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

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.