Myeloid-Derived Suppressor Cell Subset Accumulation in Renal Cell Carcinoma Parenchyma Is Associated with Intratumoral Expression of IL1β, IL8, CXCL5, and Mip-1α

Abstract

Purpose: Little is known about the association between myeloid-derived suppressor cell (MDSC) subsets and various chemokines in patients with renal cell carcinoma (RCC) or the factors that draw MDSC into tumor parenchyma.Experimental Design: We analyzed polymorphonuclear MDSC (PMN-MDSC), monocytic MDSC (M-MDSC), and immature MDSC (I-MDSC) from the parenchyma and peripheral blood of 48 patients with RCC, isolated at nephrectomy. We analyzed levels of IL1β, IL8, CXCL5, Mip-1α, MCP-1, and Rantes. Furthermore, we performed experiments in a Renca murine model to assess therapeutic synergy between CXCR2 and anti-PD1 and to elucidate the impact of IL1β blockade on MDSC.Results: Parenchymal PMN-MDSC have a positive correlation with IL1β, IL8, CXCL5, and Mip-1α, and I-MDSC correlate with IL8 and CXCL5. Furthermore, peripheral PMN-MDSC correlate with tumor grade. Given that PMN-MDSC express CXCR2 and parenchymal PMN-MDSC correlated with IL8 and CXCL5, we assessed the response of CXCR2 blockade with or without anti-PD1. Combination therapy reduced tumor weight and enhanced CD4+ and CD8+ T-cell infiltration. In addition, anti-IL1β decreased PMN-MDSC and M-MDSC in the periphery, PMN-MDSC in the tumor, and peripheral CXCL5 and KC. Anti-IL1β also delayed tumor growth.Conclusions: Parenchymal PMN-MDSC have a positive correlation with IL1β, IL8, CXCL5, and Mip-1α, suggesting they may attract PMN-MDSC into the tumor. Peripheral PMN-MDSC correlate with tumor grade, suggesting prognostic significance. Anti-CXCR2 and anti-PD1 synergized to reduce tumor weight and enhanced CD4+ and CD8+ T-cell infiltration in a Renca murine model, suggesting that CXCR2+ PMN-MDSC are important in reducing activity of anti-PD1 antibody. Finally, anti-IL1β decreases MDSC and delayed tumor growth, suggesting a potential target for MDSC inhibition. Clin Cancer Res; 23(9); 2346-55. ©2016 AACR.

©2016 American Association for Cancer Research.

Figures

Figure 1

Panel A) Representative flow cytometry graphics for RCC tumor and PBMC showing the gating scheme and subsets. Panel B) The percent of MDSC subsets from normal individuals, PBMCs from both localized and metastatic RCC patients and tumors. Compared to healthy donors the percentage of MDSC, particularly the PMN and Linage-negative subsets are significantly increased in the peripheral blood and tumor of RCC patients. The morphology of the MDSC subsets is also shown. The MDSC subsets were sorted from an RCC tumor; cytospins were made and stained with Diff-Quik Stain Set. The insets show cells at 2x.

Figure 2

Panel A) Renca tumor bearing mice were treated for 21 days with CXCR2 antagonist (40μg/100μl, once daily I.P.), anti-PD1 antibody (200μg/mouse, 2 times per week) or the combination. Untreated Renca tumor bearing mice served as controls. The tumors were harvested and wei ghed. Panel B) The tumors were digested; the resulting single cell suspensions were stained with CD4 & CD8 antibodies and acquired for flow cytometry. The results show an increase in CD4+ & CD8+ T cells in the tumors of the treated mice, with a significant (p=0.02) increase of CD4+ cells with the combination treatment. Panel C) The tumor single cell suspensions were also stained for Ly6G, CD11b, and CXCR2. There was no significant change in the PMN (CD11b+Ly6G+) or monocytic (CD11b+Ly6G-) MDSC with treatment. CXCR2 was present on the PMN-MDSC but not the monocytic MDSC. Panel D) Human RCC tumors were digested; the resulting single cell suspensions were stained for either CD66b, CXCR2 or CD15, HLADR, CD33 and CXCR2. The graph shows the mean +/− SEM (median = 51.1%, range of 0.83% to 100%) of PMN-MDSC that are CXCR2+ in the tumor.

Figure 3

Panel A) Renca tumor bearing mice were treated for 3 weeks with anti IL1b (100μg/mouse, 2 times per week). Untreated Renca tumor bearing mice and non-tumor bearing mice (NTB) served as controls. At harvest, blood was obtained by cardiac puncture. The tumors were digested using Miltenyi Mouse Tumor Dissociation Kit. Total number of viable cells was quantified. The single cell suspensions from tumor digest and whole blood were stained with Ly6G and CD11b and acquired for flow cytometry. Anti-IL1β significantly reduced both the PMN-MDSC and M-MDSC in the blood (p=0.000035 and p=0.00033 respectively) and only the PMN-MDSC in the tumor (p=0.011). Panel B & C) ELISA were done on the serum from mice for both KC and CXCL5 (LIX). Panel D) The cellularity (millions of viable cells in the tumor suspension/gram of tumor) decreased in the anti-IL1β treated mice.