STEM-20. MULTI-OMICS OF HUMAN MEDULLOBLASTOMAS AND GENETIC LINEAGE TRACING IN VIVO INDICATE CELLULAR PLASTICITY

Abstract Recent single-cell studies implicate the glutamatergic lineage of the rhombic lip (RL) as the origin of group (G) 3/4 medulloblastomas (MBs) and a stalled differentiation program as underlying malignant transformation. While the cellular compositions of G3/G4 MBs are consistent with a truncated neurodevelopmental hierarchy, it is unknown whether G3/G4 MBs exhibit cellular plasticity. We profiled tumors from 38 patients who underwent surgery for MB at UCSF via single-nucleus RNA sequencing (snRNA-seq). We developed in vivo genetic lineage-tracing systems with a single-cell readout and applied them to patient-derived G3 MB xenografts. High-complexity lentiviral barcode libraries were transduced into D283 and D425 patient-derived cell lines. Barcoded cells were injected into the brains of immunocompromised mice. Both preimplantation barcoded cultures and mature tumors were profiled by single-cell RNA-sequencing (scRNA-seq). This captured both endogenous RNA and barcode transcripts. RL neuro-developmental cell-type labels were transferred from public single-cell data from fetal cerebellum to single-cell data from our patient samples and xenograft models. Remarkably, xenografts produced similar cell types, in similar proportions to those found in human MBs. Moreover, the distribution of cell types found in the xenografts was conserved at the level of individual clones, indicating cell-intrinsic regulation of tumor composition. We developed approaches to use barcode and expressed point-mutation data to reconstruct intra-clone genetic phylogenies. We then used the transcriptional phenotypes of the observed cells to infer ancestral phenotypes for each clone’s phylogeny. We compared clade to sub-clade differences in cell type against the neural-developmental hierarchy found in the fetal RL. We observed significant rates of dedifferentiation events from unipolar brush-like cells to RL-progenitor phenotypes. These studies indicate that G3 MB cellular composition is homeostatic and conserved at the level of individual clones. Yet, these tumors exhibit plasticity, as has been observed in other brain cancers such as glioblastoma.