IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction

Abstract

Background & aims: The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy in vivo.

Methods: To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10.

Results: We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions.

Conclusions: Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases.

Impact and implications: Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

Keywords: IL-10; PD-L1; immune cells; immunotherapy; liver metastasis.

Graphical abstract

Fig. 1

Impaired IL-10 signaling protects mice against colorectal cancer-derived liver metastasis. (A) Schematic overview of intracecal LLC cancer cell injection for spontaneous liver metastasis induction. (B) Primary tumor weight in the cecum. (C) Representative pictures and (D) number of liver metastases in WT and Il10-/- mice (n ≥8 mice per group). (E) Schematic overview of MTO129 intrasplenic injection for forced liver metastasis induction. (F) Representative pictures of liver metastasis, metastatic load, including liver weight and number of macroscopic liver metastases in WT and Il10-/- mice (n ≥ 7 mice per group). (G) Schematic overview of intrasplenic MC38 cancer cell injection. (H) Representative pictures of liver metastasis, metastatic load in WT and Il10-/- mice (n ≥12 mice per group). (I) Schematic overview of WT mice receiving aIL-10Ra or isotype treatment during intrasplenic MC38 cell injection. (J) Representative pictures and analysis of liver weight and number of liver metastases (n ≥12 mice per group). Scale bar: 2 mm. Data are presented as mean ± SEM. Non-significant (n.s.): p >0.05; ∗∗p <0.01; ∗∗∗p <0.001 calculated by Mann-Whitney U test. aIL-10Ra, IL-10 receptor alpha antibody; LLC, Lewis lung carcinoma; MC38, murine colon cancer cells; MTO, mouse tumor organoid; WT, wild-type. (This figure appears in color on the web.)

Fig. 2

Foxp3+Tregs are the major IL-10-producing cells during liver metastasis formation. (A) Schematic overview of intrasplenic MC38 cancer cell injection for forced liver metastasis induction in Foxp3^RFP;Il10^GFP mice (n ≥8 mice per group). Frequency of IL-10+ cells in the fraction of (B) CD45+ cells, (C) CD3-and T cells, and in (D) CD8+ T cells and CD4+ T cells. (E) IL-10 expression in (F) Foxp3-IL-10+ cells and (G) Foxp3+Tregs. (H) General distribution of all IL-10-producing CD45+ cells in healthy liver and liver with metastasis. Data are presented as mean ± SEM. Non-significant (n.s.): p >0.05; ∗p ≤0.05; ∗∗p <0.01; ∗∗∗p <0.001, as calculated by Mann-Whitney U test. MC38, murine colon cancer cells; Tregs, regulatory T cells. (This figure appears in color on the web.)

Fig. 3

Foxp3+Treg-derived IL-10 facilitates liver metastasis formation. (A) MC38 cells were intrasplenically injected into mice with cell-specific Il10 deletion in Foxp3+Tregs and myeloid cells and their littermate controls (n ≥10 mice per group). Livers were harvested 21 days post injection. (B, C) Representative images, liver weight and number of liver metastases in mice with (B) Treg-specific or (C) myeloid cell-specific Il10 deletion and their littermates. Scale bar: 2 mm. Data are presented as mean ± SEM. Non-significant (n.s.): p >0.05; ∗∗p <0.01, as calculated by Mann-Whitney U test. MC38, murine colon cancer cells; Tregs, regulatory T cells. (This figure appears in color on the web.)

Fig. 4

Dendritic cells, myeloid cells and Foxp3+Tregs can respond to IL-10. (A–D) Representative FACS plot and ΔMFI quantification of IL-10Ra expression in immune cells isolated from (A, B) a healthy liver or (C, D) liver metastasis 21 days post intrasplenic cancer cell injection (n ≥5 mice per group). (E) Representative FACS plot and (F) ΔMFI quantification of IL-10Ra expression from different innate subsets (CD3-) in liver metastasis. Data are presented as mean ± SEM. Non-significant (n.s.): p >0.05; ∗∗∗p <0.001, as calculated by Mann-Whitney U test or one-way ANOVA (Bonferroni) with Bonferroni post hoc tests. IL-10Ra, IL-10 receptor alpha; MFI, mean fluorescence intensity; Tregs, regulatory T cells. (This figure appears in color on the web.)

Fig. 5

IL-10 signaling in Foxp3+Tregs and myeloid cells promotes colorectal cancer-derived liver metastasis. (A) Schematic overview of intrasplenic MC38 cancer cell injection in mice with distinct cell-specific Il10ra deletion (n ≥5 mice per group). Representative images, liver weight and number of liver metastases in mice with (B) Treg-, (C) myeloid-, (D) DC-, or (E) Th17-specific Il10ra deletion. Scale bar: 2 mm. Data are presented as mean ± SEM. Non-significant (n.s.): p >0.05; ∗p ≤0.05; ∗∗p <0.01; ∗∗∗p <0.001, as calculated by Mann-Whitney U test. DC, dendritic cell; Il10ra, IL-10 receptor alpha; MC38, murine colon cancer cells; Th17, T helper 17 cells; Tregs, regulatory T cells. (This figure appears in color on the web.)

Fig. 6

IL-10-mediated PD-L1 upregulation in monocytes attenuates CD8+ T-cell infiltration and antitumor immunity. (A) Schematic overview of intrasplenic MC38 cancer cell injection in mice with myeloid-specific Il10ra deletion and their littermate controls (n ≥3 mice per group). (B, C) Representative FACS plot and MFI quantification of PD-L1 expression in (B) myeloid cells, DCs and remaining innate cells, as well as in (C) indicated subpopulations within myeloid cells. 14 days post injection, (D) apoptosis of MC38 cells and (E) antitumor immunity (TNF-α and granzyme B) of CD8+ T cells were analyzed using flow cytometry. (F) Mice with myeloid-specific Il10ra deletion and their littermate controls, receiving anti-IgG or anti-PD-L1 (250 μg/mouse) were injected intrasplenically with MC38 cancer cells (n ≥9 mice per group). Number of liver metastases was quantified 21 days post liver metastasis induction. Scale bar: 2 mm. Data are presented as mean ± SEM. Non-significant (n.s.): p >0.05; ∗p ≤0.05; ∗∗p <0.01; ∗∗∗p <0.001, as calculated by one-way ANOVA (Bonferroni) with Bonferroni post hoc tests. . IL-10Ra, IL-10 receptor alpha; MC38, murine colon cancer cells; MFI, mean fluorescence intensity; (a)PD-L1, programmed cell death ligand 1 (antibody); TNF-α, tumor necrosis factor-alpha. (This figure appears in color on the web.)