Freshly frozen and formalin-fixed paraffin-embedded (FFPE) tissues were provided by the biobank of the university hospital. Histological
and clinical features including Selleckchem BMS-777607 those observed upon follow-up examinations were obtained from hospital charts. LCM was performed using the Arcturus Veritas Microdissection system (Applied Biosystems, Carlsbad, CA). From frozen tissues, serial sections of 10 μm were prepared using a Leica 3050 S cryostat (Leica Microsystems, Wetzlar, Germany) and mounted onto a PEN membrane glass slide (Applied Biosystems). Tissue sections were dehydrated by successive immersions (30 seconds, twice) in 70%, 90%, and 100% ethanol solutions. Enzymatic activity was locked by the immersion in a xylene solution (1 minute, twice) before performing LCM. Total RNA was purified using an Arcturus Picopure RNA isolation kit (Applied Biosystems). Genome-wide expression profiling was performed using human SurePrint G3 8x60K pangenomic
PLX-4720 purchase microarrays (Agilent Technologies, Santa Clara, CA) as described.[15, 17] Fifty nanograms of total RNA was purified from LCM tissues and amplified with a low-input QuickAmp labeling kit (Agilent Technologies). The amplification yield was 1.8 ± 0.7 μg complementary DNA (cRNA), and the specific activity was 5.8 ± 3.4 pmol Cy3 per μg cRNA. Gene expression data were analyzed using Feature Extraction and GeneSpring softwares (Agilent Technologies) and further analyzed using R-based ArrayTools. Microarray data are publicly available from the gene expression omnibus (GEO) database (www.ncbi.nlm.nih.gov/geo; GSE45001). Briefly, microarray data were normalized using the quantile normalization algorithm, and differentially expressed genes were identified by a two-sample univariate t test and a random variance model as described.[18] Permutation P values for significant genes were computed based on 10,000 random permutations. Clustering analysis was done using Cluster 3.0 and TreeView 1.6 with uncentered correlation and average linkage options. Enrichment for specific biological
functions or Aldol condensation canonical pathways was evaluated as described.[19, 20] Gene set enrichment analysis (GSEA) was performed using the Java-tool developed at the Broad Institute (Cambridge, MA).[21] Integration of genomic data was performed as described[22] using publicly available gene expression datasets downloaded from GEO. ChIP enrichment analysis was performed using the ChEA algorithm developed by Lachmann et al.[23] TMAs were designed with the TMADesigner software. FFPE tissues were arrayed using a Minicore 3 tissue Arrayer (Excilone, VICQ, France). After hematoxylin-eosin staining, three representative areas of stroma from each ICC tumor (T) and of fibrous tissue from portal tracts areas in the surrounding nontumor (NT) liver were selected by an experienced pathologist (B.T.).