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Background: An interaction between cancer cells and surrounded cancer associated fibroblasts (CAFs) plays an important role in the progress of cancer. Pancreatic cancer is characterized by a growth of abundant fibrous or connective tissue, called "desmoplasia", and hypovascular environment inducing hypoxic and undernutritional condition. In pancreatic cancer, CD10+ myofibroblast-like activated Pancreatic Stellate Cells (PSCs) enhance the progression of pancreatic cancer by secreting high levels of MMP3 (Ikenaga, et al. Gastroenterology 2010). Podoplanin, usually used as a lymphatic vessels marker (D2-40), had been described as a predictor of prognosis in various types of cancer when it was expressed in involved stromal fibroblasts.
Methods: We investigated Podoplanin expression in fibroblasts involved in pancreatic cancerous tissue using immunohistochemistry (IHC). We established primary cultured fibroblasts as CAFs of fresh pancreatic adenocarcinoma tissue by out-growth method, and analyzed Podoplanin expression of CAFs using qRT-PCR and flow cytometry. We sorted CAFs by Magnetic Activated Cell Sorting (MACS) according to the expression of Podoplanin, and compared Podoplanin+ CAFs with Podoplanin- CAFs by migration assay and invasion assay in co-culture with pancreatic cancer cell lines. In addition, we performed qRT-PCR to elucidate differentiation between them. We also compared Podoplanin high-expressing CAFs with Podoplanin knocked down CAFs by siRNA to clarify the own function of Podoplanin. Next, we investigated the Podoplanin inducible condition by a time course of expression analysis using total starvation medium (EBSS), and DMEM added by recombinant growth factors or several percentages of FBS.
Results: IHC showed that the frequency of Podoplanin expression (>30%) in fibroblasts was associated with lymphatic invasion, venous invasion, tumor size (>3cm), histological grade, pT, and a shorter survival time (P<0.001). Podoplanin expression in cultured CAFs showed heterogeneity (ranging from 0 to 95%) by flow cytometry. Podoplanin+ CAFs showed significantly high expression of CD10 and MMP3 compared with Podoplanin- CAFs, and co-culture experiments using sorted CAFs showed that Podoplanin+ CAFs enhanced the ability of cancer cells in migration and invasion compared with Podoplanin- CAFs (P<0.05), while knock down of Podoplanin showed no effect on migration and invasion assay. Podoplanin expression in CAFs was up-regulated in the condition of starvation and lower concentration of growth factors or FBS.
Conclusion: Despite Podoplanin in CAFs had no function of own to affect cancer cells, Podoplanin+ CAFs enhanced the progression of pancreatic cancer cells by co-expression of CD10 and MMP3. Podoplanin expression was up-regulated in the condition of starvation and lower concentration of growth factors or FBS.