Cell crowding induces TRPV4 inhibition and its relocation to plasma membranes, implicating pro-invasive cell volume reduction mechanotransduction pathway

Cell crowding is a common microenvironmental factor that affects various disease processes, but its impact on cell invasiveness into surrounding tissues is not well understood. In this study, we investigated the biomechanical changes induced by cell crowding, focusing on pro-invasive cell volume reduction. We discovered that cell crowding enhanced the invasiveness of high-grade ductal carcinoma in situ (DCIS) cells, which experienced significant cell volume reduction compared to hyperplasia-mimicking or normal cells. Mass spectrometry analyses revealed that cell crowding relocated ion channels, including TRPV4, a calcium-permeant ion channel, to the plasma membrane selectively in high-grade DCIS cells but not in less aggressive or normal cells. Cell crowding inhibited TRPV4 in high-grade DCIS cells, which led to decreased intracellular calcium levels and subsequent volume reduction. TRPV4 inhibition also prompted relocation of TRPV4 to the plasma membrane. This relocation primed inactive TRPV4 for activation, effectively counterbalancing the calcium loss from crowding-induced channel inhibition. Analyses of patient-derived breast cancer tissues validated that TRPV4 selectively associated with the plasma membrane in high-grade DCIS but not in lower-grade DCIS or less aggressive pathologies. The extent of plasma membrane TRPV4 association scaled with cell volume reduction and increased cell invasiveness and motility, suggesting its utility as an active pro-invasive mechanotransduction pathway indicator. Additionally, hyperosmotic conditions and pharmacologic TRPV4 inhibition mimicked the pro-invasive volume reduction observed under cell crowding, while TRPV4 activation reversed this effect by inducing cell volume increase. In summary, our study reveals a previously unrecognized pro-invasive mechanotransduction pathway triggered by cell crowding, which is selective in high-grade DCIS cells. This discovery offers new biophysical perspectives on cell invasiveness, highlighting the critical role of a selective mechanotransduction mechanism in the progression of breast cancer cells that are considered non-invasive but associated with high risk.