Inferring Cellular Contractile Forces and Work using Deep Morphology Traction Microscopy.

Traction Force Microscopy (TFM) has emerged as a widely used standard methodology to measure cell-generated traction forces and determine their role in regulating cell behavior. While TFM platforms have enabled many discoveries, their implementation remains limited due to complex experimental procedures, specialized substrates, and the ill-posed inverse problem where low magnitude high-frequency noise in the displacement field severely contaminates the resulting traction measurements. Here, we introduce Deep Morphology Traction Microscopy (DeepMorphoTM), a Deep Learning alternative to conventional TFM approaches. DeepMorphoTM first infers cell-induced substrate displacement solely from a sequence of cell shapes and subsequently computes cellular traction forces, thus avoiding the requirement of a specialized fiducial-marked deformable substrate or force-free reference image. Rather, this technique drastically simplifies the overall experimental methodology, imaging, and analysis needed to conduct cell contractility measurements. We demonstrate that DeepMorphoTM quantitatively matches conventional TFM results, while offering stability against the biological variability in cell contractility for a given cell shape. Without high-frequency noise in the inferred displacement, DeepMorphoTM also resolves the ill-posedness of traction computation, increasing the consistency and accuracy of traction analysis. We demonstrate the accurate extrapolation across several cell types and substrate materials, suggesting robustness of the methodology. Accordingly, we present DeepMorphoTM as a capable yet simpler alternative to conventional TFM for characterizing cellular contractility in 2D.

MAP4K4 regulates forces at cell-cell and cell-matrix adhesions to promote collective cell migration.

Collective cell migration is not only important for development and tissue homeostasis but can also promote cancer metastasis. To migrate collectively, cells need to coordinate cellular extensions and retractions, adhesion sites dynamics, and forces generation and transmission. Nevertheless, the regulatory mechanisms coordinating these processes remain elusive. Using A431 carcinoma cells, we identify the kinase MAP4K4 as a central regulator of collective migration. We show that MAP4K4 inactivation blocks the migration of clusters, whereas its overexpression decreases cluster cohesion. MAP4K4 regulates protrusion and retraction dynamics, remodels the actomyosin cytoskeleton, and controls the stability of both cell-cell and cell-substrate adhesion. MAP4K4 promotes focal adhesion disassembly through the phosphorylation of the actin and plasma membrane crosslinker moesin but disassembles adherens junctions through a moesin-independent mechanism. By analyzing traction and intercellular forces, we found that MAP4K4 loss of function leads to a tensional disequilibrium throughout the cell cluster, increasing the traction forces and the tension loading at the cell-cell adhesions. Together, our results indicate that MAP4K4 activity is a key regulator of biomechanical forces at adhesion sites, promoting collective migration.

Prostate cancer cells of increasing metastatic potential exhibit diverse contractile forces, cell stiffness, and motility in a microenvironment stiffness-dependent manner.

During metastasis, all cancer types must migrate through crowded multicellular environments. Simultaneously, cancers appear to change their biophysical properties. Indeed, cell softening and increased contractility are emerging as seemingly ubiquitous biomarkers of metastatic progression which may facilitate metastasis. Cell stiffness and contractility are also influenced by the microenvironment. Stiffer matrices resembling the tumor microenvironment cause metastatic cells to contract more strongly, further promoting contractile tumorigenic phenotypes. Prostate cancer (PCa), however, appears to deviate from these common cancer biophysics trends; aggressive metastatic PCa cells appear stiffer, rather than softer, to their lowly metastatic PCa counterparts. Although metastatic PCa cells have been reported to be more contractile than healthy cells, how cell contractility changes with increasing PCa metastatic potential has remained unknown. Here, we characterize the biophysical changes of PCa cells of various metastatic potential as a function of microenvironment stiffness. Using a panel of progressively increasing metastatic potential cell lines (22RV1, LNCaP, DU145, and PC3), we quantified their contractility using traction force microscopy (TFM), and measured their cortical stiffness using optical magnetic twisting cytometry (OMTC) and their motility using time-lapse microscopy. We found that PCa contractility, cell stiffness, and motility do not universally scale with metastatic potential. Rather, PCa cells of various metastatic efficiencies exhibit unique biophysical responses that are differentially influenced by substrate stiffness. Despite this biophysical diversity, this work concludes that mechanical microenvironment is a key determinant in the biophysical response of PCa with variable metastatic potentials. The mechanics-oriented focus and methodology of the study is unique and complementary to conventional biochemical and genetic strategies typically used to understand this disease, and thus may usher in new perspectives and approaches.