the co-purchase Vesnarinone culture model. To identify targets and mediators of the reciprocal interactions between tumor cells and their microenvironment, we performed gene expression profiling with Illumina BeadArray microarrays using RNA isolated from the mixed cell population after 48 hours of culture and RNA isolated from parallel monocultures of HBMECs and U87 cells. Because the RNA isolated from the co-cultures was derived from a mixed population of cells, measured changes in gene expression could occur as a consequence of changes in the relative numbers of HBMECs and U87 cells over the 48-hour co-culture time period, and/or as a result of changes in gene expression induced through functional interactions between the two cell types. As we were interested only in the latter, it became essential to develop computational methods to distinguish between these sources of change. To accomplish this, we developed an approach that uses gene expression data to precisely determine the ratio of HBMEC and U87 cells within a mixed culture. Previously published global expression profiling of cell-cell interactions has found that less than PDE7B in the GBM Perivascular Niche 10% of genes exhibit statistically significant differential PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674121 expression upon co-culture. Thus, we anticipated similar results and assumed that the expression levels of most genes in either cell type would remain unchanged upon co-culture, and that only a small percentage of genes would be affected by cell-cell interactions. Using expression profiling data obtained separately from HBMEC and U87 monocultures, we created one thousand computationally mixed datasets for the two cell types, from a ratio of 0.1% HBMEC/99.9% PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19672212 U87 to a ratio of 99.9% HBMEC/0.1% U87 in 0.1% increments. We then used these computed profiles to determine the precise ratio of GBM and HBMECs in the coculture at the time of mRNA isolation. To do so, we calculated the Pearson correlation coefficient between the experimentally measured co-culture expression data and the full series of computationally generated expression profiles. We concluded that the synthetic profile with the highest correlation to the measured coculture profile provided the closest approximation to the actual ratio of U87 cells and HBMECs in the co-culture. This calculation was performed for three independent sets of coculture data. In each case, normalization of the co-culture profile to the synthetic profile with the highest correlation identified those transcripts whose level of expression differed from the norm. These transcripts represented the candidate genes whose expression was either increased or decreased through functional interactions between HBMECs and U87 cells. In this manner, 45 genes with at least a two-fold change in expression were identified as significantly upregulated or downregulated as the result of functional interactions between HBMECs and U87 cells. Consistent with known effects of GBM cells on ECs and our results showing an increase in in vitro angiogenesis upon co-culture, this list of genes contained several regulators of angiogenesis such as Thrombospondin-1 and CXCL1. Also notable is the upregulation of WNT signaling genes CTHRC1 and Frizzled-9. WNT signaling is known to be involved in maintaining stemness and cell proliferation. and U87 cells under basal conditions. While CXCL6 expression in U87 cells was unaffected by any of the CM preparations, it was significantly increased in HBMECs in response to either U87 or co-culture C