Abstract 5889: Cancer associated fibroblasts regulate cancer stem cell functions in pancreatic ductal adenocarcinoma
Authors: Begum Asma , McMillan Ross , Chang Yu-tai , Penchev Vesselin , Rajeshkumar NV , Maitra Anirban , Goggins Michael G. , Eshelman James R. , Wolfgang Christopher , Rasheed Zeshaan A. , Matsui William H. 
Published: July 2017

Introduction: Despite recent advancements in treatment, the outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal due to high relapse rate and metastatic dissemination. PDAC cancer stem cells (CSCs) have been shown to play a role in these processes and are associated with worse median survival in patients. Several cell-intrinsic mechanisms of CSC regulation have been identified in PDAC, however the role of the altered tumor microenvironment remains largely unknown. We focusd on the impact of cancer associated fibroblasts (CAFs) on CSCs properties in PDAC.

Methods: Stromal fibroblast lines were established from surgically resected PDAC tissues and were marked by specific fluorophore. Following co-culture with CAFs, tumor cells from PDAC cell lines or low passage xenografts derived from primary surgical specimens cells were isolated and evaluated for colony formation and migration. The effect of Focal Adhesion Kinase (FAK) on CAF-mediated clonogenic growth was evaluated using the small molecule FAK inhibitor PF573228 or overexpression of full-length of FAK in PDAC cells.

Results: Following co-culture with CAFs, the clonogenic growth of PDAC cell lines increased by 1.3-2.5 fold compared to cells incubated alone (p ≤ 0.05). Furthermore, CAFs enhanced colony formation of cells from patient derived xenografts by at least 4 fold (p ≤ 0.001). To examine the effect of CAFs on PDAC self-renewal, tumor colonies were harvested and replated in methylcellulose. Although cells were not further exposed to CAFs, secondary colony formation of cells originally co-cultured with CAFs increased by &gt; 1.3 fold. We also found that the frequency of ALDH+ PDAC CSCs increased by 12-19 fold compared to control cells. CSCs are also highly migratory and express factors suggestive of the epithelial to mesenchymal transition (EMT) suggesting that they play an important role in metastatic disease. Following co-culture, the migration of PDAC cells increased by 1.2-2.1 fold (p ≤ 0.05) with increased expression of the EMT associated genes SNAIL1, SNAIL2, ZEB1, ZEB2, N-cadherin, and Vimentin. PDAC cells induced CAF activation as evidenced by enhanced αSMA expression. Since activated CAFs are known to secret extracellular matrix proteins, we examined the activation of FAK downstream of integrin signaling and found increased phosphorylation of FAK at Y397. To determine the importance of FAK activation on the interaction between CAFs and PDAC cells, cells were treated with the FAK inhibitor PF573228 that significantly inhibited tumor colony formation. In contrast, the overexpression of FAK-FL further increased CAF-mediated PDAC clonogenic growth.

Conclusion: CAFs enhance PDAC CSCs including clonogenic growth, migration, and self-renewal. These effects are partially mediated by FAK, and FAK inhibitors may represent a novel strategy to modulate the interaction of PDAC cells and the tumor microenviroment.

Citation Format: Asma Begum, Ross McMillan, Yu-tai Chang, Vesselin Penchev, NV Rajeshkumar, Anirban Maitra, Michael G. Goggins, James R. Eshelman, Christopher Wolfgang, Zeshaan A. Rasheed, William H. Matsui. Cancer associated fibroblasts regulate cancer stem cell functions in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5889. doi:10.1158/1538-7445.AM2017-5889