POOLED IN VIVO ASSAY TO CHARACTERIZE GLIOBLASTOMA INVASION AT THE SINGLE-CELL RESOLUTION

Tumor cell invasion is the main characteristic of glioblastoma and one of the obstacles to successful treatments. Nonetheless, molecular features of invading glioblastoma cells remain elusive and poorly characterized, such as their gene expression states and microenvironmental influences. Recently, we described cell state heterogeneity in glioblastoma by single-cell RNA-sequencing (scRNA-seq) and uncovered the contribution of immune cells to the heterogeneity. However, it is unclear whether the cell states observed in clinical specimens are implicated in invading glioblastoma cells in the surgically inaccessible brain parenchyma. It is also unclear whether the cell states are correlated to the non-immune microenvironment. As a tool for probing these questions, we developed an in vivo approach with mixtures of human glioblastoma spheroid lines for mouse intracranial xenografts. We validated this with 20 patient-derived models and identified, by scRNA-seq, in vivo models capable of infiltrating along the periphery of either neurons or blood vessels. We profiled and characterized programs, states, and clonality of invading glioblastoma cells by comparing cells from the contralateral invasive edge to their counterparts at the original bulk tumor. This comparative analysis showed evidence of a bi-directional transitioning model for cells residing in different spatial niches. Importantly, presence of an OPC-like state was significantly associated with invasion phenotype regardless of the infiltration routes. Our results suggest that cellular plasticity may be a prerequisite for the invasive phenotypes, urging further assessment to identify critical regulators of the cell states. In conclusion, this work describes pooled in vivo assays for the rapid interrogation of patient-derived xenografts, coupling the gene expression phenotypes with cellular functions such as invasion.