Abstract 2874: Pancreatic cancer associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner.
Authors: Mace Thomas A. , Ameen Zeenath , Frankel Wendy , Collins Amy , Wojcik Sylwia , Mair Markus , Young Gregory S. , Fuchs James R. , Eubank Tim D. , Bekaii-Saab Tanios , Bloomston Mark , Lesinski Gregory B. 
Published: August 2014

Pancreatic cancer is the fourth most common cause of cancer-related death in the United States with many patients that are diagnosed dying within one year. Pancreatic ductal adenocarcinoma is an inflammatory disease and includes several stromal elements such as lymphocytes and other immune cells, fibroblasts, and stellate cells. Pancreatic stellate cells (PSC), also known as cancer associated fibroblasts, can provide pro-survival signals to tumor cells, however their interactions with immune cells within the tumor microenvironment have not been explored in detail. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes immunosuppression in the tumor microenvironment. Primary PSC were harvested from human specimens and morphology was confirmed via immunofluorescent microscopy and staining for vimentin, alpha-smooth muscle actin (α SMA), and glial fibrillary acidic protein (GFAP). We analyzed soluble factors from PSC and human fetal primary pancreatic fibroblast cell line (HPPFC; negative controls) using a Luminex assay. Normal donor peripheral blood mononuclear cells (PBMC, n=3 donors) were cultured with 5 and 10% PSC supernatants, 10% HPPFC supernatants (negative control), or IL-6/GM-CSF (positive control) for 7 days, and assessed for MDSC phenotype by flow cytometry. The FLLL32 STAT3 inhibitor was used to determine whether PSC-mediated MDSC differentiation or PSC viability was STAT3-dependent. Stellate cell lines (n=7) were generated from patients and validated. Luminex analysis indicated that PSC produced cytokines involved in MDSC differentiation (IL-6, VEGF, MCSF) and chemotaxis (SDF-1, MCP-1). Culture with PSC supernatants for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype (p&lt;0.01) and a sub-population of polymorphonuclear CD11b+CD33+CD15+ cells (p&lt;0.05). Supernatants from a HPPFC were used as a negative control, which when cultured with PBMC did not induce an MDSC phenotype. The resulting CD11b+CD33+ cells were functional as they suppressed autologous T cell proliferation (p&lt;0.05). Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent (p&lt;0.01). These data demonstrate a novel role for PSC in supporting immunosuppression associated with cancer and suggest that STAT3 within both stromal and immunosuppressive cells represents a therapeutic target.

Citation Format: Thomas A. Mace, Zeenath Ameen, Wendy Frankel, Amy Collins, Sylwia Wojcik, Markus Mair, Gregory S. Young, James R. Fuchs, Tim D. Eubank, Tanios Bekaii-Saab, Mark Bloomston, Gregory B. Lesinski. Pancreatic cancer associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2013-2874