Tissue resident iNKT17 cells facilitate cancer cell extravasation in liver metastasis via interleukin-22

Abstract

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Keywords: ANPEP; IL-22; cancer cell extravasation; endothelial cells; extravasation; metastasis; metastasis formation; tissue resident cells.

Graphical abstract

Figure 1

Il22-deficient mice are protected against liver metastasis (A) Schematic overview of the experiment (i.s. injection of MC38 cells). (B) Representative pictures, number of macroscopic liver metastases, and liver weight of Il22^+/+ mice compared to Il22^−/− mice. n ≥ 11 mice per group. (C) H&E staining and metastatic foci. n = 5 mice per group. (D) Schematic overview of the experiment (i.s. injection of MC38 cells). (E) Liver weight and number of macroscopic liver metastases in mice treated with an α-IL-22 or IgG control antibody. n ≥ 8 mice per group. (F) Schematic overview of the experiment (i.s. injection of CT26CAG-Luc cells). (G) Ex vivo bioluminescent imaging of livers after hydrodynamic overexpression of eGFP or Il22eGFP plasmid. Bioluminescent scale: 2 × 10⁷–2 × 10⁸ photons/sec/cm²/sr. Liver weight and number of macroscopic liver metastases in Balb/c mice after hydrodynamic overexpression of eGFP or Il22eGFP plasmid. n ≥ 6 mice per group. (H) Bioluminescent imaging of Balb/c mice after hydrodynamic overexpression of eGFP or Il22eGFP plasmid. Bioluminescent scale: 5 × 10⁷–5 × 10⁸ photons/sec/cm²/sr. n ≥ 6 mice per group. Scale bar: 2 mm. (I) Schematic overview of the experiment (injection of LLC cells into the cecum). (J) Representative pictures and number of macroscopic liver metastases in Il22^+/+ and Il22^−/− mice. n ≥ 6 mice per group. (K) Schematic overview of the experiment (adenoviral infection of colonic epithelium for orthotopic colon cancer induction as a model of spontaneous liver metastasis). (L) Endoscopic score of primary colon cancer of Apc^15lox;Kras^G12D mice. n ≥ 6 mice per group. (M) Il22 mRNA levels in total liver of Apc^15lox;Kras^G12D mice upon hydrodynamic liver overexpression of eGFP or Il22eGFP plasmid. n = 6 mice per group. (N) Representative pictures and percentage of Apc^15lox;Kras^G12D mice that developed macroscopic metastases. n = 6 mice per group. Scale bar: 2 mm. Data presented as mean ± SEM, not significant (ns): p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by Mann-Whitney U test or Fischer’s exact test (K).

Figure 2

IL-22 affects the host endothelium, thereby promoting metastasis (A) Schematic overview of the experiment (i.s. injection of MC38shC and MC38shIl22ra1 cells in Il22tg^(wt) and Il22tg8^(Tg) mice). (B) Liver weight and number of macroscopic liver metastases. n ≥ 7 mice per group. (C) Schematic overview of the experiment (i.s. injection of MC38 cells in WT and Il22ra1−/− mice). (D) Liver weight and number of macroscopic liver metastases. n ≥ 7 mice per group. (E) IL-22RA1 expression in CD45⁺ cells and LSECs. (F) Immunostaining of LSECs with CD31 (green) and IL-22RA1 (red). Scale bar, 50 μm. (G) pSTAT3 levels in LSECs isolated from 6 WT mice and treated with recombinant murine (rm) IL-22. (H) Schematic overview of the experiment (i.s. MC38 cell injection in Il22ra1^wt/wt;Alb^Cre+ and Il22ra1^flox/flox;Alb^Cre+ mice). (I) Liver weight and number of macroscopic liver metastases. n ≥ 7 mice per group. (J) Schematic overview of the experiment (i.s. MC38 cell injection in Il22ra1^wt/wt;Cdh5^Cre+ and Il22ra1^flox/flox;Cdh5^Cre+ mice). (K) Liver weight and number of macroscopic liver metastases. n ≥ 14 mice per group. Scale bar, 2 mm (B, D, I, and K). Data presented as mean ± SEM, ns: p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by Mann-Whitney U test (C, D, I, and K) or one-way ANOVA with Bonferroni post hoc tests (B and G).

