Platelet derived growth factor receptor alpha (PDGFRA)-positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. the brain, Olig2-positive tumor cells are most comparable to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have comparable expression profiles in human tumors, and were significantly enriched in genomic deletions, suggesting an anti-proliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings demonstrate that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies. cell-specific translational profiling (17, 18), we identified all mRNAs specifically enriched in OPCs in normal mouse brain, including those likely to be important for proliferation, quiescence, and differentiation. We then contrasted this to the cell-specific translational profile of Olig2-positive cells in the mouse model of a proneural glioma, in which OPC-like cells are committed to proliferation at the expense of differentiation or quiescence. This permitted identification of candidate pathways that may serve as targets for promoting differentiation and quiescence in OPCs in mice. Examination of The Cancer Genome Atlas (TCGA) expression profiles of human gliomas established that analogous pathways are similarly regulated in human proneural GBM, suggesting their conservation as targets. Also consistent with an anti-proliferative role, many of these targets show deletions in human GBM. From this TMC 278 combined human and mouse screening, we have identified several candidate pathways for promoting quiescence and TMC 278 differentiation, which may serve as targets for complementary treatments. Material and Methods Full materials and methods are available online. All protocols involving animals were approved by the Rockefeller University and Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee. New mouse lines BACs made up of genes PDGFRA (RP23-55P22), Cnp1 (RP23-78L12), and Take25 (RP23-290A18) were modified as described (17) to insert an EGFP-L10a fusion protein into the relevant translation start site. Histology Anatomy was performed as described (17, 19). For immunofluorescence samples were incubated with the cell-specific antibodies and quantification was performed on 40 cortical fields imaged with confocal microscopy. Profiling Tumor Model Tumors were generated as described (9). Cells producing RCAS-h PDGF-B or RCAS-Cre virus were injected into the progeny of ink4a/arf?/? mice expressing tv-a receptor for RCAS under the Nes promoter, crossed to Olig2-Egfp::L10a mice. TRAP and microarray hybridizations were performed as described (17, 18), except tumors were processed individually. Microarray Analysis Microarray data were analyzed with Bionconductor module of the R statistical package, normalized as described (17, 20, 21), and deposited at GEO (“type”:”entrez-geo”,”attrs”:”text”:”GSE30626″,”term_id”:”30626″GSE30626) (70). To identify messages specific to each cell type Specificity Indices (pSI) were calculated as described (18). Transcripts with pSI<.05 Rabbit Polyclonal to MRPL54 were selected for further analysis. Heatmaps and hierarchical clustering were performed in R. All color-coded scatterplots show only top 50 transcripts for each cell type, but all statistics were performed on full lists (Supplemental Table 1). Differentiation or Quiescence (DorQ) candidates were selected as those transcripts from the OPC list which were two fold higher in the average of the normal cortex than the average of all 12 tumor samples. Statistical comparisons were conducted with LIMMA module of Bioconductor. For cross species comparisons, human and mouse homologues were mapped by Gene Symbol. For DorQ candidates, mapping was confirmed by Blating mouse protein to human genome (UCSC). Human microarray data were downloaded from TCGA and normalized as described (4). Gene Ontology Analysis For each cell type, all gene symbols for messages with a pSI <.05 were analyzed with BINGO (22). 'Biological Process' GO categories were evaluated to identify those with p <.01 using the hypergeometric test and Benjamini-Hochberg correction. Neurosphere TMC 278 Cell Culture Primary neurosphere cultures from tumors and wild type mice were generated as TMC 278 described (23). Neurospheres were produced in 20mg/ml EGF and 10mg/ml bFGF, and uridine 5-diphosphate sodium salt (UDP) (10C50uM), UDP-glucose (100uM), and leukotriene Deb4 (LTD4) (100 nM) were added on a daily basis. All data are average of cultures from four impartial mouse tumors, counted in triplicate in wells of 100C1000 spheres. Counts were normalized within each tumor to the number of spheres in 0 UDP condition. Results We recently developed the TRAP strategy, which allows profiling of all mRNAs bound to ribosomes in defined cell populations. This strategy entails using bacterial artificial chromosome (BAC) transgenesis to express EGFP fused to the ribosomal protein L10a under the control of a driver gene specific to certain cell types in the brain. Thus, the cells of interest contain ribosomes with an EGFP tag enabling affinity purification of all ribosome-associated mRNA. We have generated and characterized bacTRAP mouse lines for a variety of cell types, including the Olig2-positive oligodendroglia (17). Because Olig2 is frequently.