Conventional NK cells and tissue-resident ILC1s join forces to control liver metastasis

Abstract

The liver is a major metastatic target organ, and little is known about the role of immunity in controlling hepatic metastases. Here, we discovered that the concerted and nonredundant action of two innate lymphocyte subpopulations, conventional natural killer cells (cNKs) and tissue-resident type I innate lymphoid cells (trILC1s), is essential for antimetastatic defense. Using different preclinical models for liver metastasis, we found that trILC1 controls metastatic seeding, whereas cNKs restrain outgrowth. Whereas the killing capacity of trILC1s was not affected by the metastatic microenvironment, the phenotype and function of cNK cells were affected in a cancer type-specific fashion. Thus, individual cancer cell lines orchestrate the emergence of unique cNK subsets, which respond differently to tumor-derived factors. Our findings will contribute to the development of therapies for liver metastasis involving hepatic innate cells.

Keywords: conventional NK cells; innate lymphocytes; metastatic surveillance; tissue-resident ILC1s.

Fig. 1.

Control of hepatic metastases depends on NKp46⁺ cells. (A) Experimental schedule for NKp46⁺ depletion. NKp46⁺ cells were depleted by an injection of 250 ng DTX i.p. 48 h before, 24 h after, or 7 d after tumor injection. Depletion was maintained until the endpoint. Ncr1^DTR = Ncr1^iCre/wt.R26R^iDTR/wt; Ctrl = undepleted control mice (Ncr1^iCre/wt.R26R^wt/wt, group injected at time point −48 h); i.s. = intrasplenic. (B) Representative dot plots (Left) and quantification (Right) of cNKs and trILC1s in Ctrl and Ncr1^DTR livers. Samples were pregated on single live CD45⁺lineage⁻ cells and subsequently gated on NK1.1⁺NKp46⁺ cells. cNK = conventional NK cells, CD49a⁻CD49b⁺; trILC1 = tissue-resident ILC1s, CD49a⁺CD49b⁻. (C, Upper) Quantification of LLC liver nodules by in vivo imaging system (IVIS) at the endpoint. Bioluminescence measurements on the whole liver are shown. (Lower) Representative IVIS images of metastatic livers from each group at the endpoint. The bar represents the mean ± SD, and the symbols represent livers from individual mice. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (D, Upper) Quantification of MC38 macroscopic liver nodules and their area at the endpoint. The area of each hepatic nodule is measured manually using the drawing tool from ImageJ. Each point represented corresponds to the area of one nodule displayed in pixels (px). (Lower) Representative images of metastatic livers from each group at the endpoint. The bar represents the mean ± SD, and the symbols represent livers from individual mice. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (E–G) Representative immunofluorescence image and quantification of NKp46⁺ cell vascular localization. Ncr1^tdTomato = Ncr1^Cre/wt.R26R^Ai14/wt. Three livers per experimental condition were analyzed, and each symbol represents an individual section. The distribution of NKp46⁺ cells was determined based on their positioning relative to the liver vasculature (luminal side of the sinusoids or large vessels, or interstitial). (E) Naïve liver. (Scale bar, 50 µm.) (F) LLC metastatic liver. (G) MC38 metastatic liver. (Scale bar, 100 µm.)

Fig. 2.

Division of labor between hepatic cNKs and trILC1s in the control of metastasis. (A and B) Representative dot plots and quantification of cNKs and trILC1s. Samples were pregated on single live CD45⁺lineage⁻ cells and subsequently gated on NK1.1⁺NKp46⁺ cells. cNK = conventional NK cells, CD49a⁻CD49b⁺; trILC1 = tissue-resident ILC1s, CD49a⁺CD49b⁻. (A) Eomes^WT and Eomes^fl livers. Eomes^WT = Ncr1^iCre/wt.Eomes^wt/wt; Eomes^fl = Ncr1^iCre/wt.Eomes^fl/fl. (B) Hobit^WT and Hobit^−/− livers. (C and D) Quantification of the metastatic burden from LLC and MC38 cells in (C) Eomes^WT and Eomes^fl mice and (D) Hobit^WT and Hobit^−/− mice. Quantification of LLC liver nodules was performed by in vivo imaging system at the endpoint. Bioluminescence measurements on the whole liver are shown. Quantification of MC38 macroscopic liver nodules and their area at the endpoint. The area of each hepatic nodule is measured manually using the drawing tool from ImageJ. Each point represented corresponds to the area of one nodule displayed in pixels (px). The bar represents the mean ± SD, and the symbols represent livers from individual mice. Student’s unpaired t test. LLC: The experiment was performed twice with similar results. MC38: Pooled data from four experiments.

