Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice

Abstract

Background & aims: Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts.

Methods: We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras^G12D/+;LSL-Trp53^R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11^+/+ and Cdh11^-/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11^+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored.

Results: Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11^+/- and KPC/Cdh11^-/- mice survived significantly longer than KPC/Cdh11^+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11^+/+ mice and incompletely in KPC/Cdh11^+/- and KPC/Cdh11^-/- mice, whose lesions also contained fewer FOXP3⁺ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11^+/+ mice, tumors of KPC/Cdh11^+/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11^+/- and KPC/Cdh11^-/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11^+/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice.

Conclusions: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Keywords: Activated Stroma; Anti-Tumor Immunity; Desmoplasia; Immunomodulation; Inflammation.

Figure 1.. CDH11 is significantly increased in human and mouse PDAC CAFs and pancreatitis stroma.

(A) CDH11 mRNA in pancreatic carcinoma, ductal (d.) adenocarcinoma, and pancreatitis compared to normal pancreas. Box defines 25^th to 75^th percentiles, horizontal line defines median, and whiskers minimum and maximum. The fold change (F.C.) is indicated. P-values were determined by unpaired two-tailed t-test. Representative histology of: (B) human normal pancreas and PDAC stained with MTS and CDH11 IHC (5B2H5); (C) human pancreatitis stained with H&E and CDH11 IHC (5B2H5); (D) mouse PanIN and PDAC isolated from KPC mice stained with MTS and CDH11 IHC (5B2H5). (B-D) Black arrows represent CDH11-positive staining in CAFs; asterisks depict CDH11-negative cancer epithelial cells; red arrows represent positive MTS staining; NC – negative control for CDH11 IHC; scale bars: 50 μm. (E) CDH11 mRNA expression in human and mouse PDAC epithelial cell lines and primary human PSC and CAF cell lines. MDA-MB-231 cell line was used as a positive control for CDH11 expression. (F) Immunoblot for CDH11 (5B2H5) and E-cadherin in PDAC epithelial, PSC and CAF cell lines. (G) Immunofluorescent staining of primary human CAF cell line, 1A1399 for CDH11 (5B2H5), αSMA and NC. Scale bars: 50 μm. Single cell RNAseq analysis of (H) KPC mouse tumors (GSE114417) and (I) immune-cell-depleted s.c. mT3 pancreatic tumors. Cell clusters from 10x Genomics scRNAseq analysis visualized by Uniform Manifold Approximation and Projection (UMAP). Feature plots show Cdh11 expression in different cell types from pancreatic cancer microenvironment (CAFs in green ovals). Colors indicate clusters of various cell types.

Figure 2.. Loss of CDH11 significantly prolongs survival of PDAC-bearing mice, affects stromal activation and FOXP3⁺ cell localization.

(A) A Kaplan-Meier plot for KPC/Cdh11^+/+, KPC/Cdh11^+/− and KPC/Cdh11^−/− mice. P-values were determined by log-rank Mantel-Cox test. (B) Mass of pancreata at the time of euthanasia. Each data point represents a measurement from an individual mouse. P-values were determined by unpaired two-tailed t-test. (C) Representative pictures of αSMA IHC staining of pancreata. Green line outlines early PanIN lesions. Scale bars: 100 μm. (D) Quantification of αSMA⁺ IHC staining represented as % area of pancreata. P-values were determined by unpaired two-tailed t-tests. (E) Representative pictures of FOXP3 IHC staining of PDAC tumor center (>300 μm from the tissue margin) from KPC mice. Yellow arrows represent FOXP3-positive staining. Scale bars: 100 μm. Quantification of FOXP3⁺ cells in the (F) center, (G) periphery, and (H) ratio of FOXP3⁺ cells in center versus periphery of KPC mice. Minimum five pictures taken at 40x magnification from the center, and the periphery of each tumor were analyzed. Each data point represents an individual PDAC sample. P-values were determined by unpaired two-tailed t-tests. (I) TLS quantification in H&E stained pancreata of KPC mice using ImageJ. Each data point represents a ratio of total TLS area over pancreata area from an individual mouse. P-values were determined by unpaired two-tailed t-test.

