Loss of Cadherin-11 in pancreatic ductal adenocarcinoma alters tumor-immune microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is one of the top five deadliest forms of cancer with very few treatment options. The 5-year survival rate for PDAC is 10% following diagnosis. Cadherin 11 (Cdh11), a cell-to-cell adhesion molecule, has been suggested to promote tumor growth and immunosuppression in PDAC, and Cdh11 inhibition significantly extended survival in mice with PDAC. However, the mechanisms by which Cdh11 deficiency influences PDAC progression and anti-tumor immune responses have yet to be fully elucidated. To investigate Cdh11-deficiency induced changes in PDAC tumor microenvironment (TME), we crossed p48-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ (KPC) mice with Cdh11+/- mice and performed single-cell RNA sequencing (scRNA-seq) of the non-immune (CD45-) and immune (CD45+) compartment of KPC tumor-bearing Cdh11 proficient (KPC-Cdh11+/+ ) and Cdh11 deficient (KPC-Cdh11+/- ) mice. Our analysis showed that Cdh11 is expressed primarily in cancer-associated fibroblasts (CAFs) and at low levels in epithelial cells undergoing epithelial-to-mesenchymal transition (EMT). Cdh11 deficiency altered the molecular profile of CAFs, leading to a decrease in the expression of myofibroblast markers such as Acta2 and Tagln and cytokines such as Il6, Il33 and Midkine (Mdk). We also observed a significant decrease in the presence of monocytes/macrophages and neutrophils in KPC-Cdh11+/- tumors while the proportion of T cells was increased. Additionally, myeloid lineage cells from Cdh11-deficient tumors had reduced expression of immunosuppressive cytokines that have previously been shown to play a role in immune suppression. In summary, our data suggests that Cdh11 deficiency significantly alters the fibroblast and immune microenvironments and contributes to the reduction of immunosuppressive cytokines, leading to an increase in anti-tumor immunity and enhanced survival.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer with a limited survival rate of ~10% over five years (1).In general, by the time a patient presents with symptoms, the disease has advanced to a surgically unresectable stage and likely metastasized to other vital organs leading to rapid mortality (2).Since conventional chemotherapy (FOLFIRINOX, gemcitabine, gemcitabine + abaraxane, etc.) prolongs patients' lives for only a few months (3), it is necessary to identify new therapies or better therapeutic targets.
Our immune system, when activated, can elicit an antitumor response that can have long-term clinical benefits and could contribute to prolonged survival.Infiltration of immune cells into the tumor microenvironment (TME) has been associated with various disease prognoses depending on the type of immune cells present and has been leveraged to improve patient survival through immunotherapy in several types of cancers (4,5).For example, T cells are conventionally the focus of already approved immunotherapies (6)(7)(8)(9), and B cells show great promise for future immunotherapies as high B cell infiltration correlates with better survival in PDAC patients (10).As compared to "hot" tumors with inflammation and infiltrated T cells, PDACs are considered to be immunologically "cold" with low levels of tumorinfiltrating lymphocytes, which presents a challenge to established immunotherapies (11)(12)(13).A deeper understanding of PDAC immunobiology is necessary to make PDACs amenable to immunebased therapies.
PDAC tends to be surrounded by cells that suppress anti-tumor immune responses.Major immune suppressive cells in the TME include tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs) and regulatory T cells (Treg) (14).These immunosuppressive cells hinder CD4 and CD8 T cell response as well as the ability of natural killer (NK) and antigen-presenting cells (APC) to exert effective tumor surveillance, consequently leading to an inhibition of the anti-tumor immune responses (15).Cancer associated fibroblasts (CAFs) are another major cell type in the TME that can contribute to immunosuppression (14).CAFs are a vastly heterogeneous cell population and are the most prominent stromal cell type in pancreatic cancer (16).CAFs promote tumor proliferation, invasion and metastasis by secreting various growth factors and cytokines and by modifying the tumor extracellular matrix (ECM) (14,17,18).In addition, CAFs contribute to an immunosuppressive microenvironment through secretion of multiple cytokines and chemokines and reciprocal interactions with immune cells that mediate the recruitment and functional differentiation of these cells (16).A deeper understanding of the PDAC TME, specifically the coordinated actions of tumor supporting immune cells and CAFs against lymphocytes is necessary to make immune-based therapies feasible for effectively treating PDAC.
Cadherin 11 (Cdh11) is a mechanosensitive transmembrane protein involved in cell adhesion (19,20) and plays a role in WNT signaling by modulating b-catenin (21,22).In PDAC, it is primarily expressed by CAFs, and it was recently shown that Cdh11 deficiency induces antitumor immunity, reduces immunosuppression, and increases survival in tumor bearing mice (23).Furthermore, the administration of a small molecule inhibitor blocking Cdh11 increased the efficacy of gemcitabine, a common anti-cancer chemotherapeutic (23).However, the Cdh11 inhibitor, was ineffective in reducing tumor burden of mT3 pancreatic tumor bearing immunosuppressed Rag1-mutant mice, suggesting that T and B cells are required for immunomodulation of PDAC mediated by Cdh11 inhibition (23).Also, Cdh11 deficiency induced immune memory in Cdh11 -/-mice that cleared tumors; these mice did not form new tumors upon subsequent re-challenges with the same cancer cells (23).The cellular and molecular mechanisms behind Cdh11 deficiency-induced antitumor activity and its relationship to immunosuppression are not yet fully understood.
Understanding how Cdh11 promotes an immunosuppressive tumor microenvironment in PDAC will provide invaluable insights into developing new clinical approaches for effective eradication of cancer cells, in solid tumors that are classically immunodeficient.Using an established genetically engineered PDAC mouse model (GEMM) (p48-Cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ) that has a median survival of 5 months (known as KPC mice) (24), we investigated Cdh11-deficiency induced changes in PDAC immune microenvironment.Using single-cell RNA sequencing (scRNA-seq) we compared the tumor microenvironment of Cdh11-deficient (KPC-Cdh11 +/-) and wildtype (KPC-Cdh11 +/+ ) mice with pancreatic tumors and identified immune subpopulations that correlate with decreased tumor burden and improved survival.Our study suggests that Cdh11 deficiency alters the molecular profiles of CAFs resulting in decreased expression of immunosuppressive cytokines within the TME.We also observed an increase in T cell infiltration and a loss of TAMs/MDSCs and neutrophils in the tumor of KPC-Cdh11 +/-mice and identified several genes differentially expressed in these populations as a result of Cdh11-deficency.Knowledge of immune cell subtypes and genes found as altered in the TME as a result of a Cdh11deficiency and their relationship to tumor prognosis will provide a basis for further development of novel therapies for PDAC.

