Abstract 1608: Quantifying changes to macrophage recruitment to the breast tumor microenvironment and metastasis in response to Notch4 blockade

The Notch signaling pathway is a highly conserved pathway that regulates cell fate and cell differentiation in multicellular organisms. The Notch pathway plays a vital role in tumor angiogenesis. Notch1 is broadly expressed throughout the body, and preclinical studies demonstrate that targeting Notch1 limits tumor vessel perfusion. However, Notch1 blockade has been linked to gastric toxicity and tissue hypoxia, impeding the pharmaceutical development of Notch1 antagonists. In contrast, Notch4 is primarily expressed in the vascular endothelium and is known to be upregulated in human patients with triple-negative breast cancer (TNBC). High levels of Notch4 expression have been associated with poor overall survival in breast cancer. In mice, implantation of mammary tumors into Notch4 null animals reduces mammary tumor angiogenesis, perfusion, and volume. Notch4 inhibition could enable targeting of the Notch signaling pathway in tumor stroma without disrupting normal organ function, making it a valuable potential therapeutic target in the treatment of breast cancer. Multiple inflammatory diseases have been associated with increased Notch4 expression, suggesting that Notch4 might regulate the immune response. Although Notch4 is mainly expressed on the endothelium, some publications have observed Notch4 expression on immune cells, particularly macrophages. We found that treatment with a novel anti-Notch4 neutralizing antibody, E7011, caused a significant increase in the number of tumor-associated macrophages (TAMs). To test the functional importance of TAMs in the mechanism of E7011, we depleted TAMs with an intra-tumoral injection of liposomal chlodronate before administering E7011. Depletion of macrophages via clodronate abrogated the tumor volume reduction of E7011 treatment, suggesting that TAMs are an important player in E7011 efficacy. Therefore, we hypothesize that tumor-associated macrophage Notch4 is responsible for breast cancer metastasis and tumor progression. My project will center on visualizing the effect of Notch4 inhibition on macrophage recruitment to primary mammary carcinoma tumors and dissemination of lung metastasis by immunohistochemistry. I will section cryopreserved primary tumors and lungs from two syngeneic murine mammary carcinoma models, Py8119 and spontaneously metastatic E0771.LMB. Tumor cells from this line can be visualized by red fluorescent protein (RFP). Then, I will perform immunofluorescent staining of frozen primary tumors to quantify changes in total macrophage (CD68+) recruitment and macrophage Notch4 expression in response to E7011 or control IgG treatment. I will also perform immunofluorescent staining of frozen primary lung sections to quantify lung metastatic colonies (RFP+) from tumor-bearing mice treated with E7011 and or control IgG. Citation Format: Debdutta Mandal, Katherine A. Alexander, Jason L. Eng, Yu Kato, Bhairavi Swaminathan, Christie Kang, L A. Naiche, Yasuhiro Funahashi, Junji Matsui, Jan K. Kitajewski. Quantifying changes to macrophage recruitment to the breast tumor microenvironment and metastasis in response to Notch4 blockade [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 1608.