Abstract B33: Genomic analysis of immunosuppressive and proangiogenic genes in recombinant HE4 treated immune cells and implications for T-cell cytotoxicity in ovarian cancer cell co-culture
Authors: James Nicole , Emerson Jenna , Borgstadt Ashley , Beffa Lindsey , Urh Anze , Kim Kyukwang , Singh Rakesh , Moore Richard , DiSilvestro Paul , Ribeiro Jennifer 
Published: June 2021

Objective: To determine the effect of HE4 on expression of immune-related genes in CD8+ T cells and explore the role of HE4 in immunosuppression and angiogenesis in epithelial ovarian cancer (EOC).

Methods: Tissue HE4 levels and T cell counts were determined by immunohistochemistry (IHC). Recombinant HE4 (rHE4)-mediated gene expression in CD8+ T cells was measured by qPCR array. Cytokine levels were determined using a human cytokine array. Cell viability of ovarian cancer cells co-cultured with pooled peripheral blood mononuclear cells (PBMC) in the presence or absence of rHE4 was determined using MTS cell viability reagent. P-values were determined by unpaired 2-tailed student t test. Activation of STAT3 was evaluated by Western blot, and the interaction of STAT3 and HE4 was queried by immunoprecipitation of STAT3. Finally, HE4 and IL8 levels were measured by IHC in a microarray of human EOC and normal adjacent tissue (NAT), and levels were correlated using Spearman Rank test.

Results: Tissue HE4 levels correlated with CD8+ T cell counts in EOC. rHE4 treatment of CD8+ T cells upregulated expression of a panel of immunosuppressive and proangiogenic genes, including VEGF, HIF1A, STAT3, IDO1, FOXP3, and IL8. IL8 was most robustly upregulated (99-fold vs control). HE4 also resulted in alterations in cytokine levels in ovarian cancer cells co-cultured with PBMCs. OVCAR8 and SKOV3 ovarian cancer cell lines were treated with rHE4, and levels of phospho-STAT3—a known regulator of IL8—were measured. HE4 was found to activate STAT3 in a time-dependent manner, which occurred in the absence of a physical interaction between HE4 and STAT3. rHE4 treatment of OVCAR8 and SKOV3 cells co-cultured with PBMCs suppressed PBMC-mediated cytotoxicity. Finally, IL8 and HE4 mean levels were elevated in EOC tissue compared to NAT and significantly correlated (R=0.49332; p=0.00142).

Conclusions: HE4 upregulates expression of genes involved in promoting a proangiogenic and immunosuppressive microenvironment in EOC, which may play a role in reduced cytotoxic ability of immune cells. Upregulation of IL8 may occur via HE4 regulation of STAT3 signaling. Future directions include testing the effect of HE4 on endothelial cell angiogenesis and determining if STAT3 and VEGF inhibitors modulate HE4 suppression of immune cell cytotoxicity.

Citation Format: Nicole James, Jenna Emerson, Ashley Borgstadt, Lindsey Beffa, Anze Urh, Kyukwang Kim, Rakesh Singh, Richard Moore, Paul DiSilvestro, Jennifer Ribeiro. Genomic analysis of immunosuppressive and proangiogenic genes in recombinant HE4 treated immune cells and implications for T-cell cytotoxicity in ovarian cancer cell co-culture [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B33.