Figure 3

IL-22 signaling in endothelial cells is required for cancer cell extravasation (A) Schematic overview of the experiment (i.s. injection of MC38 GFP-labelled cells in WT, Il22^−/−, and Il22ra1^−/− mice [extravasation assay]). See also Figure S3. (B) Representative FACS (fluorescence-activated cell sorting) plots and number of extravasated cancer cells 24 h post i.s. cancer cell injection. n ≥ 4 mice per group. (C) Representative images of immunostaining of MC38 Cherry-labeled cells and endomucin (endothelial marker; green) 24 h post i.s. cancer cell injection. Scale bar, 50 μm. (D) Number of extravasated cancer cells 24 h post i.s. injection. n = 6 mice per group. (E) Schematic overview of the experiment (injection of MC38 Cherry-labeled cells in WT mice receiving an anti-IL-22 or IgG control antibody). (F) Representative FACS plots and number of extravasated cancer cells from (E). n ≥ 5 mice per group. (G) Schematic overview of the experiment (i.s. injection of MC38 GFP-labeled cells in Il22ra1^wt/wt;Cdh5^Cre+ and Il22ra1^flox/flox;Cdh5^Cre+ mice [extravasation assay]). (H) Representative FACS plots and number of extravasated cancer cells 24 h post i.s. cancer cell injection. n ≥ 8 mice per group. (I) Schematic overview of the experiment (i.s. injection of MC38 cells in Il22ra1^wt/wt;Cdh5^Cre+ and Il22ra1^flox/flox;Cdh5^Cre+ mice [extravasation assay]). (J) Immune cell infiltration in the liver 24 h post i.s. injection. n ≥ 7 mice per group. Data presented as mean ± SEM, ns p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by one-way ANOVA with Bonferroni post hoc tests (B and D) or Mann-Whitney U test (F, H, and J).

Figure 4

IL-22 increases endothelial ANPEP expression, thereby promoting endothelial permeability and cancer cell transmigration (A) Gene expression analysis of LSECs upon rm IL-22 or PBS stimulation. (B) Anpep expression in LSECs upon stimulation with rmIL-22. (C) Immunoblotting analysis and quantification of ANPEP in LSECs upon rmIL-22 stimulation. (D) Confocal microscopy pictures of in vitro cultured LSECs showing the CD31 (green) and ANPEP (red) expression. (E) In vitro extravasation model of HT29 GFP-labeled cancer cells through HUVEC cells. (F) TEER of HUVEC cell layers upon rmIL-22 stimulation. (G) ANPEP RNA levels upon rmIL-22 stimulation. (H) ANPEP RNA expression after shRNA silencing of ANPEP in different HUVEC clones. (I) In vitro extravasation model of HT29 GFP-labeled cancer cells through shC or shANPEP HUVEC cells. n = 2 independent experiments. (J) Schematic overview of the experiment (i.s. injection of MC38 Cherry cells in Cd13^+/+ or Cd13^−/− mice receiving an α-IL-22 or IgG control antibody). (K) Representative FACS plots and statistics. n ≥ 10 mice per group. Data presented as mean ± SEM, ns p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by one-way ANOVA with Bonferroni post hoc tests (D and K) or Mann-Whitney U test (B, E, F, and I).

Figure 5

IL-22 promotes lung metastasis by promoting cancer cell extravasation (A) Schematic overview of the experiment (i.v. injection of MC38 cells for forced lung metastasis), representative pictures and number of macroscopic metastases, H&E staining, and metastatic foci percentage of Il22^+/+ and Il22^−/− mice. n ≥ 10 mice per group. (B) Schematic overview of the experiment (flank injection of MC38 cells for spontaneous lung metastasis), representative pictures, and number of macroscopic metastases. n ≥ 10 mice per group. Scale bar, 2 mm. (C) Schematic overview of the experiment (i.v. injection of MC38 for forced lung metastasis in Il22ra1^wt/wt;Cdh5^Cre+ and Il22ra1^flox/flox;Cdh5^Cre+ mice). (D) Number of macroscopic lung metastases. n ≥ 5 mice per group. (E) Schematic overview of the experiment (i.v. MC38 cell injection following single left orthotopic lung transplantation using WT mice as recipients and Il22ra1^−/− mice as donors). (F) Representative pictures and quantification of lung metastases. n = 7 mice per group. (G) Schematic overview of the experiment (i.v. injection of MC38 Cherry-labeled cells in WT, Il22^−/− and Il22ra1^−/− mice [extravasation assay]). (H) Representative FACS plots and number of extravasated cancer cells 24 h post i.v. injection. n = 4 mice per group. (I) Schematic overview of the experiment (i.v. injection of MC38 GFP-labeled cells in WT mice receiving an anti-IL-22 or IgG control antibody). (J) Representative FACS plots and number of extravasated cancer cells. n ≥ 11 mice per group. (K) Schematic overview of the experiment (i.v. injection of MC38 GFP-labeled cells in Il22ra1^wt/wt;Cdh5^Cre+ and Il22ra1^flox/flox;Cdh5^Cre+ mice [extravasation assay]). (L) Representative FACS plots and number of extravasated cancer cells from Il22ra1^wt/wt;Cdh5^Cre+ and Il22ra1^flox/flox;Cdh5^Cre+ mice 24 h post i.v. injection. n = 5 mice per group. Data presented as mean ± SEM, ns p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by Mann-Whitney U test (A–D, J, and L), one-way ANOVA with Bonferroni post hoc tests (H), or two-tailed Wilcoxon matched-pairs signed rank test (F).

