Stiffness-induced EMT and Cancer Stemness in Glioblastoma Using Polyacrylamide Hydrogels

Abstract

Gliomas are the most common type of brain and are considered one of the most fatal cancer forms due to their detrimental and aggressive behaviour. Amongst these types of brain cancer is glioblastoma (GBM), classified by the World Health Organization (WHO) as grade IV, known to have characteristics like high malignancy, rapid growth and aggressiveness. As these tumours progress, the extracellular matrix (ECM) stiffness increases, influencing their growth, survivability and treatment outcomes. The induction of Epithelial to Mesenchymal transition (EMT) was associated with the production of Cancer stem-like cells (CSCs), a small subpopulation with self renewal capabilities that generates phenotypic heterogeneity comparable to the original tumour. CSCs are responsible for sustaining tumour growth and metastasis formation to other body tissues. The main focus of this study was assessing the role of brain tissue mechanical stiffness in promoting EMT and cancer of glioblastoma cells. The surface of PAAm hydrogels was modified to overcome the non-adhesiveness via covalently linked to collagen type I to facilitate the attachment of glioblastoma cells. The stiffness of Polyacrylamide Hydrogels (PAAm hydrogels) was measured using Rheology and Nanoindentation. The three stiffnesses fabricated and used were soft 305.9±16.9 Pa, which is similar to normal brain tissue, medium 10.5±0.4 kPa, comparable to glioblastoma stiffness and rigid 34.9±5.1kPa which is stiffer than glioblastoma tumours. The nanoindentation measurements were for soft 321.72±59.83 Pa, medium 8.01±0.37kPa and rigid 39.19±2.58kPa, illustrating that the stiffnesses are unfirmed across the surface and reproducible. EMT markers like N-CAD, VIMENTIN and TGF-β showed increased protein levels in the medium and rigid hydrogels compared to soft hydrogels. This response was further by increased protein expression of the EMT transcription factor SNAI1(SNAIL), which showed a significant increase in levels of SNAI1(SNAIL) (p≤0.05) on the medium and rigid hydrogels. II CSC markers showed increased protein levels highlighted by the significant increase in the protein levels of NESTIN (P≤0.001), CD133 (P≤0.0001), POU5F1(OCT-4) (P≤0.05), and EGFR (P≤0.05), respectively on the rigid hydrogels compared to soft hydrogels. Medium hydrogels showed significant increases in the protein levels of CD133 (P≤0.0001) and POU5F1(OCT-4) (P≤0.05), respectively. The findings of this research suggest that mechanical stiffness promoted EMT and cancer stemness in glioblastoma cells, underlining the influence of microenvironment stiffness in promoting invasion capabilities in glioblastoma cells.