Animal husbandry
Cdh11 -/-(https://www.jax.org/strain/023494)(25) and KPC (p48-Cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ ) mice were bred to each other to generate cohorts of KPC-Cdh11 +/+ , and KPC-Cdh11 +/-as previously described (23).Mice were housed in a pathogen-free environment under standard conditions at Georgetown University (GU) and Lawrence Livermore National Laboratory (LLNL).All animal work was conducted under approved Institutional Animal Care and Use Committee (IACUC) protocols at GU and LLNL and conformed to the National Institute of Health (NIH) guide for the care and use of laboratory animals.

scRNA-seq and data analysis
Single-cell libraries were prepared from sorted cell populations from KPC-Cdh11 +/+ , KPC-Cdh11 +/-and Cdh11 +/-mice using the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA; catalog no.1000075) on a 10x Genomics Chromium Controller following manufacturers protocol and sequenced using an Illumina (San Diego, CA, USA) NextSeq 500 sequencer as described before (26).The scRNA-seq data was demultiplexed and aligned against mouse reference genome mm10 using Cell Ranger Single-Cell Software Suite (10x Genomics, Pleasanton, CA, USA) to obtain Unique Molecular Identifier (UMI) counts.Subsequent analysis was performed in Seurat (27) as described before (26).Briefly, after pre-processing, normalization, feature selection and data scaling, data from various experimental groups were integrated to generate an integrated dataset.Subsequently, dimensional reduction by principal component analysis (PCA), clustering, Uniform Manifold Approximation and Projection (UMAP) reduction, and visualization of clusters were performed in Seurat as described before (26).Genes differentially expressed between clusters were identified using 'FindMarkers' function implemented in Seurat.For each experimental group, the cell type proportions were estimated as a ratio of the number of cells in each cell cluster relative to the total number of cells sequenced.Any CD45 + clusters found in nonimmune scRNA-seq data or CD45 -clusters found in immune data were excluded from the analysis.

