The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices.
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ABSTRACT:
SUBMITTER: Kaphle P
PROVIDER: S-EPMC8006216 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell motility under physiological and pathological conditions including malignant progression of cancer and subsequent metastasis are founded on environmental confinements. During the last two decades, three-dimensional cell migration has been studied mostly by utilizing biomimetic extracellular matrix models. In the majority of these studies, the in vitro collagen scaffolds are usually assumed to be homogenous, as they consist commonly of one specific type of collagen, such as collagen type I, isolated from one species. These collagen matrices should resemble in vivo extracellular matrix scaffolds physiologically, however, mechanical phenotype and functional reliability have been addressed poorly due to certain limitations based on the assumption of homogeneity. How local variations of extracellular matrix structure impact matrix mechanics and cell migration is largely unknown. Here, we hypothesize that local inhomogeneities alter cell movement due to alterations in matrix mechanics, as they frequently occur in in vivo tissue scaffolds and were even changed in diseased tissues. To analyze the effect of structural inhomogeneities on cell migration, we used a mixture of rat tail and bovine dermal collagen type I as well as pure rat and pure bovine collagens at four different concentrations to assess three-dimensional scaffold inhomogeneities. Collagen type I from rat self-assembled to elongated fibrils, whereas bovine collagen tended to build node-shaped inhomogeneous scaffolds. We have shown that the elastic modulus determined with atomic force microscopy in combination with pore size analysis using confocal laser scanning microscopy revealed distinct inhomogeneities within collagen matrices. We hypothesized that elastic modulus and pore size govern cancer cell invasion in three-dimensional collagen matrices. In fact, invasiveness of three breast cancer cell types is altered due to matrix-type and concentration indicating that these two factors are crucial for cellular invasiveness. Our findings revealed that local matrix scaffold inhomogeneity is another crucial parameter to explain differences in cell migration, which not solely depended on pore size and stiffness of the collagen matrices. With these three distinct biophysical parameters, characterizing structure and mechanics of the studied collagen matrices, we were able to explain differences in the invasion behavior of the studied cancer cell lines in dependence of the used collagen model.
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Project description:Cell migration is a vital process in development and disease, and while the mechanisms that control motility are relatively well understood on two-dimensional surfaces, the control of cell migration in three dimensions (3D) and in vivo has only recently begun to be understood. Vesicle trafficking pathways have emerged as a key regulatory element in migration and invasion, with the endocytosis and recycling of cell surface cargos, including growth factor and chemokine receptors, adhesion receptors and membrane-associated proteases, being of major importance. We highlight recent advances in our understanding of how endocytic trafficking controls the availability and local activity of these cargoes to influence the movement of cells in 3D matrix and in developing organisms. In particular, we discuss how endocytic trafficking of different receptor classes spatially restricts signals and activity, usually to the leading edge of invasive cells.
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Project description:CD8+ cytotoxic T lymphocytes (CTL) and natural killer cells are the main cytotoxic killer cells of the human body to eliminate pathogen-infected or tumorigenic cells (also known as target cells). To find their targets, they have to navigate and migrate through complex biological microenvironments, a key component of which is the extracellular matrix (ECM). The mechanisms underlying killer cell's navigation are not well understood. To mimic an ECM, we use a matrix formed by different collagen concentrations and analyze migration trajectories of primary human CTLs. Different migration patterns are observed and can be grouped into three motility types: slow, fast, and mixed. The dynamics are well described by a two-state persistent random walk model, which allows cells to switch between slow motion with low persistence and fast motion with high persistence. We hypothesize that the slow motility mode describes CTLs creating channels through the collagen matrix by deforming and tearing apart collagen fibers and that the fast motility mode describes CTLs moving within these channels. Experimental evidence supporting this scenario is presented by visualizing migrating T cells following each other on exactly the same track and showing cells moving quickly in channel-like cavities within the surrounding collagen matrix. Consequently, the efficiency of the stochastic search process of CTLs in the ECM should strongly be influenced by a dynamically changing channel network produced by the killer cells themselves.
