Abstract 1610: Notch4 plays distinct roles in different cell types of the breast cancer microenvironment

Abstract The Notch developmental pathway regulates cell fate in tumor angiogenesis and influences immune cell phenotypes in the tumor microenvironment (TME). While Notch receptors Notch1-3 have been extensively studied, the role of Notch4 in the tumor microenvironment is poorly understood. Previous efforts to target Notch signaling and tumor angiogenesis have been limited by broad expression of Notch receptors and their cognate ligands, leading to significant gastrointestinal toxicity. However, Notch4 is thought to be expressed primarily in the endothelium, making it a highly specific and, therefore, more attractive candidate to attenuate Notch signaling in cancer. To this end, we collaborated to develop a first-in-class Notch4 neutralizing antibody, E7011. We demonstrated that E7011 treatment caused reduced tumor growth in multiple tumor types, including mammary tumors, that express Notch4 in tumor endothelium. To specifically examine the role of endothelial Notch4 on breast cancer growth, we used a Notch4 conditional endothelial-specific knockout (ECKO) mouse and implanted orthotopic murine breast cancer cells. We found that tumors implanted into Notch4 ECKO mice were consistently and significantly larger in two distinct models of murine mammary carcinoma, Py8119 and E0771.LMB, suggesting that endothelial Notch4 has an anti-tumor role. These results indicate that the anti-tumor effect of blocking Notch4 with E7011 is mediated by Notch4 in other cell type(s). Single-cell RNA sequencing of Py8119 tumors treated with E7011 revealed that although Notch4 transcripts were found primarily in endothelial cells, the most notable effect of E7011 was a dramatic increase in the number of tissue-resident macrophages (TRMs). Confirmatory immunohistochemistry showed that E7011 alters macrophage populations during multiple stages of tumor development. Analysis of the macrophage transcriptome showed decreased expression of markers associated with anti-inflammatory, “pro-tumor” macrophage populations. To examine whether macrophages are required for treatment efficacy, we performed clodronate-based intratumoral depletion of macrophages. While treatment with E7011 or clodronate reduced tumor volume as expected, co-administration of both E7011 and clodronate was not significantly different from control tumors. This finding suggests that macrophages potentiate the anti-tumor effect of E7011. These findings support distinct and opposing roles for Notch4 on endothelial and macrophage populations in the breast cancer microenvironment. Further studies of the contrasting roles of Notch4 in breast cancer can guide considerations for tailoring anti-Notch therapies. Citation Format: Katherine A. Alexander, Jason W. Eng, Yu Kato, Bhairavi Swaminathan, Debdutta Mandal, L. A. Naiche, Yasuhiro Funahashi, Junji Matsui, Jan Kitajewski. Notch4 plays distinct roles in different cell types of the breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1610.