Analysis of human PDAC samples
Human PDAC scRNA-seq data from early and metastatic tumors (28) were obtained from the Gene Expression Omnibus database (GSE205013).The raw barcode, feature, and matrix were downloaded and analyzed using Seurat (29), as described above to identify the cell types and cell type-specific gene expression as outlined in the original analysis (28).Correlation between CDH11 expression and immune cell infiltration was determined using TIMER (http://timer.cistrome.org/).Correlation between the expression of CDH11 and other genes in human tumors was determined using TNMplot (https://tnmplot.com/).UALCAN (https://ualcan.path.uab.edu) was used to determine protein expression of CDH11 in PDAC patient tumors, using data from Clinical Proteomic Tumor Analysis Consortium (CPTAC).

Cytokine array analysis
Sera and pancreases collected at the time of euthanasia from KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice were analyzed for cytokine expression using Mouse XL Cytokine Array (R&D Systems).Equal amount of protein lysates or serum belonging to the same experimental group were pooled together before analysis.This data has been previously described (23).

Statistical analysis
Two-proportion Z-test was used to identify statistically significant differences between cell proportions.A p value <0.05 was considered as significant.

Cdh11 deficiency alters CAF profile in PDAC
To investigate the role of Cdh11 in PDAC, we isolated nonimmune (CD45 -) and immune (CD45 + ) cells from pancreases of KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice and both fractions were analyzed separately using scRNA-seq (Figure 1A).First, nonimmune scRNA-seq data was computationally analyzed to determine Cdh11 deficiency-induced changes in cancer and stromal cells.CD45 -cells were clustered using an unbiased clustering approach which identified eleven clusters with distinct gene expression profiles (Figures 1B-D).The identity of each cluster was determined based on the expression of previously established cell type-specific markers (Figures 1B, C) (23,26,30,31).
To further understand Cdh11 deficiency-induced changes in CAFs, we extracted cells from CAF clusters (cluster 0, 1 and 7; Figure 1B) and analyzed them in more detail.This analysis identified three CAF subtypes including Acta2 high myofibroblasts (myCAFs), Il33 high inflammatory CAFs (iCAFs), and a Thy1 high fibroblast cluster (Figures 2A-C).Consistent with our previous findings (23), CAFs from KPC-Cdh11 +/+ mice expressed higher levels of Acta2 than KPC-Cdh11 +/-mice (Figure 2D).Expression of other myofibroblast markers such as Tagln and Myl9 were also elevated in KPC-Cdh11 +/+ mice (Figure 2D).We also identified several other genes differentially expressed between KPC-Cdh11 +/+ and KPC-Cdh11 +/-derived CAFs.These genes included cystatin C (Cst3), inflammatory cytokines Ccl11, Il33, Il11 and Il6, insulin growth factor 1 (Igf1) and heparin binding growth factors midkine (Mdk) and pleiotrophin (Ptn), all of which had higher expression in KPC-Cdh11 +/+ mice.Proteases Adamts9 and Mmp13 and Wnt inhibitor Dkk2 were found to be upregulated in KPC-Cdh11 +/-mice (Figure 2D).Furthermore, a cytokine array analysis showed increased Il33 and Cst3 expression in both tumor and serum from KPC-Cdh11 +/+ mice compared to KPC-Cdh11 +/-mice, while Il11 was highly enriched in KPC-Cdh11 +/+ serum alone and Ccl11 was enriched in tumor alone (Figure 2E).Il33 is a member of the IL-1 family of cytokines, secreted by a variety of cells including epithelial, endothelial and fibroblast-like cells (32).It has been suggested that the increased expression of IL33 in CAFs promotes tumor growth and metastasis via modulation of the immune system (32).Consistent with the cytokine data, IHC confirmed a higher number of Il33-expressing CAFs in KPC-Cdh11 +/+ mice (Figure 3, S4).Il11, Il6, Ccl11, Mdk and Ptn have also been shown to play key roles in cancer progression and immune modulation (33-38), suggesting that altered expression of these genes in KPC-Cdh11 +/-mice may contribute to changes in tumor immune profile.Igf1 signaling has also been implicated in tumor growth, metastasis, drug resistance in PDAC (39).
Next, we analyzed publicly available human pancreatic cancer data (data from CPTAC) and found that CDH11 protein expression was significantly elevated in tumor samples compared to normal (Figure 2F).Additionally, we observed a strong positive correlation between the expressions of CDH11 and ACTA2 genes in human PDAC (Figure 2G).Consistent with our scRNA-seq data, genes such as IL33 and IGF1 also showed a positive correlation with CDH11 expression while PDGFA was negatively correlated (Figure S5).Together, these findings further confirm the role of Cdh11 in cancer progression and suggest that Cdh11 deficiency in stromal cells may significantly alter the TME.

