CNSC-70. THE ROLE OF MAP4K4 IN TUMOR CELL INVASION OBSERVED IN A 3D IN VITRO MODEL OF PEDIATRIC GLIOBLASTOMA

Abstract Several CNS tumors such as pediatric glioblastoma (pGBM), diffuse midline glioma (DMG), and medulloblastoma (MB) are characterized by local invasive spread and dissemination of tumor cells into surrounding brain tissue. As identified by our group in a genome-wide CRISPR screen and several others, MAP4K4 is a mediator of cell invasion in several cancers, including glioblastoma and medulloblastoma. Recently, we validated the pro-invasive role of MAP4K4 in a panel of pGBM cell lines. To further validate this finding in the context of pGBM cellular invasion, we performed 3D Matrigel timelapse invasion assays. We performed single- and multi-spheroid encapsulation to track spheroid invasion and cell-to-cell interactions between multiple spheroids. Tumor spheroids were cultured in a round-bottom, non-adherent well plate encapsulated in a Matrigel matrix and treated with a MAP4K4 small molecule inhibitor (PF06260933 dihydrochloride) or had a MAP4K4 knockout performed with Cas9 editing. Cell invasion was recorded over 72 hours and three-dimensional structures were stained for actin and imaged 10 days post-initial seeding. We show that MAP4K4 KO (p ~ 0.00033), small-molecule inhibition in the presence of a TGFB receptor agonist (SRI-011381), (p ~ 0.001) and with drug alone (p ~ 0.013) decreased spheroid invasion and prevented single-cell invasion of pGBM cells (SF188) into the matrix. More surprisingly, abrogation of MAP4K4 prevented individual spheroids in a multi-spheroid co-culture, from forming integrated glioma networks through the formation of tube-like structures in the matrix. Based on our initial findings, it appears that MAP4K4 is necessary for single-cell invasion into a three-dimensional matrix. It also appears that MAP4K4 is required for cell-to-cell connections to form between spheroids in the 3D environment to allow for the formation of higher-ordered and spatially organized structures. Future directions include studying how MAP4K4 allows for the formation of glioma tumor networks in pediatric GBM.