Ovarian cancer-derived IL-4 promotes immunotherapy resistance

Abstract

Ovarian cancer is resistant to immunotherapy, and this is influenced by the immunosuppressed tumor microenvironment (TME) dominated by macrophages. Resistance is also affected by intratumoral heterogeneity, whose development is poorly understood. To identify regulators of ovarian cancer immunity, we employed a spatial functional genomics screen (Perturb-map), focused on receptor/ligands hypothesized to be involved in tumor-macrophage communication. Perturb-map recapitulated tumor heterogeneity and revealed that interleukin-4 (IL-4) promotes resistance to anti-PD-1. We find ovarian cancer cells are the key source of IL-4, which directs the formation of an immunosuppressive TME via macrophage control. IL-4 loss was not compensated by nearby IL-4-expressing clones, revealing short-range regulation of TME composition dictating tumor evolution. Our studies show heterogeneous TMEs can emerge from localized altered expression of cancer-derived cytokines/chemokines that establish immune-rich and immune-excluded neighborhoods, which drive clone selection and immunotherapy resistance. They also demonstrate the potential of targeting IL-4 signaling to enhance ovarian cancer response to immunotherapy.

Keywords: CCL7; IL-4; Perturb-map; immunotherapy; intratumoral heterogeneity; macrophages; multiplex imaging; ovarian cancer; spatial CRISPR screens; spatial genomics; tumor immunology; tumor microenvironment; tumor neighborhood; type 2 immunity.

Figure 1.. Perturb-map screen of receptor-ligand genes controlling ovarian cancer growth.

A. Schematic depiction of the receptor-ligand PC/CRISPR screen in vitro. B. Log2 fold changes of each PC/CRISPR population in ID8+Cas9 versus ID8-Cas9 in vitro as measured by CyTOF. C. DepMap Chronos scores for human orthologues of the targeted genes and two control genes (MYC and PTEN) in 19 human OvCa cell lines. D. Schematic depiction of the Perturb-map experiment. ID8 carrying the PC/CRISPR library from 1B (ID8^PC/CRISPR) were injected into the peritoneal cavity of immunocompetent mice (n=8 mice). Tissue was collected at 30 days and multiplex imaging performed to spatially resolve Pro-Code expressing cancer cells. E. H&E staining of a representative ovarian tumor mass in the omentum of mice injected with the ID8^PC/CRISPR. The scale bar represents 3 mm. F. Image of the same tissue section as in E stained for the Pro-Code epitopes by multiplex imaging and colored by pseudoflorescence. G. Digital image depicting the identities of each PC tumor lesions of the same tissue section as in panels E and F. H. Volcano plot showing the Log2 fold change differences of cell frequencies for each PC/CRISPR clone in vivo versus pre-injection frequency. In red are enriched and in blue are depleted gene KO. Two-tailed t-test (n=8 mice). I. Frequencies of each PC/CRISPR tumor binned into four categories of tumor size (top right). n=5,431 tumor clones from 8 mice. See also Figure S1 and Table S1.

Figure 2.. Cancer-derived CCL7 controls immune and stromal cell recruitment into the tumor.

A. Wildtype mice were i.p. injected with equal numbers of control^KO or Ccl7^KO ID8 cancer cells (n=4–5 mice/group). After 3 weeks tissues were collected and CyCIF multiplex imaging was performed. Shown are representative images for the indicated markers. Scale bar=100μm. B. Voronoi diagrams representative of Ccl7^KO and control^KO tumors from the mice described in A. C. Comparison of cell numbers (dotplots) and Log2 fold changes of cell numbers (bar graphs) for the indicated cell types between Ccl7^KO and control^KO tumors (from A). Dotplots colored by −log(adj p-value) and sized by count of cells as depicted in the legend (n=4–5 mice/group). Fisher-exact test with Benjamini-Hochberg multiple-testing correction. D. Quantification for the normalized density of the indicated markers in Ccl7^KO and control^KO tumors (n=8–13 ROIs from n=4–7 mice/group, 2 independent experiments) based on MICSSS multiplex imaging. Two-tailed unpaired t-test. E. Heatmap showing the relative expression of the indicated CC chemokine ligand (CCL) genes (plotted by z-score) in macrophages from control^KO vs Ccl7^KO tumors. Data derived from scRNA-seq performed on CD45-enriched cells from Ccl7^KO and control^KO tumors grown in wildtype mice. F. Nearest neighbor proximity analysis for the indicated cell–cancer cell interactions in Ccl7^KO vs control^KO tumors based on CyCIF imaging data (from A). Two-tailed t-test (n=3 mice/group). G. Pearson correlation analysis between CCL7 gene expression and cytotoxic T lymphocyte (CTL) infiltration levels in human OvCa (TCGA; n=426 patients). ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant. See also Figure S2.

