Elastocapillary effects determine early matrix deformation by glioblastoma cell spheroids.

During cancer pathogenesis, cell-generated mechanical stresses lead to dramatic alterations in the mechanical and organizational properties of the extracellular matrix (ECM). To date, contraction of the ECM is largely attributed to local mechanical stresses generated during cell invasion, but the impact of "elastocapillary" effects from surface tension on the tumor periphery has not been examined. Here, we embed glioblastoma cell spheroids within collagen gels, as a model of tumors within the ECM. We then modulate the surface tension of the spheroids, such that the spheroid contracts or expands. Surprisingly, in both cases, at the far-field, the ECM is contracted toward the spheroids prior to cellular migration from the spheroid into the ECM. Through computational simulation, we demonstrate that contraction of the ECM arises from a balance of spheroid surface tension, cell-ECM interactions, and time-dependent, poroelastic effects of the gel. This leads to the accumulation of ECM near the periphery of the spheroid and the contraction of the ECM without regard to the expansion or contraction of the spheroid. These results highlight the role of tissue-level surface stresses and fluid flow within the ECM in the regulation of cell-ECM interactions.

Using Biosensors to Study Organoids, Spheroids and Organs-on-a-Chip: A Mechanobiology Perspective.

The increasing popularity of 3D cell culture models is being driven by the demand for more in vivo-like conditions with which to study the biochemistry and biomechanics of numerous biological processes in health and disease. Spheroids and organoids are 3D culture platforms that self-assemble and regenerate from stem cells, tissue progenitor cells or cell lines, and that show great potential for studying tissue development and regeneration. Organ-on-a-chip approaches can be used to achieve spatiotemporal control over the biochemical and biomechanical signals that promote tissue growth and differentiation. These 3D model systems can be engineered to serve as disease models and used for drug screens. While culture methods have been developed to support these 3D structures, challenges remain to completely recapitulate the cell-cell and cell-matrix biomechanical interactions occurring in vivo. Understanding how forces influence the functions of cells in these 3D systems will require precise tools to measure such forces, as well as a better understanding of the mechanobiology of cell-cell and cell-matrix interactions. Biosensors will prove powerful for measuring forces in both of these contexts, thereby leading to a better understanding of how mechanical forces influence biological systems at the cellular and tissue levels. Here, we discussed how biosensors and mechanobiological research can be coupled to develop accurate, physiologically relevant 3D tissue models to study tissue development, function, malfunction in disease, and avenues for disease intervention.

SPAK-dependent cotransporter activity mediates capillary adhesion and pressure during glioblastoma migration in confined spaces.

The invasive potential of glioblastoma cells is attributed to large changes in pressure and volume, driven by diverse elements, including the cytoskeleton and ion cotransporters.  However, how the cell actuates changes in pressure and volume in confinement, and how these changes contribute to invasive motion is unclear. Here, we inhibited SPAK activity, with known impacts on the cytoskeleton and cotransporter activity and explored its role on the migration of glioblastoma cells in confining microchannels to model invasive spread through brain tissue. First, we found that confinement altered cell shape, inducing a transition in morphology that resembled droplet interactions with a capillary vessel, from "wetting" (more adherent) at low confinement, to "nonwetting" (less adherent) at high confinement. This transition was marked by a change from negative to positive pressure by the cells to the confining walls, and an increase in migration speed. Second, we found that the SPAK pathway impacted the migration speed in different ways dependent upon the extent of wetting. For nonwetting cells, SPAK inhibition increased cell-surface tension and cotransporter activity. By contrast, for wetting cells, it also reduced myosin II and YAP phosphorylation. In both cases, membrane-to-cortex attachment is dramatically reduced. Thus, our results suggest that SPAK inhibition differentially coordinates cotransporter and cytoskeleton-induced forces, to impact glioblastoma migration depending on the extent of confinement.

Endothelial cell secreted metalloproteinase-2 enhances neural stem cell N-cadherin expression, clustering, and migration.

Neuroblasts have a clustered phenotype critical for their unidirectional migration, which in part is dependent on signaling from microvascular endothelial cells (EC) and pericytes (PC). Diffusible signals secreted by vascular cells have been demonstrated to increase survival, proliferation, and differentiation of subventricular zone resident neural stem cells (NSC); however, the signals that promote the necessary initiating step of NSC clustering are undefined. To investigate the role of vascular cells in promoting NSC clustering and directing migration, we created a 3-D hydrogel that mimics the biomechanics, biochemistry, and architectural complexity of brain tissue. We demonstrate that EC, and not PC, have a crucial role in NSC clustering and migration, further verified through microfluidic chamber systems and traction force microscopy. Ablation of the extended NSC aggregate arm halts aggregate movement, suggesting that clustering is a prerequisite for migration. When cultured with EC, NSC clustering occurs and NSC coincidentally increase their expression of N-cadherin, as compared to NSC cultured alone. NSC-presented N-cadherin expression was increased following exposure to EC secreted metalloproteinase-2 (MMP2). We demonstrate that inhibition of MMP2 prevented NSC N-cadherin surface expression and subsequent NSC clustering, even when NSC were in direct contact with EC. Furthermore, with exogenous activation of EGFR, which serves as a downstream activator of N-cadherin cleavage, NSC form clusters. Our results suggest that EC secretion of MMP2 promotes NSC clustering through N-cadherin expression. The insight gained about the mechanisms by which EC promote NSC migration may enhance NSC therapeutic response to sites of injury.

Investigating the effect of cell substrate on cancer cell stiffness by optical tweezers.

The mechanical properties of cells are influenced by their microenvironment. Here we report cell stiffness alteration by changing the cell substrate stiffness for isolated cells and cells in contact with other cells. Polydimethylsiloxane (PDMS) is used to prepare soft substrates with three different stiffness values (173, 88 and 17kPa respectively). Breast cancer cells lines, namely HBL-100, MCF-7 and MDA-MB-231 with different level of aggressiveness are cultured on these substrates and their local elasticity is investigated by vertical indentation of the cell membrane. Our preliminary results show an unforeseen behavior of the MDA-MB-231 cells. When cultured on glass substrate as isolated cells, they are less stiff than the other two types of cells, in agreement with the general statement that more aggressive and metastatic cells are softer. However, when connected to other cells the stiffness of MDA-MB-231 cells becomes similar to the other two cell lines. Moreover, the stiffness of MDA-MB-231 cells cultured on soft PDMS substrates is significantly higher than the stiffness of the other cell types, demonstrating thus the strong influence of the environmental conditions on the mechanical properties of the cells.