Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy

Abstract

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of Trp53, Brca1, Pten, and Nf1 and overexpression of Myc and Trp53 ^R172H, which was contrasted with an identical model carrying wild-type Brca1. For homologous recombination-proficient tumors, we constructed genotypes combining loss of Trp53 and overexpression of Ccne1, Akt2, and Trp53 ^R172H, and driven by KRAS ^G12V or Brd4 or Smarca4 overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC. SIGNIFICANCE: We engineered a panel of murine fallopian tube epithelial cells bearing mutations typical of HGSC and capable of forming tumors in syngeneic immunocompetent hosts. These models recapitulate tumor microenvironments and drug responses characteristic of human disease. In a Ccne1-overexpressing model, immune-checkpoint resistance was driven by follistatin.This article is highlighted in the In This Issue feature, p. 211.

Figure 1.. Engineering strategy and in vitro characterization of the murine FTE-derived cells.

Schema showing the strategy for generation of (A) Non-classified and HR-deficient Pax8⁺ murine FTE-derived cells, p53^−/−R172HPten^−/−Nf1^−/−Myc^OE (PPNM) and Brca1^−/−p53^−/−R172HPten^−/−Nf1^−/−Myc^OE (BPPNM) and (B) HR-proficient Pax8⁺ murine FTE-derived cell lines p53^−/−R172HCcne1^OEAkt2^OEBrd4^OE (BPCA), p53^−/−R172HCcne1^OEAkt2^OESmarca4^OE (SPCA) and p53^−/−R172HCcne1^OEAkt2^OEKRAS^G12V (KPCA). (C) Representative images showing immunofluorescence staining of nuclear Paired box 8 (PAX8) (pink) and cytoplasmic Cytokeratin-7 (CK7) (white). Cell nuclei were stained with DAPI (blue). Images were taken with 63x magnification. (D) Representative images showing immunofluorescence staining of RAD51 and γH2AX (upper panel) and the quantification of the number of Rad51 foci per nuclei (lower panel). Cell nuclei were stained with DAPI (blue). Images were taken with 63x magnification. Dose-response curves for p53^−/−, BBPNM, PPNM, BPCA, SPCA, and KPCA cells with the treatment of (E) Carboplatin, (F) Niraparib, and (G) Prexasertib. Cell viability was calculated relative to 0.01% vehicle-treated control cells, measured with Cell TiterGlo assay 72 hours after treatment. Data depicted are pooled from two independent experiments. See also Supplemental Figures 1 and 2.

Figure 2.. In vivo characterization and drug sensitivity of the engineered murine FTE-derived BPPNM, PPNM, and KPCA cells

(A) Genotypes of the engineered murine FTE-derived cell lines used for in vivo characterization. (B) Representative images showing ascites and peritoneal metastasis formation in immune-proficient C57BL/6 mice after intraperitoneal injection with engineered murine FTE-derived cells. Kaplan-Meier curves showing percent probability of survival of mice injected with (C) p53^−/−R172H, p53^−/−R172HPten^−/−, p53^−/−R172H Nf1^−/−, p53^−/−R172H Pten^−/−Nf1^−/−, PPNM, and BPPNM cells and (D) with p53^−/−R172H, p53^−/−R172HCcne1^OEAkt2^OE, BPCA, SPCA, and KPCA cells. n=5 or more/group. (E) Representative images showing Hematoxylin and eosin (H&E) staining and immunohistochemical analysis of indicated markers on harvested BPPNM, PPNM, BPCA, SPCA, and KPCA tumors. Scale bars: 200 μm. (F) Experimental treatment strategy for single-agent Carboplatin, Olaparib, and Prexasertib as two weekly doses via the intraperitoneal administration route for four weeks of duration. Kaplan-Meier curves showing percent probability of survival of mice injected with indicated engineered murine FTE-derived cell lines BPPNM, PPNM, and KPCA upon single-agent treatment with (G) Carboplatin, (H) Olaparib, and (I) Prexasertib. n=5 or more/group. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice, ns- non-significant, *p<0.05, **p<0.001, ***p<0.0001. See also Supplemental Figure 2.