Cdh11-deficient stroma promotes immune infiltration in KPC tumors
Next, we investigated how the lack of Cdh11 in the stroma impacts the tumor immune microenvironment.CD45-expressing cells purified and quantified by FACS were found in higher proportion in the KPC-Cdh11 +/-than in the KPC-Cdh11 +/+ pancreas (Figure S6A), suggesting an increase in immune infiltration when the Cdh11 is absent from the PDAC stroma.scRNA-seq analyses of these CD45 + cells from KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice identified twelve distinct immune cell subtypes including macrophages, neutrophils, dendritic cells (DCs), plasmacytoid dendritic cells (pDCs), B cells and T cells (Figure 4A).Cells in cluster 0 had high expression levels for members of the B cell receptor (BCR) signaling complex: Cd79a and Cd79b (40).This cluster also expressed Ms4a1 (CD20) and was identified as CD20 hi B cells (Figure 4B).Cluster 8 also expressed moderate levels of Cd79a and Cd79b, in addition to showing enrichment for Jun and Mef2c.This cluster was identified as Jun hi B cells.Cluster 1 had high transcript levels of monocyte/macrophage genes Cd14 and Csf1r, designating this grouping of cells as monocytes/macrophages (Mono-Mac) (41) (Figure 4B).Genes critical to T cell signaling including Thy1, Cd3e and Cd3d were highly expressed in clusters 3, 4, 5 and 10 (42).Cluster 5 had additional markers including Foxp3 and Rora and was identified as Foxp3 T cells while cluster 4 was identified as Cd8 T cells based on the expression of cytotoxic T cell markers Cd8a and Nkg7 (Figure 4B).Cluster 10 represented a small proportion of cells expressing high levels of Il22 and Ccr6 and was identified as Il22 hi T cells.Cluster 2 was identified as neutrophils based on high expression of S100a8 and S100a9 and cluster 6 was annotated as proliferating cells based on the expression of Mki67 and Top2a (Figures 4A, B).Cluster 7 expressed DC markers while cluster 11 expressed markers of pDCs.Jchain and Igkc, two genes highly expressed in plasma cells were enriched in cluster 9 (43)(44)(45)(46).
A survey of The Cancer Genome Atlas (TCGA) suggests that CDH11 expression is also elevated in many other cancers including breast cancer (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck cancer (HNSC), stomach adenocarcinoma (STAD) and glioblastoma (GBM) (Figure S9).It has previously been suggested that increased CDH11 expression indicates a poor prognosis in advanced gastric cancer (52) and triple negative breast cancer (TNBC) (22).These findings suggest that Cdh11 plays a role in multiple cancer types and Cdh11 inhibition may promote survival in these cancers.

