Abstract B75: Targeting the immune-microenvironment with combined inhibition of MEK and STAT3 in a mouse model of pancreatic cancer
Authors: Roberts Casey , VanSaun Michael N. , Lamichhane Purushottam , Messaggio Fanuel , Kovacs Krisztina , Srinivasan Supriya , Dai Xizi , Barretta Jennifer , Nagathihalli Nagaraj , Merchant Nipun B. 
Published: December 2016

Introduction: Activating KRAS mutations are commonly found in PDAC and lead to constitutive downstream activation of MEK, which results in uncontrolled proliferation. We have previously shown that MEK inhibition results in activation of STAT3 signaling which confers drug resistance and continued cancer cell growth while combined STAT3 and MEK inhibition overcomes this resistance. Since STAT3 is a critical mediator of cytokine signaling and MEK is a mediator of cytokine production, we sought to determine the effects of MEK and STAT3 inhibition on the immune tumor microenvironment (TME). Tumor infiltrating immune/inflammatory cells, such as regulatory T cells (Tregs), myeloid –derived suppressor cells (MDSCs) and macrophages support tumor growth and contribute to therapeutic resistance. We hypothesized that combined MEK and STAT3 inhibition down regulates the suppressive immune infiltrates and promotes an anti-tumor microenvironment.

Experimental procedure: To understand the effect of MEK and/or STAT3 inhibition of PDAC cells, three dimensional spheroid cultures of PDAC cells (MiaPaCa-2, Panc-1, BxPC3) were prepared. Spheroid cultures were treated with inhibitors to MEK (AZD6244) and/or STAT3 (AZD1480) for 10 days. At the end of treatment, spheroids were quantified for size and metabolic activity. To determine in vivo effects, Ptf1a Cre/+ ; LSL-KrasG12D; Tgfbr2fl/fl (PKT) mice were treated with either vehicle, MEK inhibition, STAT3 inhibition, or the combination for 2 weeks. Post-treated pancreatic tissue was extracted, weighed, and examined for pancreatic integrity using immuno-histological and enzymatic analyses. Alternately, the pancreas and spleen were extracted from the mice, cells were isolated from the tissue, and subsequently labeled with antigens for macrophages (CD45, F4/80, CD86, CD80, CD206, CD204), myeloid cells (CD45, CD11b, Ly6g, Ly6c), and T cells (CD45, CD3e, CD4, CD8, CD25, FoxP3) before assessing population percentages by flow cytometric analysis.

Results: Treatment with MEK inhibition resulted in slightly reduced spheroid size and metabolic activity; however, combined MEK/STAT3 treatment led to a significant decrease in spheroid size as well as metabolic activity. In PKT mice, treatment with combined inhibitors for MEK and STAT3 resulted in the enhanced suppression of tumor formation compared to either agent alone. Histological analysis of combined MEK/STAT3 significantly inhibited tumor size, maintained a higher percentage of pancreatic integrity, displaying increased percentage of normal acini, reduced CK-19 staining, reduced collagen deposition and minimal alcian blue stain. Analysis of the tumor immune infiltrates revealed a significant reduction in the immunosuppressive/tumor promoting myeloid derived suppressor cell (MDSC) population (CD45+CD11b+Ly6g+Ly6c+) and regulatory T cell population (CD45+CD3e+CD4+CD25+FoxP3+) in the pancreas of mice treated with combined MEK/STAT3 inhibition compared to control mice or mice treated with single agents. Alternately, combined MEK/STAT3 inhibition promoted an increased neutrophil population (7AAD-CD11b+Ly6c+Ly6g-), but a decreased inflammatory M1 macrophage population (CD45+F4/80+CD80+CD86+).

Conclusions: Combined MEK/STAT3 inhibition downregulates the tumor promoting immune infiltrates resulting in dramatically reduced tumor burden and enhanced normal pancreatic tissue in a highly aggressive mouse model of pancreatic cancer.

Citation Format: Casey Roberts, Michael N. VanSaun, Purushottam Lamichhane, Fanuel Messaggio, Krisztina Kovacs, Supriya Srinivasan, Xizi Dai, Jennifer Barretta, Nagaraj Nagathihalli, Nipun B. Merchant.{Authors}. Targeting the immune-microenvironment with combined inhibition of MEK and STAT3 in a mouse model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B75.