Abstract 4489: IL-10 blockade reactivates antitumor immunity in human colorectal cancer liver metastases

Abstract

Abstract Background: Colorectal cancer (CRC) is the 4th most common cancer in the US, and the liver is the most common site of metastatic disease. Immune checkpoint inhibitor therapy has not been successful in achieving a clinical response in most patients with CRC liver metastases (CRCLM). The liver is known to induce tolerance to foreign antigens as a result of immunosuppressive cytokines including IL-10. We hypothesized that blockade of IL-10 signaling in CRCLM would potentiate tumor infiltrating lymphocyte (TIL)-mediated tumor cell death. Methods: We performed single-cell RNA sequencing (scRNAseq) of CRCLM using the 10x platform to evaluate for expression of IL-10 or IL-10 receptor (IL-10R) RNA within the tumor (n=8). To confirm if the IL-10R protein was present within the tumor microenvironment (TME), we also performed immunohistochemistry (IHC) (n=3). In order to study the functional effects of IL-10 blockade, we utilized a tumor slice culture (TSC) model, which allows for the study of cancers with their intact TME including immune cells. For TSCs, cores (6 mm diameter) were taken from freshly resected sterile human CRCLM and cut to 250 µm thick slices using a vibratome (n=3). Duplicate slices were treated with either IgG control or anti-IL-10 monoclonal antibodies and cultured for up to 6 days. To evaluate for histological evidence of necrosis and cell apoptosis within the tumor slice, we stained slides with either hematoxylin and eosin (H&E) or cleaved-Caspase-3 (CC3). To gain insight into the activation state of TIL after treatment, we measured levels of cytokines within the culture supernatants. Results: We found by scRNAseq that that IL-10 was expressed by a subset of tumor-associated macrophages, and IL-10R was expressed by both CD4+ and CD8+ T cells as well as macrophages. We confirmed that IL-10R protein was present within the CRCLM TME by IHC, and IL-10R expression was distributed throughout the stroma in non-tumor cells. In TSC treated with anti-IL-10 antibody, CC3+ cells were found to be 82.8% of total cells, compared to 36.1% of control (p = 1 x 10-6) at day 6. These findings were consistent across all human tumor samples treated with IL-10 blockade versus control at all time points examined. Furthermore, IL-10 blockade led to histologic evidence of generalized necrosis compared to an intact TME seen in the control group. Analysis of cytokines released into the media confirmed that IL-10 was present in controls, but absent in slices blocked with anti-IL-10 antibody. We also found increased levels of granzyme B, IL-2, GM-CSF, and IL-18, as well as a reduction in the immune checkpoint receptor TIM3, after one day of IL-10 blockade in culture. Conclusion: Treatment of human CRCLM TSCs with anti-IL-10 antibody leads to a marked increase in immune-mediated cell death within the tumor. Our data suggest that IL-10 serves as a critical regulator of anti-tumor immunity in the CRCLM TME and may serve as an important immunotherapeutic target. Citation Format: Kevin M. Sullivan, Xiuyun Jiang, Yongwoo David Seo, Heidi L. Kenerson, Xiaowei Yan, Chris Lausted, Changting Meng, Neda Jabbari, Kevin P. Labadie, Sara K. Daniel, Qiang Tian, Teresa S. Kim, Raymond S. Yeung, Venu G. Pillarisetty. IL-10 blockade reactivates antitumor immunity in human colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4489.