Abstract P037: Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis
Authors: Kim Teresa S. , Sullivan Kevin , Jiang Xiuyun , Hsu Cynthia , Labadie Kevin , Kohli Karan , Kenerson Heidi , Daniel Sara , Abbasi Arezou , Yeung Raymond , Pillarisetty Venu 
Published: January 2022

Background: Colorectal cancer liver metastasis (CRLM) causes major morbidity and mortality. Improved systemic therapies are crucial. We previously reported that macrophages infiltrated CRLM and expressed the immunosuppressive cytokine interleukin-10 (IL-10). In patient-derived CRLM tumor slice cultures, we found that a neutralizing antibody against IL-10 (anti-IL-10) caused tumor apoptosis. Here, we investigated the efficacy and immune-dependent mechanisms of IL-10 blockade in a larger cohort of CRLM patients.

Methods: Tumor specimens were obtained from consenting patients at the time of surgery and cut into 250mm-thick tumor slice cultures as previously described. Tumor slices were treated with control or neutralizing antibodies against programmed cell death protein 1 (PD-1, EH12.1), IL-10 (JES3-9D7), IL-10 receptor alpha (IL-10RA, 3F9), major histocompatibility complex (MHC) class I (G46-2.6) or class II (Tu39). After 4-6 days of treatment, tumor apoptosis was evaluated by cleaved caspase-3 (CC3) immunohistochemistry (IHC) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Tumors were evaluated by IHC and in situ hybridization (ISH) for immune markers. Student's t-test, paired t-test, or 1-way ANOVA was used as indicated. p &lt; 0.05 was defined as significant.

Results: We generated tumor slice cultures from 34 unique CRLM patients, 76% (26/34) of whom received preoperative chemotherapy, and all of whom had either microsatellite stable tumors (76%, 26/34) or unknown microsatellite status. Anti-PD-1 did not generate tumor apoptosis (n = 4 patients' tumors). In contrast, anti-IL-10 caused nearly 2-fold increased tumor apoptosis compared with control, in the majority of 34 CRLM patients' tumors evaluated (median 50.1% versus 27.4% apoptotic cells, p &lt; 0.0001). IL-10 receptor blockade generated a similar but non-significant increase in apoptosis. Tumors treated with anti-IL-10 demonstrated increased frequency of CD8+ T cells (median 17.8% versus 7.0% of total cells, n = 5 patients' tumors, p = 0.02) and a non-significant increase in activated PD-1+CD3+ T cells. IL-10 blockade also increased tumor IFNG expression (median 2.9 versus 2.3 counts per total cell number, n = 6 patients' tumors, p &lt; 0.05). Macrophage frequency did not increase, but MHC class II expression nearly doubled (median 19.5% versus 11.5% of total cells, n = 5 patients' tumors, p = 0.02). To test which immune cells were required for anti-IL-10 effect, we treated human CRLM slices with anti-IL-10 +/- blocking antibodies against MHC class I or II. In the 3 of 4 patients' tumors that responded to anti-IL-10, the effect was nearly completely reversed by aMHC-I or aMHC-II (p = 0.0005).

Conclusion: IL-10 is a dominant and reversible mechanism of immune evasion in human CRLM. IL-10 blockade nearly doubled tumor apoptosis in a heterogeneous cohort of CRLM patients' tumors, through a mechanism that required intact CD8+ T cell and antigen-presenting cell function. IL-10 is a compelling immunotherapeutic target for CRLM.

Citation Format: Teresa S. Kim, Kevin Sullivan, Xiuyun Jiang, Cynthia Hsu, Kevin Labadie, Karan Kohli, Heidi Kenerson, Sara Daniel, Arezou Abbasi, Raymond Yeung, Venu Pillarisetty. Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P037.