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Project description:In addition to its role in controlling cell cycle progression, the tumor suppressor protein p53 can also affect other cellular functions such as cell migration. In this study, we show that p53 deficiency in mouse embryonic fibroblasts cultured in three-dimensional matrices induces a switch from an elongated spindle morphology to a markedly spherical and flexible one associated with highly dynamic membrane blebs. These rounded, motile cells exhibit amoeboid-like movement and have considerably increased invasive properties. The morphological transition requires the RhoA-ROCK (Rho-associated coil-containing protein kinase) pathway and is prevented by RhoE. A similar p53-mediated transition is observed in melanoma A375P cancer cells. Our data suggest that genetic alterations of p53 in tumors are sufficient to promote motility and invasion, thereby contributing to metastasis.
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Project description:Membrane ruffling and lamellipodia formation promote the motility of adherent cells in two-dimensional motility assays by mechano-sensing of the microenvironment and initiation of focal adhesions towards their surroundings. Lamellipodium formation is stimulated by small Rho GTPases of the Rac subfamily, since genetic removal of these GTPases abolishes lamellipodium assembly. The relevance of lamellipodial or invadopodial structures for facilitating cellular mechanics and 3D cell motility is still unclear. Here, we hypothesized that Rac1 affects cell mechanics and facilitates 3D invasion. Thus, we explored whether fibroblasts that are genetically deficient for Rac1 (lacking Rac2 and Rac3) harbor altered mechanical properties, such as cellular deformability, intercellular adhesion forces and force exertion, and exhibit alterations in 3D motility. Rac1 knockout and control cells were analyzed for changes in deformability by applying an external force using an optical stretcher. Five Rac1 knockout cell lines were pronouncedly more deformable than Rac1 control cells upon stress application. Using AFM, we found that cell-cell adhesion forces are increased in Rac1 knockout compared to Rac1-expressing fibroblasts. Since mechanical deformability, cell-cell adhesion strength and 3D motility may be functionally connected, we investigated whether increased deformability of Rac1 knockout cells correlates with changes in 3D motility. All five Rac1 knockout clones displayed much lower 3D motility than Rac1-expressing controls. Moreover, force exertion was reduced in Rac1 knockout cells, as assessed by 3D fiber displacement analysis. Interference with cellular stiffness through blocking of actin polymerization by Latrunculin A could not further reduce invasion of Rac1 knockout cells. In contrast, Rac1-expressing controls treated with Latrunculin A were again more deformable and less invasive, suggesting actin polymerization is a major determinant of observed Rac1-dependent effects. Together, we propose that regulation of 3D motility by Rac1 partly involves cellular mechanics such as deformability and exertion of forces.
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Project description:BackgroundThe behaviour of tumour cells depends on factors such as genetics and the tumour microenvironment. The latter plays a crucial role in normal mammary gland development and also in breast cancer initiation and progression. Breast cancer tissues tend to be highly desmoplastic and dense matrix as a pre-existing condition poses one of the highest risk factors for cancer development. However, matrix influence on tumour cell gene expression and behaviour such as cell migration is not fully elucidated.ResultsWe generated high-density (HD) matrices that mimicked tumour collagen content of 20?mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1?mg/cm3. Live-cell imaging showed breast cancer cells utilizing cytoplasmic streaming and cell body contractility for migration within HD matrix. Cell migration was blocked in the presence of both the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugs individually. This suggests roles for ROCK1 and MMP in cell migration are complicated by compensatory mechanisms. ROCK1 expression and protein activity, were significantly upregulated in HD matrix but these were blocked by treatment with a histone deacetylase (HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirect and relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA or DAPT abrogated the inhibition of ROCK1 by MS-275.ConclusionIncreased matrix density elevates ROCK1 activity, which aids in cell migration via cell contractility. The upregulation of ROCK1 is epigenetically regulated in an indirect manner involving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1 expression.
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Project description:The mechanical environment of a cell is not constant. This dynamic behavior is exceedingly difficult to capture in (synthetic) in vitro matrices. This paper describes a novel, highly adaptive hybrid hydrogel composed of magnetically sensitive magnetite nanorods and a stress-responsive synthetic matrix. Nanorod rearrangement after application of (small) magnetic fields induces strain in the network, which results in a strong (over 10-fold) stiffening even at minimal (2.5 wt %) nanorod concentrations. Moreover, the stiffening mechanism yields a fast and fully reversible response. In the manuscript, we quantitatively analyze that forces generated by the particles are comparable to cellular forces. We demonstrate the value of magnetic stiffening in a 3D MCF10A epithelial cell experiment, where simply culturing on top of a permanent magnet gives rise to changes in the cell morphology. This work shows that our hydrogels are uniquely suited as 3D cell culture systems with on-demand adaptive mechanical properties.
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