Abstract A53: Chemotherapy treated pancreatic cancer tumor-associated fibroblasts are protumorigenic
Authors: Toste Paul A. , Nguyen Andrew H. , Kadera Brian E. , Duong Mindy , Li Luyi , Donahue Timothy R. 
Published: October 2015

Introduction: The dense tumor-associated stroma (TAS) characteristic of pancreatic ductal adenocarcinoma (PDAC) plays an important role in disease progression and treatment resistance. PDAC tumor-associated fibroblasts (TAFs), the predominant cell type of the TAS, enhance tumor growth, invasion, metastasis, and chemoresistance. However, the impact of cytotoxic chemotherapy on TAF biology has not yet been reported.

Methods: Immortalized human TAFs were grown in either standard media or media containing IC50 doses of gemcitabine (GEM) for 4 weeks to generate chemotherapy-naïve and chemotherapy-resistant lines. Conditioned media (CM) was produced from both naïve and resistant TAFs. MTT assay was used to assess tumor cell (TC) viability in naïve versus resistant CM. TC migration and invasion as well as endothelial cell migration were assessed using modified Boyden chambers with naïve or resistant CM. For in vivo studies, TCs were subcutaneously co-implanted with naïve or resistant TAFs in immunodeficient mice. Gene expression microarray analysis was performed to compare naïve and chemoresistant TAFs. Expression of selected targets was confirmed by qRT-PCR and/or antibody-based cytokine array. IL-8 expression was quantified by ELISA. Expression of phospo- and total forms of ATM, AKT, and S6 were assessed by immunofluorescence and/or Western blot.

Results:TCs (Miapaca-2 and Panc1) demonstrated increased viability when grown in the presence of resistant as compared to naïve TAF CM (p&lt;0.001). TCs also showed increased migration (Miapaca-2, p&lt;0.001 and Panc1, p=0.002) and invasion (Panc-1, p&lt;0.001) in the presence of resistant CM. In addition, resistant TAF CM increased endothelial cell migration (p=0.01). In vivo, TCs (Miapaca-2) implanted along with chemoresistant TAFs grew larger tumors compared to those implanted with naïve TAFs (p&lt;0.05). Microarray analysis demonstrated that TAFs undergo a large number of molecular changes in response to GEM. Molecules involved in important TAF functions such as stellate cell activation, cellular movement, extracellular matrix production and remodeling, and cytokine signaling were altered. Of note, the expression of multiple protumorigenic inflammatory molecules was increased. Cytokine array and qRT-PCR confirmed elevated levels of multiple inflammatory cytokines (IL-6, IL-8, CXCL-1, SPP1) in chemoresistant TAFs. Furthermore, ELISA demonstrated that IL-8 concentration was 6-fold higher in chemoresistant TAF CM compared to naïve TAF CM. TAFs activated a DNA damage response (DDR) upon treatment with GEM as evidence by phosphorylation of ATM. Treatment with NVP-BEZ235, a pan-PI3K related kinase (PIKK) inhibitor that inhibits the DDR effector kinases ATM and ATR as well as PI3K and mTOR, attenuated the induction of inflammatory factors by GEM.

Conclusions: Chemotherapy treated TAFs support tumorigenesis and angiogenesis in vitro and in vivo via paracrine signaling. After treatment with GEM, TAFs activate a DDR and manifest a pro-inflammatory gene expression signature. Inhibition of PIKK attenuates this inflammatory response to chemotherapy in TAFs.

Citation Format: Paul A. Toste, Andrew H. Nguyen, Brian E. Kadera, Mindy Duong, Luyi Li, Timothy R. Donahue. Chemotherapy treated pancreatic cancer tumor-associated fibroblasts are protumorigenic. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A53.