Similarly, our lineage tracing analysis indicated that we can successfully examine TIC cell division at an individual cell level. were crossed with Sox2CEGFP mice, and tumors were initiated that contained a subpopulation of Sox2CEGFP-high cells enriched for tumor-initiating cell properties such as self-renewal, multilineage differentiation potential, and perivascular localization. Results Following implantation into recipient mice, Sox2CEGFP-high cells generated tumors containing Sox2CEGFP-high and Sox2CEGFP-low cells. Kinomic analysis of Sox2CEGFP-high cells revealed activation of known glioma signaling pathways that are strongly correlated with patient survival including platelet-derived growth factor receptor beta, phosphoinositide-3 kinase, and vascular endothelial growth factor. Our functional analysis identified active feline sarcoma (Fes) signaling in Sox2CEGFP-high cells. Fes negatively correlated with glioma patient survival and was coexpressed with Sox2-positive cells in glioma xenografts and primary Satraplatin patient-derived tissue. Conclusions Our RCAS-tva/Sox2-EGFP model will empower closer examination of cellular heterogeneity and will be useful for identifying novel glioma pathways as well as testing preclinical treatment efficacy. values are detailed in the text and figure legends. Patient survival analysis was performed using the NCI REMBRANDT database, where survival was calculated by log-rank analysis. Results Mouse models of glioma have been limited in highlighting distinct cell populations within the tumor. Because the GFAP-tva system can reproducibly generate de novo Rabbit Polyclonal to MRPS30 gliomas in mice and Sox2 is characterized as a TIC marker important for glioma growth and tumorigenic potential, we sought to generate a system to model intratumoral heterogeneity using Sox2-EGFP in GFAP-tva mice. We crossed GFAPCtva-positive and Sox2CEGFP-positive mice (Fig.?1A) and confirmed the presence Satraplatin of both tva and EGFP in Satraplatin resulting progeny (Fig.?1B). GFAP-tva and Sox2-EGFP could be distinguished from wild-type GFAP and Sox2 by differences in PCR amplicon size when separated on an agarose gel. Tumors were initiated in GFAP-tva/Sox2-EGFP mice by orthotopic injection with chicken fibroblast (DF1) cells containing RCAS-PDGF virus. Open in a separate window Fig.?1. PDGF overexpression induces tumor formation in GFAPCtva-positive/Sox2CEGFP-positive mice. (A) Schematic of breeding strategy yielding GFAPCtva-positive/Sox2CEGFP-positive mice bearing gliomas initiated via PDGF overexpression. The Holland GFAPCtva-positive and RCAS-PDGF model system was modified to include a transgene in which one copy of the Sox2 allele was replaced with EGFP. (B) GFAPCtva-positive/Sox2CEGFP-positive mice were genotyped to confirm the presence of EGFP and tva. Sox2CEGFP-high glioma cells isolated from mice bearing neurological symptoms were orthotopically injected into immunocompromised mice to confirm their tumorigenic capacity and generate allografts for further use (Fig.?2A and B). Sox2-EGFP cells were also confirmed to express endogenous Sox2 both in vivo and in vitro (Supplementary material, Fig.?1). Heterogeneity in resulting allografts was confirmed by fluorescence-activated cell sorting analysis and fluorescent microscopy, which showed 22%C35% Sox2CEGFP-positive glioma cells (Fig.?2CCE), which is within the range of Sox2-positive cells in xenografts27 as well as in GFAP-tva mice.28 Additionally, this heterogeneity appeared to be intrinsic to the tumor, as we did not observe Sox2-EGFP cells in the normal brain parenchyma outside of the previously reported neurogenic niche locations.25 To determine if Sox2CEGFP-positive cells may be supported by specific tumor microenvironments, we determined whether the EGFP-positive population was found adjacent to blood vessels in a perivascular niche.12 We stained tumor sections for the endothelial cell marker CD31 and examined Sox2-EGFP localization by fluorescent microscopy. Sox2CEGFP-high glioma cells preferentially associated with the vasculature compared with Sox2CEGFP-low cells, and 20% of Sox2CEGFP-positive cells were located <10 m from a blood vessel (Fig.?2F and G). These results indicate that PDGF overexpression leads the formation of gliomas in GFAPCtva-positive/Sox2CEGFP-positive mice and that Sox2CEGFP-high cells give rise to Sox2CEGFP-high and Sox2CEGFP-low tumor cells. Additionally, these data indicate that Sox2CEGFP-high cells preferentially localize adjacent to the vasculature. Open in a separate window Fig.?2. Sox2CEGFP-high glioma cells give rise to heterogeneous tumors and display perivascular localization. (A) Schematic demonstrating propagation of Sox2CEGFP-high glioma cells through intracranial injection into athymic nude mice to generate an allograft. (B) Representative histology of an allografted tumor showed by hematoxylin and eosin staining. (C) Analysis of tumor cells immediately after dissociation demonstrated that Sox2CEGFP-positive glioma cells make up a fraction of the bulk tumor. (D) Representative immunofluorescent images of allografts resulting from Sox2CEGFP-positive glioma cells. Images demonstrate intratumoral heterogeneity with respect to Sox2-EGFP expression. (E) Quantification of the percentage.