Abstract 3437: Dynamic waves of anti-and pro-tumoral myeloid and Treg cells are generated in response to cyclophosphamide

Abstract In addition to its direct cytotoxic effects on cancer cells, chemotherapy exerts profound influences on immune cells. These immunological effects must be carefully considered when designing clinically effective combination regimens that include both chemotherapeutic and immunotherapeutic agents. Cyclophosphamide (Cy), a widely used alkylating chemotherapeutic agent, is known to affect various components of the adaptive immune system and natural killer (NK) cells. Based on the observation that a single dose of Cy can lead to increased tumor growth in EMT6 triple-negative breast cancer-bearing mice with predominant myeloid infiltration (but not in 4T1- bearing mice with predominant lymphoid infiltrate), we investigated the immune transcriptional programs triggered by Cy. Using flow cytometry and single-cell transcriptome analysis in tumor-free, tumor-bearing, and Cy-treated mice, we identify novel effects of Cy on neutrophils—innate immune cells increasingly recognized for their roles in both cancer immune tolerance and rejection. One day after Cy administration, a subset of neutrophils expressing an interferon (IFN) signature and associated with anti-tumor activity is expanded, while neutrophils displaying a potentially pro-tumoral Il1b/TNF inflammatory signature and immunosuppressive regulatory T (Treg) cells are reduced. However, in the absence of additional Cy treatment, these effects are reversed by day 10, with the re-emergence of the pro-tumoral neutrophil population. To functionally validate these single-cell findings and directly test the role of myeloid cells in mediating tumor rebound, we performed in vivo immune cell depletion experiments in the EMT6 model. Mice were injected intraperitoneally with neutralizing antibodies against CD11b or Ly6G, targeting myeloid cells and (more selectively) neutrophils, respectively. Depletion efficiency was confirmed by flow cytometry analysis of peripheral blood. Following depletion, mice were orthotopically transplanted with EMT6 cells and treated with a single dose of Cy. Tumor growth in mice receiving either Cy alone or neutralizing antibodies alone was comparable to that of untreated controls, indicating that neither intervention alone was sufficient to control tumor progression. In contrast, the combination of Cy with broad myeloid-cell and selective neutrophil depletion produced a marked anti-tumor effect, resulting in a significant reduction in tumor size compared with all other groups. This experiment represents the functional validation of our single-cell transcriptomic data, suggesting that neutrophils actively drive tumor rebound following transient Cy exposure. These findings highlight the dynamic and time-dependent nature of Cy’s immunomodulatory effects and have implications for the design of effective combinatorial anti-cancer therapies. Citation Format: Andrea Franceschini, Giovanna Talarico, Davide Lombardi, Stefania Orecchioni, Giulia Bravetti, Iros Barozzi, Paolo Falvo, Francesco Bertolini. Dynamic waves of anti-and pro-tumoral myeloid and Treg cells are generated in response to cyclophosphamide abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3437.