Abstract 1564: Endothelial Notch activation is essential for the education of tumor associated macrophages
Authors: Mülfarth Ronja , Alsina-Sanchis Elisenda , Moll Iris , Böhn Sarah , Stögbauer Adrian , Rigotti Francesca De Angelis , Wiedmann Lena , Taylor Jacqueline , Fischer Andreas , Vita Juan Rodriguez 
Published: June 2022

Abstract
Epithelial ovarian cancer (EOC) is one of the most lethal gynecological cancers worldwide. Notch1 receptors (N1ICD) are frequently activated in Endothelial cells (ECs) of human tumors to facilitate metastasis. Using VE-Cadherin-CreERT2 transgenic mice to induce gene recombination exclusively in ECs, we explored the role of Notch signaling in ECs in vivo. RbpjiΔEC (VE-Cad-CrERT2xRbpjflox) mice inhibit the canonical Notch signaling upon tamoxifen administration. We modeled metastatic epithelial ovarian cancer (EOC) by injecting the mouse ovarian cancer cell line ID8 intraperitoneally. After four weeks of tumor growth, we observed a significant decreased tumor burden and reduced infiltration of monocyte-derived macrophages into the TME upon EC-specific Rbpj deletion. In vitro, we also confirmed a decreased infiltration of human monocytes through an EC barrier upon Rbpj deletion in human ECs. We isolated tumor-associated macrophages (TAMs) from RbpjΔEC tumor bearing mice and their littermate controls and performed microarray analysis. TCs educate TAMs through CD44-mediated cholesterol depletion. Several bioinformatic analyses showed that TAM from RbpjiΔEC mice were not able to induce the expected signature of TAMs in metastatic EOC because cholesterol depletion cannot take place. This indicates that the TC-induced macrophage education requires the presence of Rbpj in ECs. In vitro experiments showed that CD44 expression was downregulated in monocytes co-cultured with ECs lacking RBPJ. Analysis of the angiocrine factors regulated by Notch signaling revealed CXCL2 as a novel Rbpj-mediated Notch target gene: activation of Notch1 in ECs in vitro induced the production of the chemotactic cytokine CXCL2, whereas Rbpj-deletion (CRISPR-Cas9 technology) in N1ICD transduced ECs abolished CXCL2 expression. In addition, CXCL2 stimulation increased CD44 expression. In keeping with this, the analysis of publicly available ovarian cancer patient’s datasets we found that CXCL2 expression correlates with CD44 expression, suggesting that CXCL2 induces CD44 also in patients. CD44 is a co-receptor of a complex involving CXCR2 and CD74, which without CD44 it is not possible to signal. CD74 expression has been associated with worse prognosis in metastatic ovarian cancer and associated with an immunosuppressive phenotype in macrophages. Bioinformatic analyses of publicly available data showed that Rbpj in ECs is necessary for the immunosuppressive role of CD74. Finally, T cells sorted from RbpjiΔEC tumor bearing mice showed stronger cytotoxic activity (LDH-cytotoxicity assay) than those sorted from their littermate controls. In summary, ovarian cancer cells employ the tumor endothelium to secrete CXCL2 in order to facilitate an immunosuppressive microenvironment.
Citation Format: Ronja Mülfarth, Elisenda Alsina-Sanchis, Iris Moll, Sarah Böhn, Adrian Stögbauer, Francesca De Angelis Rigotti, Lena Wiedmann, Jacqueline Taylor, Andreas Fischer, Juan Rodriguez Vita. Endothelial Notch activation is essential for the education of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1564.