Matrix protease production, epithelial-to-mesenchymal transition marker expression and invasion of glioblastoma cells in response to osmotic or hydrostatic pressure

Abstract

Both hydrostatic and osmotic pressures are altered in the tumour microenvironment. Glioblastoma (GBM) is a brain tumour with high invasiveness and poor prognosis. We hypothesized that physical and osmotic forces regulate glioblastoma (GBM) invasiveness. the osmotic pressure of GBM cell culture medium was adjusted using sodium chloride or water. Alternatively, cells were subjected to increased hydrostatic force. The proteolytic profile and epithelial-mesenchymal transition (EMT) were investigated using zymography and real-time qPCR. The EMT markers assessed were Snail-1, Snail-2, N-cadherin, Twist and vimentin. Invasion was investigated in vitro using extracellular matrixcoated Transwell inserts. In response to osmotic and mechanical pressure, GBM cell lines U87 and U251 and patient-derived neural oncospheres upregulated the expression of urokinase-type plasminogen activator (upA) and/or matrix metalloproteinases (MMps) as well as some of the eMt markers tested. the adherent cell lines invaded more when placed in media of increased osmolality. therefore, GBM respond to osmotic or mechanical pressure by increasing matrix degrading enzyme production, and adopting a phenotype reminiscent of eMt. Better understanding the molecular and cellular mechanisms by which increased pressure promotes GBM invasiveness may help to develop innovative therapeutic approaches. Physical solid and fluid forces play a key role when solid tumours grow, progress and also respond to therapy 1. Compressive stresses affect cancer cells by promoting invasiveness and metastasis 2. Tumours are generally hypoperfused, and interstitial fluid pressure is increased compared to normal tissue 1,3,4 with both increased hydrostatic pressure 4 and oncotic pressure 5,6. Increased interstitial fluid pressure results from abnormal blood and lymphatic vessels, fibrosis and contraction of the matrix by stromal cells 7. In addition to these stresses common to most solid tumours, brain tumours experience pressure when the tumour grows within a space limited by the skull 8. Glioblastoma is the most common primary brain cancer. The average survival time is approximately one year after diagnosis. A major feature of GBM that contributes to its poor prognosis is its high invasiveness. The urokinase-type plasminogen activator (uPA) derives its name from its ability to activate plasminogen into plasmin. While tissue-type plasminogen activator (tPA) plays a role in the fibrinolytic process, uPA is involved in cell migration and tissue remodelling, thereby playing a major role in cancer development and spreading. This role is especially crucial in glioblastoma 9-11. Equally important and complementary to the uPA system, MMPs play a key role in the control of the tumour microenvironment and ECM, thereby modulating tumor growth, angiogenesis, invasion and metastasis. Recently reports showed that the MMPs play pivotal roles in the invasiveness of GBM by degrading surrounding tissue, activating signal transduction, releasing ECM-bound growth factors, activating

A role for caveola‐forming proteins caveolin‐1 and CAVIN1 in the pro‐invasive response of glioblastoma to osmotic and hydrostatic pressure

“…We tested the mRNA expression of CAV1 and CAVIN1 in GBM cells exposed to hyperosmotic or hypo‐osmotic medium for 48 hours (Figure A). The osmolality was adjusted by adding sodium chloride as previously described . Hyperosmolality (440 mOsmol/kg) caused a slight increase (around 2‐fold) in mRNA of CAV1 and CAVIN1 in the U251 cell line.…”

“…Previous work has shown that GBM respond to hydrostatic pressure by increasing matrix proteases and EMT marker expression, and caveolae have been proposed to serve mechanosensing roles. Therefore, in the following set of experiments, we assessed whether CRISPR ablation of caveola‐forming proteins CAV1 or CAVIN1 altered the pro‐invasive response of U251 cells to hydrostatic pressure, that is 30 mm Hg applied to the cells over 48 hours, as previously described . The mRNA expression of matrix proteases (Figure a,c) and EMT markers (Figure b,d) in response to increased hydrostatic pressure was significantly lower in cells lacking caveola‐forming proteins CAV1 or CAVIN1 for the majority of the genes assessed.…”

“…The osmolality was adjusted by adding sodium chloride as previously described. 19 Hyperosmolality (440 mOsmol/kg) caused a slight increase (around 2-fold) in mRNA of CAV1 and CAVIN1 in the U251 cell line. A similar trend was seen in the U87 and U118 cell lines for CAV1 mRNA, albeit non-statistically significant, and for CAVIN1 in U118 cells.…”

“…We have previously demonstrated that the expression of caveolin-1 and CAVIN1 correlates with GBM invasiveness 12 and that GBM cells respond to osmotic pressure by increased production of matrix-degrading enzymes, EMT markers and invasion. 19 We asked whether caveolae may be involved in the cellular response to pressure. We tested the mRNA expression of CAV1 and CAVIN1 in GBM cells exposed to hyperosmotic or hypo-osmotic medium for 48 hours ( Figure 1A).…”

“…Previous work has shown that GBM respond to hydrostatic pressure by increasing matrix proteases and EMT marker expression, 19 and caveolae have been proposed to serve mechanosensing roles. Therefore, in the following set of experiments, we assessed whether CRISPR ablation of caveola-forming proteins CAV1 or CAVIN1 altered the pro-invasive response of U251 cells to hydrostatic pressure, that is 30 mm Hg applied to the cells over 48 hours, as previously described.…”