ANGI-06. CRISPRI SCREEN USING 3D BIOMIMETICS TO IDENTIFY NOVEL MARKERS OF GLIOBLASTOMA INVASION

Abstract

Abstract Despite the aggressive treatment regimen, patients with GBM have a very poor prognosis, with a median survival of less than 2 years. The main reason behind this poor survival is the infiltration of tumor cells into the surrounding normal tissue. These infiltrative cells escape both surgical resection and radiation therapy, leading to inevitable tumor recurrence. Current approaches have failed to recognize that infiltrating GBM cells extending beyond the tumor edge have evolved a unique adaptive cellular machinery due to local stressors in their microenvironment. Unfortunately, these cells at the invasive tumor front are often not the ones sampled in studies analyzing banked tumor tissue, Second, most studies of invasion have used two-dimensional (2D) culture systems coated with a thin layer of extracellular matrix proteins, which fail to capture the dimensionality, mechanics, and heterogeneity of GBM invasion. We addressed these limitations by using an advanced CRISPRi druggable genome screen to identify novel druggable markers of GBM invasion in a 3D engineered biomimetic model comprised of Hyaluronic Acid (HA) based matrix. To better understand druggable pathways underlying GBM invasion, we performed a large-scale CRISPRi screen with a sgRNA library that targets the druggable human genome (2,550 genes). In the primary screen, sgRNAs targeting 12 genes were enriched in the non-invasive fraction. Out of these 12 genes, 8 genes are associated with cellular metabolism or chromosome assembly. We then validated these 8 genes using small molecule inhibitors in our 3D-gel sphere cultures, from which we identified four druggable targets, SOD-1 and NDUFV2 associated with mitochondrial metabolism and AURKB and ACP1 related to centromere assembly and chromosome segregation. In follow-up to these novel findings linking mitochondrial metabolism and centromere assembly to GBM invasion, our next steps are to identify the mechanism linking these genes to GBM cell invasion followed by validation in vivo.