Figure 3.. Effect of CCL7 loss on tumor growth and TME is not compensated by CCL7-expressing tumors.

A. Schematic of the clonal competition experiments. Control^KO and Ccl7^KO ID8 were mixed in a 50:50 or 80:20 ratio and i.p. injected into mice (n=3 mice/group, 2 independent experiments). B. Representative image of diaphragm, mesentery, and omentum tissue stained for tags indicative of Ccl7^KO or control^KO cancer cells in mice injected as (A). C. Normalized fold change of Control^KO vs Ccl7^KO ID8 cell abundance in omentum and diaphragm in mice injected with 50:50 mix of cells. Two-tailed t-test (n=3 mice/group, 2 independent experiments). D. Image of omentum tissue stained for tags indicative of control^KO and Ccl7^KO cancer cells along with the indicated markers (representative image from n=3 mice, 2 independent experiments). Scale bar=50μm. E. Immune infiltration scores for the indicated immune cell types in the mixed Ccl7^KO:control^KO tumors in the omentum and diaphragm at 3 weeks post-transplantation. Two-tailed t-test. F. Image of tumor sections stained for CCL7 protein and tags indicative of Ccl7^KO or control^KO cancer cells. Shown is a representative image from n=3 mice. Scale bar=50μm. ****p<0.0001, **p<0.01, *p<0.05. See also Figure S3.

Figure 4.. IL-4 promotes resistance to anti-PD-1 treatment in clonally heterogeneous tumors.

A. Schematic depiction of the Perturb-map experiment with anti-PD-1 treatment. Mice were i.p. injected with ID8^PC/CRISPR cells as in Figure 1 and treated as shown. Tissues were collected from the peritoneal cavity and multiplex imaging was performed to spatially resolve Pro-Code expressing cancer cells. B. Volcano plot showing the differences of tumor frequencies (x-axis) for each PC/CRISPR between anti-PD-1 treated and control tumors. Two-tailed t-test (n=8 mice/group). C. Relative size of each PC/CRISPR tumor in mice treated with anti-PD-1 antibody (outer ring) versus control tumors (inner ring). Il4^KO is in red. Two-tailed t-test (n=8 mice/group, >5000 tumors). D. Schematic depiction of the validation experiment for individually transplanted Il4^KO and control^KO tumors treated with an anti-PD-1 or isotype control antibody. E. Ascites development curve comparison between the indicated mouse cohorts. Mantel-Cox test (n=6–7 mice/group; 3 independent experiments). F. Overall survival curve comparison between the indicated mouse cohorts. Mantel-Cox test (n=6–7 mice/group; 3 independent experiments). Median number of days per group shown in parenthesis. **p<0.01, *p<0.05, ns=not significant. See also Figure S4.

Figure 5.. Cancer cells are the major IL-4 producers in ovarian tumors.

A. Schematic of the CRISPR tdTomato reporter knock-in to the Il4 gene locus. B. Flow cytometry dotplots for tdTomato (Il4-reporter) expression in ID8 cancer cells from ovarian tumors from the omentum and diaphragm. Mice were i.p. injected with ID8^Il4-tdTomato cells and tissue was collected for flow cytometry and imaging analysis. C. Quantification of the percent of tdTomato+ cancer cells (mean+/−standard deviation) from mice injected in B (n=3 mice/group; 2 independent experiments). GFP-positivity was used to delineate cancer cells. D. Immunofluorescence images of tumors from mice i.p. injected with ID8^Il4-tdTomato cells. Images are representative from n=3 mice. Scale bars=100μm (left) and 50μm (right). E. Mice were i.p. injected with Il4^KO or control^KO ID8 cells and after 3 weeks diaphragm and omentum were collected and stained for IL-4 protein by immunohistochemistry (n=5 mice/group). Shown are representative images colored by pseudoflorescence. Inserts shows a 2-fold magnification of the indicated areas. Scale bar=50μm. F. IL-4 (left) and PAX8 (center) protein staining by immunohistochemistry on human ovarian tumor specimens. Merged image (right) colored by pseudofluorescence. The insert shows a 2-fold magnification of indicated area. Scale bar=100 μm. The image is representative from analysis of tumor specimens from 10 different patient tumors. G. Heatmap showing Log2 fold changes of secreted IL4 protein detected by ELISA in the supernatants of OV90 human OvCa cells with KO of the indicated genes. H. ELISA quantification of IL4 concentration in supernatants of OV90 cells with indicated gene KO (n=4 replicates; 2 independent experiments). ****p<0.0001 two-tailed t-test. See also Figure S5.