Figure 3.. Cellular microenvironment analyses of omental tumors and ascites from BPPNM, PPNM, and KPCA tumor-bearing mice

. (A) UMAP plot of unbiased clustering the cellular components of merged BPPNM, PPNM, and KPCA omental tumors, where each color-coded cluster represents one cell type/state. Inset relative composition of the clusters. Each point represents one cell that is colored by its cell type/state. (B) Markers used to classify the clusters in panel 3A (see Supplemental Table 3 for the details). Immunophenotypic analysis by multi-parameter flow-cytometry showing the frequency of live adaptive and innate immune cells of representative (C) Omental tumors and (D) Ascites derived from HGSC mouse models. (E) Cytokine analysis of ascites supernatant HGSC mouse models. Data are presented as mean ± SEM, * BPPNM vs KPCA, + PPNM vs KPCA and # BPPNM vs PPNM, *p<0.05, **p<0.001, ***p<0.0001, Multiple t-test. n=5 or more/group. (F) Depicts the transcript level cytokines and chemokine expression within the omental tumors of the HGSC mouse models by scRNA seq cluster analysis (panel 3A and 3B). See also Supplemental Figures 3 and 4.

Figure 4.. Evaluating optimal combination treatment strategies in BPPNM and PPNM tumor models.

Synergy analysis of Prexasertib and Olaparib treatment in (A) BPPNM and (B) PPNM cell lines. Synergy and antagonism between the drugs were determined using SynergyFinder (see material and methods section for more details). Cell viability was calculated relative to 0.01% vehicle-treated control cells, measured with Cell TiterGlo assay 72 hours after treatment. Data depicted are pooled from two independent experiments. (C) Experimental treatment strategy. Kaplan-Meier curves showing percent probability of survival of mice injected with engineered murine FTE-derived cell lines (D) BPPNM and (E) PPNM upon indicated treatment. n=5 or more/group. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice, *p<0.05, **p<0.001, ***p<0.0001. Cartoon models depicting (F) BPPNM and (G) PPNM tumor immune-microenvironment and sensitization strategies. (H) Bulk-tumor RNA-seq analysis showing enrichment of distinct hallmark terms in Brca1-null HR-deficient ovarian model, BPPNM, and non-classified Brca1 wild-type, PPNM model. The figure shows the categories enriched in each comparison. The x-axis shows the normalized enrichment score. Gene sets are shown on the y-axis. The dot size represents the number of genes from the ranked list present on each gene set, and the color indicates the FDR q-value.

Figure 5.. The sensitivity of HR-proficient Ccne1-overexpressing HGSC models to cell cycle checkpoint kinase- and immune checkpoint- inhibitors.

(A) Experimental treatment strategy. Kaplan-Meier curves showing percent probability of survival of mice injected with KPCA cell line (B and C) and KPCA.C cell line (D and E) upon indicated treatment. Data depicted are pooled from three independent experiments. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice. n=5 or more/group, *p<0.05, **p<0.001, ***p<0.0001. (F) Experimental treatment strategy for the depletion of CD8 T-cells using the anti-CD8 antibody. (G) KPCA (H) KPCA.C tumor-bearing mice with triple agents of Prexasertib plus anti-PD-L1 and anti-CTLA-4 combination therapies with or without anti-CD8 treatment. Data depicted are pooled from two independent experiments. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice. n=5 or more/group, *p<0.05, **p<0.001, ***p<0.0001. (I) Experimental treatment strategy for mid-point immune analysis of omental tumor of KPCA and KPCA.C tumor-bearing mice treated with Prexasertib plus anti-PD-L1 and anti-CTLA-4 combination therapies compared to vehicle-matched control mice. Immunophenotypic analysis by multi-parameter flow-cytometry shows the frequency of live adaptive and innate immune cells (J) KPCA (K) KPCA.C omental tumors at mid-point mice treated with Prexasertib plus anti-PD-L1 and anti-CTLA-4 combination therapies compared to vehicle-matched control mice. Data are presented as mean ± SEM, t-test, *p<0.05, **p<0.001, ***p<0.0001. Data depicted are pooled from two independent experiments. See also Supplemental Figures 5 and 6.

Figure 6.. Identification and ablation of Follistatin in Ccne1-overexpressing KPCA.C model to sensitize the tumor to cell cycle checkpoint kinase- and immune checkpoint-inhibitors.