Fig. 3.

cNKs but not trILC1s control advanced metastatic disease. (A) Kinetics of NKp46⁺ cell depletion. trILC1s in Eomes^fl.R26R^iDTR mice were depleted by i.p. injection of DTX. cNKs in Hobit^WT and Hobit^−/− were depleted by i.p. injection of anti-Asialo-GM1. Depletion was performed 48 h before, 24 h after, or 7 d after tumor injection and maintained until the endpoint (day 18). i.s. = intrasplenic; Eomes^WT Ctrl = Ncr1^iCre/wt.Eomes^wt/wt.R26R^wt/wt; Eomes^fl.R26R^iDTR Ctrl = Ncr1^iCre/wt.Eomes^fl/fl.R26R^wt/wt; Eomes^fl.R26R^iDTR NKp46⁺ depleted = Ncr1^iCre/wt.Eomes^fl/fl.R26R^iDTR/wt. (B and C, Upper) Quantification of LLC liver nodules was performed by ex vivo in vivo imaging system imaging at the endpoint. Bioluminescence measurements on the whole liver are shown. (Lower) Representative measurements of metastatic livers at endpoint. The bar represents the mean ± SD, and the symbols represent livers from individual mice. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (D and E, Upper) Quantification of MC38 macroscopic liver nodules and area at the endpoint. The area of each hepatic nodule is measured manually using the drawing tool from ImageJ. Each point represented corresponds to the area of one nodule displayed in pixels (px). (Lower) Representative images of metastatic livers at endpoint. The bar represents the mean ± SD, and the symbols represent livers from individual mice. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (F and G) Representative immunofluorescence images of MC38 metastatic livers from (F) Ncr1^iCre/wt.Eomes^fl/fl.R26R^Ai14/wt mice and (G) Ncr1^iCre/wt.Hobit^−/−.R26R^Ai14/wt mice. (H) Quantification of the localization of cNKs and trILC1s. Groups contained three to four MC38 metastatic livers from Ncr1^iCre/wt.Eomes^fl/fl.R26R^Ai14/wt mice (trILC1s) and Ncr1^iCre/wt.Hobit^−/−.R26R^Ai14/wt mice (cNKs), each symbol represents a liver section. (I) trILC1s and (J) cNKs were sorted from naïve, LLC metastatic, or MC38 metastatic livers and incubated with the respective cell lines at different effector:target ratios for 12 h. The percentage of specific killing was calculated as follows: [(% cell death in the presence of NKp46⁺ cells) − (% spontaneous cell death)] ÷ (100 − % spontaneous cell death). The results are displayed as mean of 3 replicates ± SD. Student’s unpaired t test: **P = 0.0076, ****P < 0.0001. The experiment was performed twice with similar results.

Fig. 4.

Metastatic livers drive the differentiation of unique cNK populations. NKp46⁺ cells from naïve and day 21 metastatic (nodules and adjacent tissue) livers were analyzed by multiparameter single-cell mapping using flow cytometry. (A–G) LLC metastatic and control livers. (H–N) MC38 metastatic and control livers. Samples were pregated on single live CD45⁺lineage⁻ cells and subsequently gated on NK1.1⁺NKp46⁺ cells. cNK = conventional NK cells, CD49a⁻CD49b⁺; trILC1 = tissue-resident ILC1s, CD49a⁺CD49b⁻. (A and H) UMAP projections overlaid with FlowSOM-guided manual metaclusters displaying cNKs and trILC1s from all samples. (B and I) UMAP projections overlaid with FlowSOM-guided manual metaclusters separated by sample category (naïve, adjacent, nodules). (C and J) Relative frequency of each cluster in the different sample categories (naïve, adjacent, nodules). (D and K) Heat map summary of median marker expression values of the different markers analyzed for each cluster. (E and L) The frequency of unique metastasis-induced subsets. (E) CD49a⁻Eomes⁻ NKp46⁺ cells in LLC metastasis. (L) CD49a⁺Eomes⁺ NKp46⁺ cells in MC38 metastasis. The bar represents the mean ± SD, and the symbols represent livers from individual mice. One-way ANOVA with Tukey’s multiple comparisons test. Experiments were performed at least twice with similar results. (F and M) Representative dot plots of cNKs and trLC1s cells for each sample category based on their expression of CD49a and Eomes. (F) LLC metastasis. Highlighted in yellow is the CD49a⁻Eomes⁻ population observed in LLC-adjacent tissue and nodules. (M) MC38 metastasis. Highlighted in green is the CD49a⁺Eomes⁺ population observed in MC38 nodules. (G and N) Representative dot plots of cNKs and trILC1s isolated from naïve liver or metastatic nodules from Hobit^−/− and Eomes^fl mice based on their expression of CD49a and Eomes. (G) LLC metastasis. Highlighted in yellow is the CD49a⁻Eomes⁻ population observed in LLC-adjacent tissue and nodules. (N) MC38 metastasis. Highlighted in green is the CD49a⁺Eomes⁺ population observed in MC38 nodules. Groups contained three to five mice. Experiments were performed at least twice with similar results.