Figure 3.. PDAC tissues from KPC/Cdh11^+/− mice are associated with increased anti-tumor immunity, and decreased immunosuppression in comparison to KPC/Cdh11^+/+.

(A-H) Differential gene expression analysis of pooled PDAC samples from KPC/Cdh11^+/− mice (n=5) normalized to KPC/Cdh11^+/+ (n=7). Top 20 genes with (A) increased, and (B) decreased expression; and expression of (C) antigen processing and presentation, (D) cDC, (E) M1 macrophage, (F) M2 macrophage, (G) B cell, and (H) MDSC markers. (I) IMC for CD4, CD8a and CD19 proteins coupled with RNAscope for Ccl21a mRNA performed on PDAC samples from KPC/Cdh11^+/+ and KPC/Cdh11^+/− mice. Bigger panels represent merge staining, and smaller panels represent single staining of the same region. DapB was used as a negative control (NC) for mRNA detection by RNAscope. Six regions of interest per PDAC sample (n=3 mice per genotype) were analyzed and quantified by ImageJ using integrated density (IntDen) of the signal. P-values were determined by unpaired two-tailed t-test. Arrowheads point to positive staining; scale bars: 50 μm. Cytokine analysis of pooled (J) serum, and (K) PDAC samples of KPC/Cdh11^+/+ (n=3 serum; n=7 tumor) and KPC/Cdh11^+/− mice (n=3 serum; n=6 tumor), performed in duplicates. P-values were determined by unpaired two-tailed t-test, and reported as *P<.05, **P<.01, ***P<.001.

Figure 4.. Loss of Cdh11 promotes response to gemcitabine.

A Kaplan-Meier plot of KPC/Cdh11^+/+ versus KPC/Cdh11-deficient mice, KPC/Cdh11⁻ (KPC/Cdh11^+/− and KPC/Cdh11^−/−) treated with: (A) PD1 mAb; or (B) gemcitabine (GEM). Duration, dose and treatment schedule are indicated on the graphs. P-value was determined by log-rank Mantel-Cox test. Cytokine analysis of pooled (C) serum, and (D) PDAC samples of KPC/Cdh11^+/+ (n=3 serum; n=4 tumor) and KPC/Cdh11^+/− mice (n=3 serum; n=5 tumor) treated with gemcitabine, performed in duplicates. P-values were determined by unpaired two-tailed t-test, and reported as *P<.05, **P<.01, ***P<.001, ****P<.0001. (E) Representative pictures of CD19 IHC staining of pancreata from gemcitabine treated KPC/Cdh11^+/+ and KPC/Cdh11^+/− mice. Black arrowheads represent CD19-positive staining; PC – positive control; NC – negative control; scale bars: 100 μm. (F) Quantification of CD19⁺ staining of pancreata of 3 KPC/Cdh11^+/+ and 3 KPC/Cdh11^+/− mice. Minimum of eight pictures taken at 40x magnification from each tumor were analyzed. P-value was determined by unpaired two-tailed t-test.

Figure 5.. Effective attenuation of pancreatic cancer growth upon CDH11 inhibition requires the presence of T and B cells.

C57BL/6J mice engrafted with mT3 cells (derived from a PDAC of a KPC C57BL/6 mouse) treated with (A) gemcitabine (GEM), (B) CDH11 mAb, and (C) CDH11 mAb + GEM. (D) Binding model of small molecule CDH11-inhibitor, SD133 (green) with the extracellular domain 1 of CDH11. C57BL/6J mice engrafted with mT3 cells treated with (E) SD133 for 2 weeks, (F) SD133 for 2 weeks + GEM, (G) SD133 for 5 weeks. (H) C57BL/6J mice with pre-existing mT3 tumors treated with SD133 at 40 mg/kg or 10 mg/kg. (I) Immunocompromised Rag1-mutant mice (on C57BL/6J background) with pre-existing mT3 tumors treated with SD133 at 40 mg/kg. The dose, route and treatment schedule are indicated on graphs. P-values for interaction were determined by two-way ANOVA.