(A) Bulk-tumor RNA-seq analysis showing enrichment of distinct hallmark terms in Ccne1-overexpressing ovarian models. The figure shows the categories enriched in each comparison. The x-axis shows the normalized enrichment score. Gene sets are shown on the y-axis. The dot size represents the number of genes from the ranked list present on each gene set, and the color shows the FDR q-value. (B) UMAP plot of unbiased clustering the cellular components of merged KPCA and KPCA.C omental tumors, where each color-coded cluster represents one cell type/state. Inset relative composition of the clusters. Each point represents one cell that is colored by its cell type/state. (C) sc-RNA-seq analysis showing enrichment of distinct hallmark terms in Ccne1-overexpressing ovarian models. The figure shows the categories enriched in each comparison. The x-axis shows the normalized enrichment score. Gene sets are shown on the y-axis. The dot size represents the number of genes from the ranked list present on each gene set, and the color shows the FDR q-value. (D) Cancer cluster identified by sc-RNA seq of KPCA.C and KPCA omental tumors, depicting genes upregulated in KPCA.C tumors versus KPCA tumors. (E) Follistatin concentration in cell culture supernatants taken from KPCA and KPCA.C cell lines and, KPCA. sgFstKO single-cell clones. (F) In situ hybridization of Follistatin on omental tumor sections derived from KPCA and KPCA.C ovarian models using RNA-scope methodology (Pink= Follistatin). Scale bars: 200 μm. (G) Experimental treatment strategy for mid-point analysis (H) Comparing omental tumor weights of untreated and Prexasertib plus anti-PD-L1 and anti-CTLA-4 combination therapy treated KPCA.C or KPCA.C sgFstKO cohorts. (I) Kaplan-Meier curves showing percent probability of survival of mice injected with KPCA.C or KPCA.C sgFstKO cell line upon indicated treatment. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice. n=5 or more/group, *p<0.05, **p<0.001, ***p<0.0001. The data depicted are pooled from two independent experiments. (J) Follistatin concentration in cell culture supernatants taken from KPCA and KPCA.A cell lines, and KPCA.A sgFstKO single-cell clones. In situ hybridization of Follistatin on omental tumor section derived from KPCA.A ovarian model using RNA-scope methodology (Pink= Follistatin). Scale bar: 200 μm. (K) Experimental treatment strategy for mid-point analysis. (L) Comparison of omental tumor weights of untreated and Prexasertib plus anti-PD-L1 and anti-CTLA-4 combination therapy treated KPCA.A or KPCA.A sgFstKO cohorts. (M) Kaplan-Meier curves showing the percent probability of survival of mice injected with KPCA.A or KPCA.A sgFstKO cell line upon indicated treatment. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice. n=5 or more/group, *p<0.05, **p<0.001, ***p<0.0001. The data depicted are pooled from two independent experiments. See also Supplemental Figure 7.

Figure 7.. Epigenetic features and Follistatin overexpression in Ccne1-overexpressing model

(A) Schema depicting ATAC-seq experimental setup using KPCA, KPCA.C, and KPCA.A cell lines. (B) Chromatin accessibility (ATAC-seq read pileups) in the Fst genomic region. Comparisons are shown for each peak and all four peaks together. (C) Follistatin concentration in cell culture supernatants taken from KPCA and KPCA Fst overexpression (OE) cell lines. (D) Kaplan-Meier curves showing percent probability of survival of mice injected with KPCA or KPCA Fst OE cell line upon indicated treatment. A log-rank test compared the survival curves of individual groups to vehicle-matched control mice. n=5 or more/group, *p<0.05, **p<0.001, ***p<0.0001. The data depicted are pooled from three independent experiments. (E) Bulk-tumor RNA-seq analysis showing enrichment of distinct hallmark terms in KPCA Fst OE and KPCA tumors. The figure shows the categories enriched in each comparison. The x-axis shows the normalized enrichment score. Gene sets are shown on the y-axis. The dot size represents the number of genes from the ranked list present on each gene set, and the color shows the FDR q-value. Cartoon models of (F) KPCA- exceptional responder and (G) KPCA.C-partial responder tumors depicting tumor immune-microenvironment and sensitization strategies. (H) Kaplan-Meier curves depicting progression-free survival (PFS) in patients with CCNE1-amplified HGSCs grouped to High and Low by median FST mRNA expression). (I) Kaplan-Meier curves depicting progression-free survival (PFS) in patients with BRCA1/2-deficient HGSCs grouped to High and Low by median FST mRNA expression (see material and methods for details). See also Supplemental Figure 7.