Figure 6

iNKT cells promote cancer cell extravasation via IL-22 (A) Schematic overview of the experiment (i.v. injection of MC38 cells in IL-22 reporter mice). (B) Il22 mRNA levels in the liver at steady state, 12 and 24 h post i.s. MC38 cell injection. (C) Representative concatenated FACS plots indicating mean and statistics of CD45⁺, CD3⁻, CD3⁺CD4⁺, CD3⁺CD8⁺, and CD3⁺CD4⁻CD8⁻ IL-22-producing cells in the liver at 0 and 12 h post i.s. MC38 cell injection. (D) Il22 mRNA levels from indicated cell populations sorted from the liver at 0 and 12 h post i.s. MC38 cell injection. (E) Schematic overview of the experiment (i.s. injection of MC38 GFP-labeled cells in WT and Rag1^−/− mice, receiving an α-IL-22 or IgG control antibody). (F) Representative FACS plots and statistics. n ≥ 4 mice per group. (G) Schematic overview of the experiment (i.s. injection of MC38 GFP-labeled cells in Il22^wt/wt;CD4^Cre+ and Il22^flox/flox;CD4^Cre+ mice). n ≥ 5 mice per group. (H) Representative FACS plots and number of extravasated cancer cells 24 h post i.s. injection. n ≥ 4 mice per group. (I) Schematic overview of the experiment (i.s. injection of MC38 GFP-labeled cells in Rag^−/−Il22^−/− mice engrafted with Il22^+/+or Il22^−/− iNKT cells). (J) Representative FACS plots and number of extravasated cancer cells 24 h post i.s. injection. n ≥ 10 mice per group. (K) Schematic overview of the experiment (i.s. injection of MC38 Cherry-labeled cells in Ja18^−/− mice engrafted with Il22^+/+or Il22^−/− iNKT cells). (L) Representative FACS plots and number of extravasated cancer cells 24 h post i.s. injection. n ≥ 10 mice per group. Data presented as mean ± SEM, ns p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by one-way ANOVA with Bonferroni post hoc tests (B and F) or Mann-Whitney U test (C, D, H, J, and L).

Figure 7

IL-22-producing iNKT17 cells are tissue resident and facilitate cancer cell extravasation (A) Injection of an α-CD45 antibody intravenously after i.s. injection of MC38 cells. (B) Representative FACS plots and statistics 0 and 12 h post i.s. injection. n ≥ 10 mice per group. (C) Pie charts showing the proportion of infiltrating and circulating immune subsets among CD3⁺IL-22⁺ cells in murine liver 0 and 12 h post i.s. injection. (D) Concatenated FACS plots of iNKT and γδ T cells gated on CD45⁺IL-22⁺ cells, including in vivo and in vitro CD45 staining. n ≥ 8 mice per group. (E) Statistics of iNKT and γδ T cells gated on CD45⁺IL-22⁺ cells, including in vivo and in vitro CD45 staining. n ≥ 8 mice per group. (F) Representative FACS plots showing the immune characterisation of iNKT cells gated on CD1d-tetramer⁺IL-22⁺ cells 12 h upon i.s. cancer cell injection. (G) Representative FACS plots and statistics showing the immune subtypes (iNKT1, iNKT2, and iNKT17) of iNKT cells gated on CD1d-tetramer⁺IL-22⁺ cells 12 h upon i.s. cancer cell injection. (H) CD45.2 reporter mice were connected together with CD45.1 (n = 3 mice per group) mice and the established blood chimerism was checked 14 days later. (I) CD45.2 reporter mice were connected together with CD45.½ mice (n = 12 mice per group), followed by intraportal cancer cell injection after established blood chimerism. (J) Representative FACS plots of CD45.½ and CD45.2 cells from parabiotic pairs. (K) The proportion of CD45.½ and CD45.2 cells of parabiotic pairs was quantified. (L) WT mice were connected with WT or Il22^−/− mice (n ≥ 8 mice per group), followed by intraportal injection of MC38 GFP-labeled cells. (M and N) Mice were sacrificed and the number of extravasated cancer cells was quantified. Data presented as mean ± SEM, ns p > 0.05; ^∗p < 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001 as assessed by Mann-Whitney U test (B, E, and K) or two-tailed Wilcoxon matched-pairs signed rank test (N).