Mechanosensitive expression of the mesenchymal subtype marker connective tissue growth factor in glioblastoma.

Mechanical forces created by the extracellular environment regulate biochemical signals that modulate the inter-related cellular phenotypes of morphology, proliferation, and migration. A stiff microenvironment induces glioblastoma (GBM) cells to develop prominent actin stress fibres, take on a spread morphology and adopt trapezoid 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. Recurrent GBM have been reported to transition to mesenchymal. We therefore sought to test the hypothesis that stiffer 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 classical) on polyacrylamide (PA) hydrogels with controlled stiffnesses spanning the healthy and pathological tissue range. We then assessed the canonical mesenchymal markers 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 can stimulate expression of mesenchymal-associated molecules in GBM.

Advanced Spheroid, Tumouroid and 3D Bioprinted In-Vitro Models of Adult and Paediatric Glioblastoma.

The life expectancy of patients with high-grade glioma (HGG) has not improved in decades. One of the crucial tools to enable future improvement is advanced models that faithfully recapitulate the tumour microenvironment; they can be used for high-throughput screening that in future may enable accurate personalised drug screens. Currently, advanced models are crucial for identifying and understanding potential new targets, assessing new chemotherapeutic compounds or other treatment modalities. Recently, various methodologies have come into use that have allowed the validation of complex models-namely, spheroids, tumouroids, hydrogel-embedded cultures (matrix-supported) and advanced bioengineered cultures assembled with bioprinting and microfluidics. This review is designed to present the state of advanced models of HGG, whilst focusing as much as is possible on the paediatric form of the disease. The reality remains, however, that paediatric HGG (pHGG) models are years behind those of adult HGG. Our goal is to bring this to light in the hope that pGBM models can be improved upon.