Significant differences in messenger RNA (mRNA) profiles (T stroma versus matched NT fibrous tissue) were evaluated at a protein level using a validating set which consisted of 40 FFPE ICC equally distributed in cases with or without recurrence (Table 1). IHC was done using TMA to reduce experimental noise. Finally, the prognostic value of selected proteins was estimated using clinical records and patient follow-up reports (Fig. 1). LCM was performed to isolate RNA from the stromal compartment of freshly frozen ICC tissues. For each ICC tumor, fibrous tissue from portal areas within the surrounding nontumor

liver was also isolated and used as a reference (Fig. 2A). Following hybridization on microarrays, the statistical Alvelestat purchase analysis focused on identifying genes for which expression was significantly altered selleck chemicals llc in the stroma of ICC. As shown in Fig. 2B, applying stringent criteria (P < 0.001 and fold change FC > 2) resulted in the identification of 1,073 nonredundant genes differentially expressed between the NT fibrous tissue and the T stroma, demonstrating that the stroma of

ICC displayed substantial genomic changes (Supporting Table 3). Thirty-one percent of the stromal signature included genes that were up-regulated relative to NT fibrous tissue. Supporting the gene selection, a hierarchical clustering analysis based on this signature efficiently discriminated the NT fibrous tissue from the T stroma (Fig. 2C). Fully supporting this observation, integrative genomics demonstrated that the LCM-derived stroma signature also discriminated cholangiocarcinoma tumors from surrounding NT livers in an independent

genomic dataset (GSE26566) established from whole MCE tissue sections (Fig. 2D). Further validating the enrichment of the stromal compartment by LCM, both up- and down-regulated genes were categorized into functional modules associated with the extracellular region, including ECM (Supporting Table 4F). Down-regulated genes were enriched in gene sets known to be down-regulated in liver cancers, including genes involved in metabolism (Supporting Table 4, Supporting Fig. 2). Up-regulated genes were related to entry into the cell cycle, ECM organization, and cell signaling pathways, namely, p38, p53, and TGFβ. Accordingly, the stroma signature of ICC coincided with a discrete enrichment of transcription factor (TF)-associated gene sets (Supporting Table 4D). Indeed, up-regulated genes were known to be transcriptionally regulated by E2F(s) and SMAD(s), two families of TF involved notably in the regulation of cell cycle and TGFβ signaling pathway, respectively. Down-regulated genes were regulated by hepatocyte nuclear factors known to control the expression of numerous metabolic genes (Supporting Table 4, Supporting Fig. 2). These results were confirmed by an unsupervised GSEA (Supporting Fig. 2).