(135f) Fluid Forces Inhibit Confined Cell Migration

Mechanical abnormalities in cancer have been implicated in the progression of disease and metastasis, the deadliest part of cancer. Understanding what drives metastasis and developing ways to inhibit it is of upmost importance. Most tumors exhibit elevated interstitial fluid pressure, which increases with tumor aggressiveness and drops abruptly at the tumor margins. This pressure gradient generates fluid flow from the tumor interior into the surrounding matrix. Despite its potential significance, the role of pressure-driven flow in promoting cancer cell dissemination remains unclear.

To begin addressing this, we investigate how pathophysiologically relevant pressure-driven flow influences cell migration. We focused on confined migration because metastatic cells disseminate from the primary tumor by migrating through confined spaces, including tissue-like tracks typically 3-30 μm wide and up to few hundred micrometers long. Using photolithography and replica molding we created PDMS-based microfluidic devices, containing extracellular matrix (ECM)-coated confined microchannels with dimensions of 10 μm wide, 3 μm high, and 200 μm long. Cells were introduced into the devices, allowed to attach and spread and then subjected to hydrostatic pressure differentials (ΔP) ranging from 0 to 500 Pa, applied across the microchannels to mimic interstitial flow conditions in the tumor environment. To assess the effects of flow on cell entry into confined microchannels, ΔP was applied immediately after cell adhesion. For cell reversal experiments, cells were allowed to enter the confined microchannels prior to application of ΔP. Time-lapse microscopy was used to monitor cell invasion and migration, with imaging carried out for 8- and 16-hour in entry experiments and 3 hours in reversal experiments.

Regardless of the type of ECM used to coat the microchannels, fluid forces counterintuitively inhibit invasion in the direction of flow and induce confined migration toward regions of higher pressure. Interestingly, this is not seen in moderately confined channels (width x height = 20 x 10 μm²), where fluid convection enables the formation of pericellular chemokine gradients that inhibit upstream motility. It has been previously demonstrated that the nucleoskeleton, cytoskeleton, and mechanosensitive ion channels (MICs) are important for transmitting mechanical signals through the cell and initiating cell response. We therefore visualized actin and myosin IIA in confined cells under different ΔP. Cells respond to fluid forces by repolarizing actin and myosin IIA to the new leading and trailing edge, respectively. Interestingly, there was a marked delay in reversal time and actin repolarization as ΔP increased. Actomyosin reorganization is essential for efficient cell reversal, as demonstrated through mathematical modeling, chemical inhibitors, knockdown strategies and optogenetics. The cytoskeleton is connected to the nucleus via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Disruption of the LINC complex and knockdown of the nuclear envelope protein lamin A/C decrease fluid force-induced cell reversal, triggering downstream motility. Moreover, highly invasive breast cancer cell lines with naturally lower lamin A/C levels are less responsive to fluid forces, exhibiting a greater tendency to migrate toward regions of lower pressure. This behavior may help explain how invasive tumor cells migrate down the pressure gradient when escaping the primary tumor. Nuclear stiffening, overexpression of the MIC TRPM7 and intracellular calcium rise restores fluid force sensing and upstream migration of lamin A/C^low cancer cells, suggesting that these interventions may help suppress cancer cell escape from high pressure environments.

Collectively, these results underscore the interplay between the nucleus, cytoskeletal elements and ion channels in fluid force-dependent migration and further suggest that pressure gradients are negative regulators of metastasis. Future studies will focus on investigating how higher pressure differentials (ΔP > 500 Pa), as observed in highly aggressive tumors, influence the cellular migration machinery and directional migration under confinement.