Cancer associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic adenocarcinomas (PDACs). chemotherapy for overcoming PDAC chemoresistance. reduced Snail expression in co-cultured epithelial cancer cells and reduced survival of drug-resistant cancer cells, suggesting that blocking exosome communication may be a promising new therapeutic strategy for patients receiving gemcitabine-based treatment regimens. RESULTS Pancreatic Fibroblasts are Innately Chemoresistant We first compared 301836-41-9 the innate drug resistance of cancer-associated fibroblast (CAF) cell lines created from patient-derived tumor samples with that of epithelial cancer cell lines. Patient-derived fibroblasts were grown out of tumor samples obtained from patients who had undergone surgical resection. The CAFs displayed an elongated, mesenchymal morphology, and stained positively for fibroblast markers vimentin and -SMA (17) (Figure 1a). Sequencing revealed no mutation, indicating that these CAF cell lines were truly of fibroblast origin (Supplementary Figure S1). CAFs and normal fibroblasts had greater survival rates than chemoresistant epithelial cells (PANC1) and chemosensitive epithelial cells (L3.6) when treated with the same dosage of the chemotherapeutic agent, gemcitabine (GEM) (Figure 1b). Having shown that CAFs are resistant to GEM, we next assessed if the increased survival of CAFs exposed to GEM could be a result of CAFs undergoing 301836-41-9 senescence and not incorporating the drug. Therefore, we Sele analyzed cell proliferation of GEM-treated CAFs and epithelial cells. The most chemoresistant CAF cell line, CAF1, also retained the most proliferation during GEM treatment, while the second leading resistant CAF cell line, CAF2, showed dramatically decreased proliferation (Figure 1c). To further elucidate the role of proliferation on chemoresistance, we compared the survival 301836-41-9 rate of CAFs and epithelial cells with similar proliferation rates (CAF2 and PANC1 cell lines, respectively). Although CAF2 and PANC1 cells both demonstrate a relatively low proliferation rate following exposure to GEM, CAF2 cells still showed more than a 2-fold higher cell survival rate compared to PANC1 cells following GEM treatment (Figure 1d). Taken together, these data demonstrate that fibroblasts have an innate resistance to GEM instead of a growth-dependent resistance mechanism. Figure 1 Pancreatic fibroblasts are innately chemoresistant. (a) Immunofluorescence stain for SMA and vimentin of cancer-associated fibroblasts (CAF1) and wild-type (WT) fibroblasts. (b) Cells were treated with 1M gemcitabine for 2C6 … Pancreatic CAF-Conditioned Media Increases Proliferation and Survival of Epithelial Cancer Cells Considering the important role of cell extrinsic factors on cell growth and survival, we next assessed whether factors secreted by the innately chemoresistant fibroblasts could affect proliferation and survival of epithelial cancer cells. We first determined the effect CAF-conditioned media 301836-41-9 had on the proliferation of chemosensitive L3.6 cells. An equivalent number of L3.6 or CAF cells were plated and incubated in DMEM for 24 hours. Conditioned cell media from either the L3.6 or the CAF cells was then transferred onto recipient L3. 6 cells each day for six days. CAF-conditioned media increased proliferation of L3.6 cells by more than 50% compared to L3.6-conditioned media (Figure 2aCb). Having demonstrated that media from GEM-resistant CAF cells could increase the proliferation of GEM-sensitive L3.6 cells, we next assessed if this effect was CAF specific or if GEM-resistant epithelial cancer cells 301836-41-9 could also elicit this change in proliferation. We observed that conditioned media from the chemoresistant PANC1 epithelial cancer cell line did not elicit a significant increase in proliferation (Supplementary Figure S2). Next, we determined if CAF-conditioned media also affected the chemoresistance of epithelial cells. L3.6 cells were grown in either L3.6 or CAF cell-conditioned media for 6 days then treated with 100nM gemcitabine for 3 days, and cell survival was assessed. We observed that L3.6 cells grown in CAF-conditioned media and subsequently treated with gemcitabine showed a significant increase in cell survival compared to L3.6 cells grown in L3.6 conditioned media (Figure 2cCd). Taken together, these data show that CAF-secreted factors affect proliferation and drug resistance of epithelial cancer cells. Figure 2 Pancreatic CAF1-conditioned media increases proliferation and survival.