The role of the local microenvironment during early astrocytoma development remains elusive. We previously showed that Rb and Pten inactivation and Kras activation (TRP) transforms Gfap + astrocytes and induces low-grade astrocytoma tumorigenesis throughout the adult brain. To confirm that astrocytes are the cell of origin, we targeted them using an alternative astrocyte-specific promoter, Glast. In the absence of oncogenic mutations, genetic lineage tracing with Glast-CreER; floxed TdTomato

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... e produced recombination in 8-38% of Gfap/Blbp + astrocytes in the cortex, diencephalon, brainstem, and olfactory bulb. EdU labeling showed ,0.04% proliferated at 7 days. Astrocyte proliferation (EdU + ) increased 8-29-fold 3 weeks and TdTomato + cells increased 3-7-fold by 8 weeks after TRP transformation. Glast;TdTomato + astrocytes were rare in the brainstem and their transformation produced minimal tumorigenesis. In contrast, hGFAP-CreER uniformly induced recombination in 50% of astrocytes in all regions, including the brainstem. Whereas TdTomato + astrocytes increased 6-11-fold in hGFAP-TRP mice after 8 weeks, Glast-targeted transformation was 1-4 fold lower. Differences in growth rates were most pronounced in the brainstem (P,0.001), but olfactory bulb growth rates were similar (P.0.05). Both CreER driver and brain region significantly affected astrocyte growth rate (ANOVA P , 0.0003). In both models, transformed astrocytes maintained Gfap/Blbp expression, gained expression of the stem cell marker Nestin, and formed increasingly dense perineuronal satellites over time. Ki-67 labeling showed clonal expansion, as hypercellular foci with 11-fold higher proliferation relative to less-cellular areas of tumor developed by 16 weeks. Glast-, but not hGFAP-driven tumors contained dividing, but untransformed (TdTomato;T121 2 ) BLBP + astrocytes, IBA1 + microglia, and PDGFRa + oligodendrocyte progenitors (OPC). Proliferating microglia (6-0%) and OPC (18-5%) decreased over time, while dividing, untransformed astrocytes increased (57-85%). These findings support the notion that regional microenvironment and astrocyte heterogeneity contribute to astrocytoma development. The Glast-TRP model may be useful in dissecting the role of regional microenvironment during astrocytoma tumorigenesis.