Engineered In Vitro Tumor Model Recapitulates Molecular Signatures of Invasion in Glioblastoma

Glioblastoma stem cells (GSCs) play an important rolein the invasivenature of glioblastoma (GBM); yet, the mechanisms driving this behaviorare poorly understood. To recapitulate tumor invasion in vitro, wedeveloped a GBM tumor-mimetic hydrogel using extracellular matrixcomponents upregulated in patients. We show that our hydrogel facilitatesthe infiltration of a subset of patient-derived GSCs, differentiatingsamples based on phenotypic invasion. Invasive GSCs are enriched forinjury-responsive pathways while noninvasive GSCs are enriched fordevelopmental pathways, reflecting established GSC stratifications.Using small molecule inhibitors, we demonstrate that the suppressionof matrix metalloprotease and rho-associated protein kinase processesresults in a significant reduction of cell invasion into the hydrogel,reflecting mesenchymal- and amoeboid-dependent mechanisms. Similarreduction in cell invasion was observed by siRNA knockdown of ITGB1and FAK focal adhesion pathways. We elucidate the transcriptomic profileof cells invading in the hydrogel by performing bulk RNA sequencingof cells cultured in the hydrogel and compare these to cells culturedin conventional tissue culture polystyrene (TCP). In our 3D hydrogelcultures, invasion-related molecular signatures along with proliferationand injury response pathways are upregulated while development processesare downregulated compared to culture on 2D TCP. With this validatedin vitro model, we establish a valuable tool to find therapeutic interventionstrategies against cellular invasion in glioblastoma.