CANCER CELL MIGRATION AND DECISION-MAKING IN PHYSIOLOGICALLY RELEVANT CONFINING MICROENVIRONMENTS

In metastasis, cancer cells break away from a primary tumor and travel through the blood or lymph system to distant parts of the body forming new secondary metastatic tumors. Metastatic disease is responsible for ~90% of cancer-related deaths. Cell migration is central to the metastatic cascade and recent technological advances have elucidated the importance of cancer migration through confining open tracks around the cell body. Thus, extensive study and characterization of confined cell migration is critical for the development of future cancer treatments. Following an introduction to the in vitro devices used in confined migration and the mechanisms of cell locomotion that they have revealed, we present a novel tool, a hydrogel encapsulated microchannel array (HEMICA), used for studying cell migration in physiologically relevant microenvironments. HEMICA is made out of accessible, inexpensive materials based on established techniques and emulates a set of physical properties found in native tissue. HEMICA can be used for live-cell imaging, confocal imaging, immunofluorescence, Foster Resonance Energy Transfer (FRET), actin flow measurements, chemotactic gradient studies, apicobasal stiffness anisotropy studies and 3D Traction Force Microscopy. In addition, it can create microenvironments that support collective and single cell-file migration. Subsequently, we use HEMICA to characterize MDA-MB-231 adenocarcinoma cell migration through various confining and non-confining microchannels, and compare confined migration characteristics in stiff versus soft substrates. We observe the impact of substrate stiffness on the morphodynamic changes of cells, and distinguish the role of confinement in accentuating cell polarization and diminishing changes in cell spreading. We demonstrate the dependence of contact guidance to substrate stiffness and via 3D TFM we showcase that the increase of substrate stiffness leads to higher traction forces in confinement. Furthermore, we affirm that MDA-MB-231 confined migration in soft substrates is dependent on myosin IIA (MIIA), phosphorylated myosin light chain (pMLC), actin, integrins and adhesions, and juxtapose the osmotic engine effects of sodium hydrogen exchanger 1 (NHE1) observed in stiff substrates with its function in more physiologically relevant ones. Finally, we correlate MIIA-based contractility to the polarization of pMLC, NHE1 and actin flow. We next use microfabrication techniques to create microenvironments of varying hydraulic resistance and analyze the decision-making process of MDA-MB-231 and HT1080 fibrosarcoma cells. We confirm that hydraulic resistance dictates decision-making in confinement, characterize the decision-making process based on the persistent growth of a leading protrusion of a cell at a channel intersection, showcase the effects of actin nucleation on decision-making time, and identify MIIA and transient receptor potential melastatin-7 (TRPM7) as the main mechanosensors of hydraulic resistance in confinement. We then establish the relationship between hydraulic resistance, actin and myosin and build a theoretical framework for confined cell decision-making. Overall our results elucidate the importance of the physical microenvironment in regulating cell migration

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

International audienceActin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensi-tive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca 2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2. mechanotransduction | calcium signaling | RhoA | actin stress fibers | cance

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2

Downregulation of YAP Activity Restricts P53 Hyperactivation to Promote Cell Survival in Confinement

Abstract Cell migration through confining three dimensional (3D) topographies can lead to loss of nuclear envelope integrity, DNA damage, and genomic instability. Despite these detrimental phenomena, cells transiently exposed to confinement do not usually die. Whether this is also true for cells subjected to long‐term confinement remains unclear at present. To investigate this, photopatterning and microfluidics are employed to fabricate a high‐throughput device that circumvents limitations of previous cell confinement models and enables prolonged culture of single cells in microchannels with physiologically relevant length scales. The results of this study show that continuous exposure to tight confinement can trigger frequent nuclear envelope rupture events, which in turn promote P53 activation and cell apoptosis. Migrating cells eventually adapt to confinement and evade cell death by downregulating YAP activity. Reduced YAP activity, which is the consequence of confinement‐induced YAP1/2 translocation to the cytoplasm, suppresses the incidence of nuclear envelope rupture and abolishes P53‐mediated cell death. Cumulatively, this work establishes advanced, high‐throughput biomimetic models for better understanding cell behavior in health and disease, and underscores the critical role of topographical cues and mechanotransduction pathways in the regulation of cell life and death

