Ovarian cancer (OvCa) preferentially metastasizes in association with mesothelial cell-lined surfaces. We sought to determine if mesothelial cells are required for OvCa metastasis and detect alterations in mesothelial cell gene expression and cytokine secretion upon interaction with OvCa cells. Using omental samples from patients with high-grade serous OvCa and mouse models with Wt1-driven GFP-expressing mesothelial cells, we validated the intratumoral localization of mesothelial cells during human and mouse OvCa omental metastasis. Removing mesothelial cells ex vivo from human and mouse omenta or in vivo using diphtheria toxin mediated ablation in Msln-Cre mice, significantly inhibited OvCa cell adhesion and colonization. Human ascites induced angiopoietinlike 4 (ANGPTL4) and stanniocalcin 1 (STC1) expression and secretion by mesothelial cells. Inhibition of STC1 or ANGPTL4 via RNAi, obstructed OvCa cell-induced mesothelial cell to mesenchymal transition while inhibition of ANGPTL4 alone obstructed OvCa cell-induced mesothelial cell migration and glycolysis. Inhibition of mesothelial cell ANGPTL4 secretion via RNAi prevented mesothelial cell induced monocyte migration, endothelial cell vessel formation and OvCa cell adhesion, migration, and proliferation. In contrast, inhibition of mesothelial cell STC1 secretion via RNAi prevented mesothelial cell induced endothelial cell vessel formation and OvCa cell adhesion, migration, proliferation, and invasion. Additionally, blocking ANPTL4 function with antibodies reduced the ex vivo colonization of three different OvCa cell lines on human omental tissue explants and in vivo colonization of ID8 p53-/-Brca2-/cells on mouse omenta. These findings indicate that mesothelial cells are important to the initial stages of OvCa metastasis, and that the crosstalk between mesothelial cells and the tumor microenvironment, promotes OvCa metastasis through the secretion of ANGPTL4.
Nicotinamide N-methyltransferase (NNMT) is highly expressed in the stroma of several malignancies and has been shown to drive the transformation of resting fibroblasts into cancer-associated fibroblasts (CAFs) through epigenetic changes. Given that CAFs can promote immunosuppression in cancer, the aim of our study was to investigate the effect of NNMT-expressing CAFs on the tumor-immune environment. Using a co-culture system of normal fibroblasts and activated PBMCs, we found that IFN-γ produced by PBMCs upregulates NNMT expression in fibroblasts. We confirmed this mechanism in vivo in IFN-γ knockout mice and identified T cells as the main source of IFN-γ in the tumor. Gene expression and protein analysis showed that upregulation of NNMT in CAFs induces the secretion of the chemokine CXCL1, a critical chemoattractant for immunosuppressive myeloid-derived suppressor cells (MDSCs). Upregulation of NNMT induces CXCL1 secretion directly through promoter hypomethylation, and in vitro migration assays show that NNMT-expressing CAFs recruit high numbers of MDSCs. Using spectral flow cytometry, we analyzed the tumor-immune cell infiltration in whole-body NNMT-knockout mice and discovered that NNMT knockout of the stroma significantly reduced the abundance of MDSCs and increased the number of functional CD8+ T cells. Increased T cell cytotoxicity was associated with a significant reduction in tumor burden in the syngeneic ID8 ovarian and MC38 colon cancer models. These findings were confirmed after adoptive transfer of NNMT-wildtype immune cells into irradiated whole-body NNMT-knockout mice. Moreover, in scRNA-seq data of various human malignancies, we found a strong correlation between NNMT and CXCL1 expression in CAFs. In summary, our results support a model in which cancer cells induce IFN-γ secretion by T-cells, thereby upregulating NNMT expression in fibroblasts, leading to CXCL1 secretion. CXCL1 secreted by CAFs recruits MDSCs to the tumor, which in turn suppress the anti-tumor T cell response. Citation Format: Janna Heide, Andras Piffko, Agnes J. Bilecz, Ethan A. Teich, Lisa Schweizer, Sayed R. Alhunayan, Kaiting Yang, Ernst Lengyel. Immunoregulatory effects of NNMT-expressing cancer-associated fibroblasts. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4462.
Introduction: High-grade serous ovarian cancer (OvCa) remains the deadliest gynecologic cancer in the United States because it often presents at an advanced stage when the disease is incurable. Despite the impressive clinical success of cancer immunotherapy in patients with lung cancer and melanoma, it is largely ineffective in OvCa patients. Resistance to cancer immunotherapy is mediated by the apoptosis of T-cells, which can be prevented by blocking Fas ligand (FasL), a death gene. However, little is known about the expression and function of FasL in OvCa. Therefore, the aims of this study were to identify the cells in the OvCa tumor microenvironment that produce and secrete FasL, and to delineate the functional role of FasL in T-cell apoptosis within the OvCa tumor microenvironment. Methods: FasL protein localization and levels were evaluated in OvCa tumors by immunohistochemistry (IHC) using a FasL antibody on tissue microarrays (TMAs) containing primary and matching metastases from 72 OvCa patients. In-situ hybridization (ISH) using a FasL specific probe was performed on omental OvCa tumors from pre- (n=12) and post-chemotherapy (n=12) patients to localize FasL mRNA. To define the role of OvCa and endothelial cell derived FasL on T cell apoptosis, co-culture experiments were completed. First, FasL was knocked down with a FasL siRNA or neutralized with a FasL antibody in OvCa (OVCAR8 and OVCAR5) cells or endothelial (HUVEC) cells. Next, the OvCa or endothelial cells were co-cultured with primary human blood mononuclear cell-derived T cells. Subsequently, the level of FasL secreted was measured using a FasL specific enzyme-linked immunoassay, and early (4h; cleaved-caspase 3) and late (24h; TUNEL assay) T cell apoptosis were analyzed by flow cytometry. Results: FasL protein and mRNA are present in OvCa and endothelial cells in the tumor microenvironment of OvCa patients. Higher levels of FasL are detected in the endothelial cells when compared to the OvCa cells in these OvCa tumors. These results were confirmed in vitro as primary human endothelial cells secrete higher levels of FasL when compared to the OVCAR8 and OVCAR5 ovarian cancer cell lines. Functionally, both OvCa and endothelial cells stimulate early and late apoptosis in primary human blood mononuclear cell-derived T cells after a four hour co-culture. Knockdown or neutralization of FasL significantly inhibits the ability of OvCa or endothelial cells to induce early and late T cell apoptosis. In summary, FasL is localized to OvCa and endothelial cells in the OvCa tumor microenvironment, and both OvCa and endothelial cell derived FasL induce T cell apoptosis. Discussion: These findings indicate that blocking FasL may be an effective approach for reducing T-cell apoptosis and improving cancer immunotherapy response in OvCa patients. Citation Format: Grace Keegan, Hilary Kenny, Agnes Bilecz, Ricardo Lastra, Ernst Lengyel. Exploring the role of FasL in the ovarian cancer tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3655.
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