Molecular Profiling of Pancreatic Cancer Stroma
JR Brody, J Kline, CJ Yeo, S Peiper, P McCue, AK Witkiewicz. Thomas Jefferson University, Philadelphia, PA
Background: Treatment strategies targeting epithelial cells in pancreatic ductal adenocarcinoma (PDA) have yielded disappointing results. Since the bulk of PDA tumor volume consists of stroma, investigating the stromal cells may elucidate pathways responsible for PDA aggressiveness and lead to the identification of new treatment targets. A characteristic of prior studies of gene expression in the stromal compartment of human PDA is that these studies have focused on a limited number of genes identified by serial analysis of gene expression as highly expressed in invasive pancreatic cancer tissues but not in pancreatic cancer cell lines. The goal of our study was to perform global gene expression analysis of PDA associated stroma, obtained thorough laser microdissection (LCM), from frozen PDA tissue.
Design: Qiagen RNAeasy Micro kit (Qiagen, Inc., Valencia, CA). The purity of the obtained stromal RNAs was confirmed by quantitative RT-PCR for epithelial marker keratin 18. RNA amplification and labeling was performed by the WT-Ovation Pico RNA amplification system (NuGen Technologies, Inc.). Expression analysis was carried out using Affymetrix gene chip human Gene 1.0 ST array (Affymetrix, Santa Clara, CA). Background correction and normalization was done using Robust Multichip Average (RMA) with Genespring V 10.0 software (Agilent, Palo Alto, CA, USA). Selected genes showing > 3 fold difference (p<0.001) were validated by Q RT-PCR and immunohistochemistry (IHC).
Results: showed significant changes in a subset of genes with 49 genes showing > 5 and 260 genes >3 fold difference when compared to normal cultured fibroblasts (p<0.01) Ingenuity Pathway Analysis showed that differentially expressed genes were involved in “Cell Death, Immunological Response and Post-Translational Modification” (score 53), “Cell signaling, actin cytoskeleton remodeling/ signaling and cell adhesion” (score 43) and 'Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry (score 31). Three up regulated (UIMC1, SPARCL1, NCOR2) and three downreagulated genes (CAV-1, LDHB, MMP1) were validated by QRT-PCR using amplified and unamplified RNA. Additionally we demonstrated expression of SPARCL1 in stroma and lack of Caveolin-1 expression by IHC.
Conclusions: Our preliminary results show that a number of genes and pathways are specifically altered in PDA associated stroma and may lead into greater insight into pathophysiology of PDA.
Category: Liver & Pancreas
Tuesday, March 23, 2010 1:45 PM
Platform Session: Section C, Tuesday Afternoon