Mechanosensitive expression of the mesenchymal subtype marker Connective Tissue Growth Factor in Glioblastoma

Abstract

Mechanical forces created by the extracellular environment regulate biochemical signals that modulate the inter-related cellular phenotypes of morphology, proliferation, and migration. A rigid microenvironment induces glioblastoma (GBM) cells to develop prominent actin stress fibres, take on a spread morphology and adopt trapezoid cells shapes when cultured in 2D, which are phenotypes characteristic of a mesenchymal cell program. The mesenchymal subtype is the most aggressive among the molecular GBM subtypes and recurrent GBM of other subtypes have been reported to transition to mesenchymal. We therefore sought to test the hypothesis that rigid microenvironments - such as those found in different brain anatomical structures and induced following treatment - contribute to the expression of markers characterising the mesenchymal subtype. We cultured primary patient-derived cell lines that reflect the three common GBM subtypes (mesenchymal, proneural and classic) on polyacrylamide (PA) hydrogels with controlled stiffnesses spanning the healthy and pathological tissue range. We then assessed the canonical mesenchymal marker Connective Tissue Growth Factor (CTGF) and yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) expression, via immunofluorescence. Replating techniques and drug-mediated manipulation of the actin cytoskeleton were utilised to ascertain the response of the cells to differing mechanical environments. We demonstrate that CTGF is induced rapidly following adhesion to a rigid substrate and is independent of actin filament formation. Collectively, our data suggest that microenvironmental rigidity may stimulate the GBM mesenchymal signalling program.