Abstract PS2-10-29: Tumor emboli in inflammatory breast cancer reveals macrophage-TNF-a signaling networks as targetable pathways

Abstract Background: Despite multidisciplinary treatment strategies, overall survival rates for patients with inflammatory breast cancer (IBC) remain dismal. IBC tumors are characterized by diffuse clusters of cells found in dermal tissue and lymphatic vessels, known as tumor emboli, believed to contribute to disease lethality. Yet, the mechanisms behind tumor embolus formation and their interaction with the immune-rich microenvironment (TiME) remain poorly understood. We hypothesized that the presence of tumor-associated macrophages (TAMs) in the TiME of IBC tumor emboli leads to the activation of the TNFα-NFκB-XIAP survival signaling, causing immune evasion and therapy resistance. Methods: Ex vivo tumor emboli/organoids were generated from patient-derived cell lines cultured in lymphatic-like conditions for transcriptomic and proteomic analysis. To validate these preclinical datasets, spatial immunophenotyping was performed on clinical IBC samples. A transgenic CX3cr1 GFP murine model was generated for visualization of macrophage infiltration and tumor emboli within the TiME via a surgically implanted window chamber, enabling intravital imaging and targeting. Results: Firstly, to overcome the scarcity of annotated tumor emboli biospecimens, a novel technique was established to culture tumor emboli organoids recapitulating the physiological conditions of the dermal lymphatics. Using these organoids, transcriptomic and proteomic profiling were performed, revealing differentially expressed genes and proteins compared to monolayer cell cultures, which resembled drug resistance (JI = 0.402, P 0.001). Gene Set Enrichment Analysis of organoid versus monolayer culture revealed significant activation of TNFα and NF-κB signaling, as well as upregulation of inflammatory cytokines and chemokines, including IL-6, CCL20, and CXCL10, implicating enhanced leukocyte chemotaxis. Next, to validate the ex vivo data, 24 biospecimens with tumor emboli underwent spatial immunohistochemical analysis, for immune cell infiltration, revealing TAM enrichment (19/23). Notably, the ratio of cell densities between emboli and stroma was the highest for TAMs (P = 0.1). Other immune cell types less frequently observed in emboli included cytotoxic T cells (12/24), activated B cells (11/24), and regulatory T cells (6/23). Moreover, In the World IBC Consortium cohort (n = 78), 73% of patients with dermal lymphatic emboli failed to achieve pathological complete response (pCR) to neoadjuvant chemotherapy (Odds Ratio = 2.093; Chi-square P = 0.145) suggesting a potential association between the presence of tumor emboli and a drug-resistant phenotype in IBC patients. Finally, to further study macrophage infiltration, a novel murine model with GFP-tagged macrophages (CX3cr1 GFP Nu/Nu) was developed. Intravital imaging of these mice using a dorsal skin window chamber revealed rapid macrophage recruitment to the implanted tumor embolus stroma (P 0.05), suggesting active macrophage infiltration in TiME. Additionally, to target TNFα-XIAP signaling, birinapant-treated IBC cell clusters were implanted in mice, resulting in significant inhibition of tumor spread (P 0.01), despite unchanged macrophage migration, indicating potential benefit of TNFα-XIAP blockade in IBC. Conclusion: Using both preclinical models and patient-derived biospecimens, our research showed that IBC tumor emboli contribute to TAM infiltration in TiME, driving pro-survival TNFα-NFκB-XIAP signaling leading to drug resistance. We identified birinapant as a potential therapeutic agent to disrupt this signaling axis, which paves a path towards the development of new treatments for IBC. Funding: W81XWH2010153, MBG-20-141-01-MBG, R01CA264529. Prevalence of BC-relevant mutations by age cohort Citation Format: P. PAI, C. Van Berckelaer, S. Van Laere, A. Bennion, T. Charity, J. Yang, F. Bertucci, P. Van Dam, G. M. Palmer, S. J. McCall, L. Y. Dirix, N. T. Ueno, G. R. Devi. Tumor emboli in inflammatory breast cancer reveals macrophage-TNF-a signaling networks as targetable pathways abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-10-29.