Cell Membrane Potential Regulates Spatial-Specific Mechanoptosis Resistance and Metastasis in Colorectal Cancer Via Mechanotransduction and the PVR/CD96 Axis

Plain-language summary

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.

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