Figure 6.. Cancer-derived IL-4 promotes the immunosuppressive environment of ovarian tumors.

A. CyCIF image of Il4^KO and control^KO tumors from mice treated with anti-PD-1 or isotype (IgG) antibody and stained with the indicated markers. Image is representative from n=3 mice, 2 independent experiments. Scale bar=50μm. B. Heatmap showing the relative fold difference in density of the specific cell population in the indicated tumor and treatment condition (anti-PD-1 or IgG) compared to control^KO tumors from animals treated with IgG, as determined by CyCIF. Fisher’s exact test with p=0.05 cutoff (n=3 mice/group, 2 independent experiments). C. Ratio of Tregs:CD8 T cells in indicated groups as determined by CyCIF imaging of the tumors. Two-tailed t-test (n=3 mice/group). D. Ratio of CD4+PD1+ T cells:macrophages in indicated groups as determined by CyCIF imaging of the tumors. Two-tailed t-test (n=3 mice/group). E. UMAP plot of macrophages colored by macrophage subtype. scRNA-seq was performed on CD45-enriched cells collected from Il4^KO and control^KO tumors. Subtypes were assigned based on genes defined in human ovarian tumors by Vázquez-García et al. (PMID: 36517593) F. UMAP plot of macrophages profiled by scRNA-seq colored by the sample type. G. Kernel density estimates of macrophages from Il4^KO or control^KO tumors in UMAP space. H. UMAP plot of macrophages with relative expression levels of the indicated macrophage marker genes. I. Heatmap showing z-score for the expression levels of the indicated genes (selected based on their induction by IL-4) in macrophages of control^KO versus IL4^KO tumors. J. Voronoi diagram representation of lymphoid aggregates in Il4^KO and control^KO tumors treated with anti-PD-1 or isotype antibody. Image is representative from n=3 mice, 2 independent experiments. K. Histogram comparing the distribution of lymphoid aggregates by size across the indicated sample groups (n=3 mice/group, 2 independent experiments). L. Cell type density heatmap for the indicated cell types comparing lymphoid aggregates in Il4^KO and control^KO tumors treated with anti-PD-1 or isotype antibody, as determined by CyCIF. Scale bar indicates Log2 fold changes. Fisher’s exact test with p=0.05 cutoff (n=3 mice/group). See also Figure S6.

Figure 7.. Ovarian cancer resistance to anti-PD-1 is via IL-4R-mediated macrophage control.

A. UMAP representation of the IL4 response gene signature in the TME of human OvCa tumors. scRNA-seq data is from human ovarian tumors in the MSK SPECTRUM cohort and described previously. B. UMAP of the macrophage populations of the human OVCA scRNA-seq data (from A). C. UMAP of the IL-4 response gene signature in the macrophage populations of the human OVCA scRNA-seq data (from A). D. Schematic of the treatment regimen of ID8 tumor bearing Il4ra^ΔMs4a3 or wildtype littermate (WT) mice. E. Ascites development curve in the indicated mouse cohorts. Mantel-Cox test (n=6 mice/group; 2 independent experiments). ***p<0.001, ns=not significant. F. UMAP of macrophage subsets in ID8 tumors grown in Il4ra^ΔMs4a3 or WT mice and profiled by scRNA-seq. G. Kernel density estimates of macrophages from indicated tumors in UMAP space (from data in F). H. UMAP plot of macrophages with relative expression levels of the indicated genes. I. Number (top) and fraction (bottom) of cells per each macrophage subtype comparing tumors from Il4ra^ΔMs4a3 or wildtype littermate (control) mice. J. Heatmap showing z-score for the expression levels of the indicated genes in macrophages of tumors from Il4ra^ΔMs4a3 or WT mice. Genes shown represent top differentially expressed mRNAs. K. Ascites development curve in the indicated cohorts of ID8 tumor-bearing mice. Mantel-Cox test (n=5 mice/group). **p<0.01. See also Figure S7.