Cell sensing and decision-making in confinement: The role of TRPM7 in a tug of war between hydraulic pressure and cross-sectional area

How cells sense hydraulic pressure and make directional choices in confinement remains elusive. Using trifurcating Ψ-like microchannels of different hydraulic resistances and cross-sectional areas, we discovered that the TRPM7 ion channel is the critical mechanosensor, which directs decision-making of blebbing cells toward channels of lower hydraulic resistance irrespective of their cross-sectional areas. Hydraulic pressure-mediated TRPM7 activation triggers calcium influx and supports a thicker cortical actin meshwork containing an elevated density of myosin-IIA. Cortical actomyosin shields cells against external forces and preferentially directs cell entrance in low resistance channels. Inhibition of TRPM7 function or actomyosin contractility renders cells unable to sense different resistances and alters the decision-making pattern to cross-sectional area-based partition. Cell distribution in microchannels is captured by a mathematical model based on the maximum entropy principle using cortical actin as a key variable. This study demonstrates the unique role of TRPM7 in controlling decision-making and navigating migration in complex microenvironments.This line of research was supported by the NIH through grants R01-CA183804 (to K.K.), U54-CA210173 (to K.K. and S.X.S.), and R01-GM114675 (to S.X.S. and K.K.), as well as by the Spanish Ministry of Economy and Competitiveness through grants SAF2015-69762R and RTI2018-099718 (to M.A.V.), and an institutional “Maria de Maeztu” Programme for Units of Excellence in R&D (MDM-2014-0370 to M.A.V.) and FEDER funds (to M.A.V.)

Migration and 3D Traction Force Measurements inside Compliant Microchannels

Cells migrate in vivo through channel-like tracks. While polydimethylsiloxane devices emulate such tracks in vitro, their channel walls are impermeable and have supraphysiological stiffness. Existing hydrogel-based platforms address these issues but cannot provide high-throughput analysis of cell motility in independently controllable stiffness and confinement. We herein develop polyacrylamide (PA)-based microchannels of physiological stiffness and prescribed dimensions for high-throughput analysis of cell migration and identify a biphasic dependence of speed upon confinement and stiffness. By utilizing novel four-walled microchannels with heterogeneous stiffness, we reveal the distinct contributions of apicolateral versus basal microchannel wall stiffness to confined versus unconfined migration. While the basal wall stiffness dictates unconfined migration, apicolateral stiffness controls confined migration. By tracking nanobeads embedded within channel walls, we innovate three-dimensional traction force measurements around spatially confining cells at subcellular resolution. Our unique and highly customizable device fabrication strategy provides a physiologically relevant in vitro platform to study confined cells

Cell sensing and decision-making in confinement: The role of TRPM7 in a tug of war between hydraulic pressure and cross-sectional area

How cells sense hydraulic pressure and make directional choices in confinement remains elusive. Using trifurcating Ψ-like microchannels of different hydraulic resistances and cross-sectional areas, we discovered that the TRPM7 ion channel is the critical mechanosensor, which directs decision-making of blebbing cells toward channels of lower hydraulic resistance irrespective of their cross-sectional areas. Hydraulic pressure-mediated TRPM7 activation triggers calcium influx and supports a thicker cortical actin meshwork containing an elevated density of myosin-IIA. Cortical actomyosin shields cells against external forces and preferentially directs cell entrance in low resistance channels. Inhibition of TRPM7 function or actomyosin contractility renders cells unable to sense different resistances and alters the decision-making pattern to cross-sectional area-based partition. Cell distribution in microchannels is captured by a mathematical model based on the maximum entropy principle using cortical actin as a key variable. This study demonstrates the unique role of TRPM7 in controlling decision-making and navigating migration in complex microenvironments.This line of research was supported by the NIH through grants R01-CA183804 (to K.K.), U54-CA210173 (to K.K. and S.X.S.), and R01-GM114675 (to S.X.S. and K.K.), as well as by the Spanish Ministry of Economy and Competitiveness through grants SAF2015-69762R and RTI2018-099718 (to M.A.V.), and an institutional “Maria de Maeztu” Programme for Units of Excellence in R&D (MDM-2014-0370 to M.A.V.) and FEDER funds (to M.A.V.)