ID #1034 Tumeroid models for NF1 low-grade glioma: validation and comparison to primary tumor using scRNAseq

Abstract

About 10-15% of individuals with Neurofibromatosis Type 1 develop a low-grade glioma (LGG) within the optic pathway, and an additional 3-5% of individuals develop one outside of the optic pathway. In recent years molecular analysis has greatly enhanced our understanding of what drives these NF1 LGG, however, to date in vitro models do not exist and in vivo models are scarce. Here, we present the first in vitro tumor derived spheroid cultures (tumeroids) of 3 NF1 associated low grade gliomas grown for 4 weeks. These tumeroids can be passaged and banked. To determine how accurately the NF1 LGG tumeroid cultures reflect human disease, and how much and how quickly they drift from the primary tumors, we performed single cell RNAseq (scRNAseq) at different timepoints (primary tumor and 1, 2 and 4 weeks in culture). Observations were confirmed with immunofluorescence staining. As expected, the few CD3 T-cells present in the primary tumor are lost by day 7 (IF) and not detected on scRNAseq; macrophages/microglia remain present for at least 14 days, but are largely depleted by week 4. Additionally, we observe a shift from more homeostatic microglia in the primary tumor to CD163 positive tumor associated macrophages/microglia (TAM) in vitro. Neoplastic clusters uniquely associated with the primary tumor had clear upregulation of gene signatures associated with neuronal communication. Our analysis also shows that primary NF1 LGG rely on the metabolism of Fatty Acids (FA) for their energy, while in vitro glycolysis is the primary energy source. Additionally, instead of metabolizing FA, both neoplastic and immune cells switch to FA synthesis in vitro. Finally, we observe a slightly increased proliferative index in vitro versus in vivo, both in the neoplastic and immune cells. This is the first description of NF1 derived tumeroid models, that open opportunities for in vitro drug sensitivity and other studies.