Discussion
Here we describe several key cellular and molecular changes in stromal and immune cell populations associated with the loss of Cdh11 in PDAC.Particularly, we show significant changes in CAFs in response to the Cdh11 deficiency.In both human and mouse PDAC, CDH11 was primarily expressed by CAFs.CAFs from KPC-Cdh11 +/-mice had significantly lower expression of myofibroblast markers and several immune modulatory factors including Il6, Il11, Il33, Ccl11, Mdk and Ptn.Consistent with this, we observed a reduction in monocyte, macrophage, DC and neutrophil infiltration in these Cdh11-deficient tumors while the proportion of T cells increased.This suggests that Cdh11 deficiency in CAFs may alter the tumor's immune profile.
While changes in infiltrating T cell populations have been previously observed as a result of Cdh11 deficiency ( 23), here we further highlight CD8 + T cells to be correlative with Cdh11 deficient tumors.These cytotoxic T cells may be directly responsible for the reduced tumor burden, and enhanced response to gemcitabine treatment and extended survival of KPC-Cdh11 +/-mice relative to KPC-Cdh11 +/+ (23).Consistent with previously reported findings by Peran et al., we also observed a significant decrease in the expression of genes such as Foxp3, Il2rg, Il4ra, Ctla4 and Tnfrsf18 that play a role in differentiation, activation or function of Tregs (47)(48)(49)53), in KPC-Cdh11 +/-pancreases (Figure 4E).
MDSCs in tumors have been identified to block the recruitment of anti-tumor T/NK cells (54,55).Reduction in myeloid cells (Figure 4C) associated with Cdh11 deficiency may have contributed to increased T cell infiltration and enhanced survival observed in these mice (23).Furthermore, several chemokines including Ccl2, Ccl4, Ccl6, Ccl8, Ccl9 and Osm were upregulated in monocyte/macrophages of KPC-Cdh11 +/+ mice, suggesting possible candidates that can be therapeutically targeted to reduce and antibody targeting of CDH11 inhibited EMT and suppressed metastasis in breast cancer (62).Interestingly, KPC-Cdh11 +/+ mice had significantly more EMT cells than KPC-Cdh11 +/-mice.The reduced presence of EMT cells in Cdh11-deficient mice may have also contributed to the enhanced survival observed in these mice (23).In addition to PDAC, increased Cdh11 expression was observed in many other cancers including breast cancer, stomach and colon cancer.These findings suggest that targeting Cdh11 with small molecule inhibitors or function-blocking antibodies may be an effective strategy in treating aggressive tumors including PDACs.
Cdh11 transcripts have been previously found in the peripheral blood as indicators of severe disease as in rheumatoid arthritis (63).The increased presence of CDH11 in the peripheral blood of cancer patients may be indicative of an advanced disease state.The set of immune cell signatures identified in Cdh11-deficient mice may represent hallmarks of positive disease prognosis in pancreatic cancer, and maybe other solid tumors such as breast, head and neck and colorectal cancers.Targeting these specific immune cell subtypes or genes differentially expressed in these immune subpopulations as a result of Cdh11 deficiency may be an effective therapeutic strategy to treat Cdh11expressing cancers and fibrotic disease.

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FIGURE 1Single-cell level analysis of Cdh11 deficiency-induced changes in stromal cells.(A) Experimental design.CD45 + (immune) and CD45 -(non-immune) cells from pancreases of KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice were isolated and both fractions were analyzed separately using scRNA-seq.(B) Non-immune cell clusters from both KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice visualized by Uniform Manifold Approximation and Projection (UMAP).Each color represents a cell type/subtype with distinct transcriptomic profiles.(C) Dot plot showing the expression of cell type markers corresponding to the cell clusters shown in panel (A).Dot size represents the fraction of cells expressing a gene in a cluster and intensity of color indicates the average expression level in that cluster.(D) UMAP plot colored by experimental condition.(E) Graph showing the proportion of various cell types in each experimental group, calculated using scRNA-seq data.(F) Feature plot showing the enrichment of Pdgfra, Cdh11 and myofibroblast marker Acta2 in CAF clusters.(G) IHC showing co-expression of Cdh11 and Pdgfra in KPC-Cdh11 +/+ mice.The dotted boxed area is shown on the bottom with higher magnification.

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FIGURE 4 Characterization of pancreas-derived immune cells.(A) Immune cell clusters from KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice visualized by UMAP.(B) Dot plot showing the expression of immune cell markers in scRNA-seq data from both KPC-Cdh11 +/+ and KPC-Cdh11 +/-mice.Dot size signifies the percentage of cells in that cluster that express a particular gene, while strength of color denotes average expression in that cluster.(C) Bar graph showing the proportion of each cell type in both experimental groups, calculated from the scRNA-seq data.(D) IHC showing CD8 T cell infiltration in tumors.(E) Violin plot showing the differential expression of selected T cell genes in various T cell clusters.