Abstract 4150: Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma
Authors: Nywening Timothy M. , Belt Brian A. , Panni Roheena Z. , Cullinan Darren , Sanford Dominic E. , Fields Ryan C. , Hawkins William G. , DeNardo David G. , Gillanders William E. , Goedegebuure Peter , Linehan David C. 
Published: July 2016

Abstract
Introduction: Pancreatic cancer (PC) is characterized by a dense tumor stroma with a heavy leukocytic infiltrate, comprised predominately of immunosuppressive bone marrow (BM) derived cells. We have previously demonstrated in a phase Ib clinical trial that CCR2 inhibition (CCR2i) prevents inflammatory monocyte (IM) recruitment from the BM and results in a significant reduction of tumor associated macrophages (TAM) and an increase in treatment efficacy. However, granulocytic myeloid derived suppressor cells (G-MDSC) remain in the tumor microenvironment (TME) following CCR2i. Herein, we explored the impact of targeting G-MDSC recruitment to PC tumors both alone and in combination with CCR2i.
Methods: Human BM, blood, and tumor was collected under an IRB approved protocol. A tissue microarray (TMA) from resected PC patients was analyzed for immune infiltrate. Mice were injected orthotopically with 2.5×106 syngeneic PC cells. CXCR2 and CCR2 inhibitors (Tocris) were given twice daily. Tumor growth was assessed and specimens obtained for analysis by flow cytometry, RNAseq, and IHC.
Results: Human PC overexpresses CXCL5 and CXCL8, corresponding with an abundance of tumor infiltrating CXCR2+ G-MDSC. Furthermore, the ratio of CD8 to G-MDSC correlates with survival in human PC patients. In an orthotopic murine model that recapitulates human disease, ΣCXCL ligands were also increased. Either Ly6G depletion or targeted blockade with a CXCR2 inhibitor decreased G-MDSC and reduced tumor burden. Intriguingly, blockade of IM from the BM did not reduce G-MDSC and paradoxically resulted in a modest increase in this population within the tumors from human patients following CCR2i. Thus, we explored the combination of CCR2/CXCR2 blockade both with and without FOLFIRINOX chemotherapy. This resulted in a synergistic impact when both BM derived populations were targeted and dual therapy was further enhanced by FOLFIRINOX. RNAseq analysis of tumors following monotherapy or dual inhibition revealed alterations in the TME favoring an anti-tumor immune response. To test the hypothesis that this effect was mediated by restoration of anti-tumor immunity we analyzed the tumor infiltrating lymphocyte (TIL) populations and found a significant increase in the relative and absolute numbers of CD8+ and C4+ TIL. Analysis of the activation status of these cells demonstrated an increase in effector CD8+ T-cell phenotype (IFNγ+, CD69+, CD44+). Using Nur77GFP T-cell receptor reporter mice, we showed an increase in GFP expressing CD8+ TIL following dual blockade. CD8 depletion resulted in a loss of therapeutic efficacy of myeloid blockade, further confirming our hypothesis.
Conclusion: These findings suggest that combinatorial blockade strategies preventing tumor infiltration by myeloid cells may restore anti-tumor immunity in PC.
Citation Format: Timothy M. Nywening, Brian A. Belt, Roheena Z. Panni, Darren Cullinan, Dominic E. Sanford, Ryan C. Fields, William G. Hawkins, David G. DeNardo, William E. Gillanders, Peter Goedegebuure, David C. Linehan. Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4150.