Fig. 5.

The MC38- but not the LLC-derived metastatic microenvironment preserves functional features of cNKs and trILC1s. Flow cytometry analysis of (A) Granzyme B (GrzB), (B) CD107a and (C), IFN-γ expression by cNKs and trILC1s cells from naïve and metastatic livers. Metastatic livers were manually dissected to separate the nodules from the adjacent tissue, and tissues were enzymatically processed into a single-cell suspension. Granzyme B was stained intracellularly directly ex vivo; CD107a and IFN-γ were stained after 4 h of stimulation by plate-bound anti-NK1.1. Samples were pregated on single live CD45⁺lineage⁻ cells and subsequently gated on NK1.1⁺NKp46⁺ cells. cNK cells (CD49a⁻ cells) were divided into CD49b⁺Eomes^high/int cells (named here cNKs and present in naïve and both LLC and MC38 metastatic livers), CD49b⁻Eomes⁻ cells (present in LLC-derived metastatic livers), and CD49b⁺Eomes⁺ cells (present in MC38-derived nodules). trILC1s were gated as CD49a⁺CD49b⁻ cells. (Left) Representative dot plots showing the expression and gating strategy for GrzB⁺ (A), CD107a⁺ (B), and IFN-γ⁺ (C) cells in cNKs (blue), trILC1s (red), CD49a⁻Eomes⁻ cells (yellow), and CD49a⁺Eomes⁺ cells (green). (Right) Percentages of GrzB⁺ (A), CD107a⁺ (B), and IFN-γ⁺ (C) cells. The bar represents the mean ± SD, and the symbols represent livers from individual mice. One-way ANOVA with Tukey’s multiple comparisons test. Groups contained five and six mice. Experiments were performed at least twice with similar results.

Fig. 6.

Single-cell RNA-seq reveals unique transcriptional signatures of cNKs in the hepatic metastatic niche. Single-cell RNA-seq was performed on NKp46⁺ cells sorted from naïve, LLC metastatic, and MC38 metastatic livers (six mice per condition pooled into one sample for droplet encapsulation and library preparation). (A–C) UMAP projections identifying the different cNK/trILC1 clusters in (A) all samples or (B) individual samples with (C) bar plots showing the relative frequency of the different clusters in each sample. (D) Top up-regulated genes in cluster cNK_4, cNK_5, and cNK_6 arranged in functional categories. (E) Pathway analysis of significantly up-regulated genes in cluster cNK_6. (F) cNKs and trILC1s were sorted from naïve livers and cultured in vitro for 48 h with 25 ng/mL mouse IL-15/IL-15R complex and/or 10 ng/mL human TGF-β1. The transcripts of cNK_6 cluster-specific genes were quantified by qPCR. The heat map shows the average fold change relative to the untreated condition. The data represent three biological and three technical replicates, and the experiment was performed three times with similar results.

Fig. 7.

IL-15 and TGF-β modulate the fate of CD49a⁺Eomes⁺ cNKs in metastatic nodules. (A and B) Quantification of transcripts of (A) Tgfb1, (B) Il15, and (C) Il15ra in lysates of naïve, LLC and MC38 metastatic livers. Bars show the mean ± SD. Each symbol represents an individual mouse. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (D, Upper) Quantification of LLC liver nodules from Tgfbr2^WT and Tgfbr2^fl mice by in vivo imaging system (IVIS) 21 d after tumor cell injection. Bars show the mean ± SD. Each symbol represents an individual mouse. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (Lower) Representative IVIS images of metastatic livers from each group at the endpoint. Tgfbr2^WT = Ncr1^iCre/wt.Tgfbr2^wt/wt ; Tgfbr2^fl = Ncr1^iCre/wt.Tgfbr2^fl/fl. (E, Upper) Quantification of macroscopic MC38 liver nodules (number and area) from Tgfbr2^WT and Tgfbr2^fl mice 21 d after tumor cell injection. Bars show the mean ± SD. Each symbol represents an individual mouse. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (Lower) Representative images of metastatic livers from each group at the endpoint. Tgfbr2^WT = Ncr1^iCre/wt.Tgfbr2^wt/wt; Tgfbr2^fl = Ncr1^iCre/wt.Tgfbr2^fl/fl. (F, Upper) Percentage of CD49a⁺Eomes⁺ NKp46⁺ cells in MC38 nodules from Tgfbr2^WT and Tgfbr2^fl mice. Bars show the mean ± SD. Each symbol represents an individual mouse. One-way ANOVA with Tukey’s multiple comparisons test. The experiment was performed twice with similar results. (Lower) Representative dot plots of cNKs and trILC1s in MC38 nodules from Tgfbr2^WT and Tgfbr2^fl mice with the CD49a⁺Eomes⁺ population highlighted in green.