi-chamber biomimetic peristaltic CRC organ-on-a-chip. This microfluidic platform integrates a tumor chamber, vascular channel, immune cell compartment, and cyclic peristaltic mechanical actuation (0.2 Hz, 8-12 kPa) to mimic in vivo mechanical forces. Dual membrane potential-sensitive dye staining (DiOC₂(3) for hyperpolarization; DiSBAC₂(3) for depolarization) demonstrated that the central tumor region is hyperpolarized, while the peripheral region is depolarized-establishing membrane potential as a direct electrophysiological readout of regional mechanical pressure.

Pharmacological modulation of membrane potential revealed that depolarization (via ouabain) promotes YAP nuclear translocation, enhances Ki-67 expression, strengthens F-actin cytoskeletal condensation, and reduces Cleaved Caspase-3 and TUNEL-positive apoptotic cells, thereby reinforcing mechanoptosis resistance. Hyperpolarization (via valinomycin) or YAP inhibition (via verteporfin) reversed these phenotypes: YAP was retained in the cytoplasm, proliferation was suppressed, cytoskeletal tension was reduced, and mechanoptosis was restored. Depolarization also upregulated ABCG2 expression and increased 5-FU chemoresistance, particularly in the central tumor region, while hyperpolarization resensitized CRC cells to chemotherapy. In migration and invasion assays, depolarization strongly promoted EMT (downregulated E-cadherin; upregulated N-cadherin) and DiI-labeled tumor cell invasion into the vascular channel, whereas hyperpolarization suppressed metastatic phenotypes.

Immunological characterization in the organ-on-a-chip demonstrated that mechanically active peripheral tumor cells highly express PVR, which binds to CD96 on infiltrating CD8+ T cells to induce exhaustion marked by elevated PD-1 and TIM-3 and reduced IFN-γ and Granzyme B secretion. Depolarization amplified PVR expression and exacerbated T cell dysfunction. In contrast, hyperpolarization or PVR neutralizing antibody blockade restored CD8+ T cell cytotoxicity, increased pro-inflammatory cytokine secretion, and reduced exhaustion marker expression. Pearson correlation analysis confirmed a strong positive correlation between PIEZO1 mechanical signal intensity and PD-1 expression in CD8+ T cells (R² > 0.85, P < 0.001), verifying that mechanosignaling directly drives immune suppression via the PVR/CD96 axis.

Rescue experiments using YAP or PVR overexpression showed that restoring YAP activity under hyperpolarization partially recovered PVR expression, T cell exhaustion, and tumor cell proliferation; PVR overexpression directly reversed hyperpolarization-mediated immune restoration without affecting upstream Hippo signaling. These results define a linear signaling cascade: membrane potential → Hippo/YAP mechanotransduction → PVR/CD96 immune checkpoint → T cell function.

In vivo validation using C57BL/6 mouse subcutaneous MC38 CRC xenografts replicated key in vitro findings. Depolarization accelerated tumor growth, suppressed apoptosis, activated YAP, and elevated PVR expression, while hyperpolarization inhibited tumor progression, induced apoptosis, activated the Hippo pathway (increased p-MST1/2, p-LATS1/2, p-YAP), and reduced PVR/CD96 interaction. Overexpression of YAP or PVR partially reversed the therapeutic effects of hyperpolarization, confirming the in vivo relevance of the identified axis. Serum ELISA and tissue immunofluorescence confirmed that hyperpolarization restored IFN-γ and Granzyme B levels and reduced CD8+ T cell exhaustion.

Collectively, this study reveals a previously unrecognized mechano-electro-immune regulatory axis that governs spatial heterogeneity in CRC: low peripheral mechanical pressure induces membrane potential depolarization, which inhibits Hippo signaling to promote YAP nuclear translocation and PVR expression; PVR then engages CD96 on CD8+ T cells to trigger exhaustion and immune evasion. High central pressure induces hyperpolarization, which activates Hippo signaling, sequesters YAP in the cytoplasm, reduces PVR expression, and promotes a stress-adaptive, mechanoptosis-resistant phenotype.

This work establishes membrane potential as a master regulator that integrates mechanical signals, transcriptional programs, and immune checkpoint activity to spatially pattern CRC malignancy. Targeting this axis-via pharmacologically induced hyperpolarization, YAP inhibition, or PVR/CD96 blockade-represents a novel therapeutic strategy to overcome chemoresistance, restore anti-tumor immunity, and inhibit metastasis in CRC. These findings provide a mechanistic framework for developing next-generation combination therapies that target the tumor mechanical microenvironment and immune checkpoints simultaneously.

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