373 Exploring Principles of the Interplay Between Cancer Stem Cells and Cancer Activated Fibroblasts Towards Advanced Therapies Against Glioblastoma

Abstract

INTRODUCTION: The extremely poor response to therapy, resulting in relapse of patients with GBM, is attributed to the existence of Cancer Stem Cells (CSCs). Lately we have come to understanding that regulation of CSCs comes also from the tumour microenvironment (TME), making the attempts to therapeutically target CSCs solely, largely ineffective. METHODS: Using Fluorescent Activated Cell Sorting (FACS) of six individual patient-derived GBM samples, we obtained fraction of CAFs based on their FAP+/COL1A1+/CD90+/CD31-/EPCAM- marker signature and the remaining fraction of the sample was cultured to enrich for CSCs. The functional tumour aggressiveness assays as well as gene expression analysis were performed using the optimized GBM organoid system with integrated fluorescent, CAF and/or normal fibroblast (nF) component to mimic the in vivo setting. RESULTS: 3D live-cell analysis of the organoid growth time-course demonstrated nF recruitment and their activation into CAFs by glioma cells in the co-culture setting which was confirmed by increased CAF-specific gene and protein expression levels compared to control with p < 0.001 and p < 0.0001 at 25% and 50% of co-cultured nFs, respectively. The recruited nFs enhanced proliferation (p < 0.05) at day 9 and CSC marker expression (p < 0.01) in GBM cells when compared to appropriate controls. We found that CAFs control changes of the chemical composition of TME of glioblastoma with upregulation of hyaluronan and Vinculin (p < 0.05; both) and post-treatment changes in invasion, in comparison to CAF-depleted culture controls. CONCLUSIONS: Our data demonstrates that TME is an essential player in the GBM resistance to therapy and tumour recurrence and that normal fibroblast component is a potential source of CAFs in brain tumours warranting further investigation.