Abstract B56: CXCR2 blockade reduces granulocytic myeloid cell compensation in response to macrophage targeted therapy and further enhances the efficacy of chemotherapy in pancreatic ductal adenocarcinoma
Authors: Han Booyeon J. , Nywening Tim M. , Belt Brian A. , Ye Jian , Hawkins William G. , DeNardo David G. , Linehan David C. 
Published: February 2019

Background: The incidence of pancreatic ductal adenocarcinoma (PDAC) is rising, yet patient survival rates following conventional treatment remain abysmal. A unique feature of PDAC is the presence of a prominent tumor stroma with a dense network of fibrotic material and abundant inflammatory immune infiltrate. Bone marrow derived myeloid cells of both monocytic and granulocytic origin, including tumor associated macrophages (TAM) and neutrophils (TAN) respectively, are the most prevalent immune cells in the tumor microenvironment (TME) of PDAC. PDAC tumors co-opt chemokine signaling pathways to attract myeloid cells from the bone marrow to the TME where they suppress anti-tumor immune responses, support tumor progression, and confer resistance to therapy. In a previous clinical study, we demonstrated that targeted blockade of CCR2+ TAM with a small molecule CCR2 inhibitor (PF-04136309) nearly doubled responses to the FOLFIRINOX chemotherapy regimen in patients with locally advanced PDAC. Here, we show that tumor infiltrating CXCR2+ TAN compensate for TAM depletion in response to CCR2 blockade, so we hypothesized that dual targeting of both CCR2+ TAM and CXCR2+ TAN would further enhance chemotherapy efficacy for treating PDAC.

Methods: Human blood, bone marrow, and tumor samples were acquired under an IRB approved protocol. PDAC cell lines (KCKO or KP) derived from spontaneous tumors were subcutaneously or orthotopically implanted into C57BL/6 mice. Tumor bearing mice were randomized and treated with vehicle, FOLFIRINOX, CCR2i (PF-04136309 or RS504393), CXCR2i (SB225002), CCR2i+CXCR2i, CCR2i+FOLFIRINOX, CXCR2i+FOLFIRINOX, or CCR2i+CXCR2i+FOLFIRINOX. Mice were sacrificed at fixed time points for tissue collection or followed for survival. Resected tissues were snap frozen for RNA extraction to perform qRT-PCR, fixed in formalin for histological staining and immunohistochemistry, or digested into single cell suspensions for flow cytometry analysis.

Results: The prevalence of peripheral blood and bone marrow CXCR2+ TAN is prognostic in patients with PDAC. Tumors expressed elevated levels of the ELR+ chemokines including CXCL1, CXCL2, CXCL5, and CXCL8 and were infiltrated with significantly more CXCR2+ TAN compared to normal pancreas. Tumor expression of the ELR+ chemokines correlated with tumor infiltrating TAN and their levels were prognostic in human disease. Analysis of longitudinally paired tumor biopsies from patients treated with PF-04136309 had significantly increased levels of CXCR2+ TAN (p &lt; 0.05). In mice, orthotopically implanted PDAC tumors significantly upregulated the ELR+ chemokines resulting in increased levels of CXCR2+ TAN in the peripheral blood, bone marrow, and TME compared to normal pancreas controls, recapitulating human disease. CXCR2i significantly decreased tumor infiltrating CXCR2+ TAN and reduced overall tumor burden. Single agent targeting of either TAM or TAN, using CCR2i or CXCR2i respectively, resulted in a compensatory influx of the other myeloid cell subset. However, dual targeting of both CCR2+ TAM and CXCR2+ TAN blocked myeloid compensation and further enhanced the efficacy of FOLFIRINOX. Dual CCR2/CXCR2 blockade reprogrammed the TME and generated a more robust anti-tumor T cell response compared to either agent alone.

Conclusions: The ELR+ CXC/CXCR2 chemokine pathway plays a key role in PDAC tumor progression and CXCR2+ TAN compensation is observed with depletion of TAM in response to CCR2 blockade. Duel targeting of CCR2+ TAM and CXCR2+ TAN prevents myeloid plasticity to single agent therapy and further enhances the efficacy of chemotherapy. Thus, further studies exploring complementary immune targeting strategies are warranted in PDAC.

Citation Format: Booyeon J. Han, Tim M. Nywening, Brian A. Belt, Jian Ye, William G. Hawkins, David G. DeNardo, David C. Linehan. CXCR2 blockade reduces granulocytic myeloid cell compensation in response to macrophage targeted therapy and further enhances the efficacy of chemotherapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B56.