Notch4 blockade reprograms tumor-associated macrophages and enhances anti-tumor inflammatory signature in breast cancer

Macrophages play a crucial role in the tumor microenvironment (TME) and significantly influence pro- or anti-tumor immunity in breast cancer. Though macrophages often participate in pro-inflammatory tumor surveillance and elimination during early phases of tumor development, during later stages, they may become immunosuppressive, pro-angiogenic, pro-metastatic, and/or facilitative of a pro-tumor TME. The Notch pathway is essential for the differentiation of tumor-associated macrophages (TAMs) through the core canonical transcription factor, RBPJκ. However, individual Notch receptors (Notch1-4) have been shown to have varying effects on TAM polarization, with Notch1 and Notch2 displaying both pro- and anti-inflammatory activity. In peritoneal macrophages, Notch4 responds to inflammatory cues by limiting pro-inflammatory cytokine production and is upregulated during M2 induction. Notch4 promotes breast tumor initiation and maintains breast cancer stem cells. We have previously demonstrated that Notch4 inhibition with a novel, first-in-class neutralizing antibody, E7011, caused reduced tumor growth in mouse models of multiple tumor types, including mammary carcinoma. However, it is currently unclear whether the efficacy of anti-Notch4 treatment is mediated by Notch4 regulation of TAM function in breast cancer. We have demonstrated longitudinal increases in total macrophage populations following Notch4 blockade, accompanied by a decrease in myeloid-derived suppressor cells (MDSCs), which contribute to an immunosuppressive TME. Single-cell RNA sequencing of Py8119 mammary tumors treated with a murine analog of E7011 (6-3-A6) revealed a significant increase in the number of macrophages, and particularly Stab1+ TAMs, a subpopulation of TAMs that exhibits strong immunosuppressive activity and supports tumor growth by dysregulating antigen presentation and inducing resistance to checkpoint inhibitors. Analysis of the Stab1+ TAM transcriptome from tumors treated with 6-3-A6 showed decreased expression of markers associated with anti-inflammatory and “pro-tumor” macrophage activity, and an increase in pro-inflammatory mediators, notably the Dectin-2 pathway. This suggests that Notch4 blockade via 6-3-A6 functionally alters the traditionally pro-tumor Stab1+ TAM cluster towards a tumor-destructive, phagocytic macrophage phenotype. Taken together, these new insights into the functional reprogramming of pro-tumor Stab1+ TAMs towards a pro-inflammatory phenotype and decrease in immunosuppressive MDSCs following Notch4 blockade underscore a role for Notch4 in shaping a pro-tumor microenvironment. Ongoing genetic studies aim to elucidate the mechanism of Notch4-induced TAM immunosuppression and identify opportunities to enhance the efficacy of current immunotherapies by targeting Notch4 blockade in breast cancer.