Abstract Tumor necrosis factor-α (TNFα) is a central inflammatory cytokine with paradoxical functions in cancer, capable of promoting robust innate immune activation while simultaneously inducing tissue damage that facilitates metastatic dissemination. Our preliminary data show that TNFα triggers membrane localization and secretion of HSP70 in murine EMT6 breast cancer cells, enabling HSP70 to function as a potent tumor-associated antigen that activates innate immunity and mediates clearance of disseminated tumor cells. To extend these in vivo, we investigated the pleiotropic effects of TNFα in EMT6 orthotopic models, where TNFα-treated EMT6-conditioned medium (EMT6-CM) unexpectedly induced both early systemic immunization and late development of brain metastases.Our preliminary data reveal that EMT6-CM derived from TNFα-treated cells contains high concentrations of soluble TNFα that compromise blood-brain barrier (BBB) integrity, generating a permissive niche for metastatic colonization. Besides, the same CM harbors HSP70-rich extracellular vesicles that promote anti-tumor immune priming in peripheral tissues. Thus, TNFα exerts a paradoxical influence: beneficial through HSP70-mediated innate immune activation, yet detrimental through TNFα-driven BBB disruption that facilitates brain metastatic outgrowth.To mechanistically separate these opposing functions in our ongoing studies, we are selectively depleting TNFα from EMT6-CM using immunoabsorption approaches while preserving HSP70-containing vesicles and soluble fractions. TNFα-depleted CM will be used to immunize BALB/c mice for 1-2 weeks, followed by orthotopic challenge with EMT6-Luc cells. We hypothesize that mice receiving TNFα-depleted, HSP70-enriched CM will reject EMT6-Luc tumors, demonstrating that HSP70-driven immunogenicity is sufficient for tumor protection in the absence of TNFα-induced BBB damage. Successful rejection will validate our model in which TNFα is necessary for BBB disruption but dispensable for HSP70-mediated anti-tumor immunity.Collectively, our findings reveal a previously unrecognized dichotomy in TNFα biology, with significant implications for cytokine-based immunotherapies and mechanisms underlying breast cancer brain metastasis. Understanding how to uncouple TNFα’s immunostimulatory and pathological effects may guide development of safer and more effective immune-modulating strategies. Citation Format: Neva Celiker, Fulya Koksalar Alkan, Hilmi K. Alkan, Ahmet Caglayan, Aminal Lawal, Morhaf Al Achkar, Wicha S. Max, Hasan Korkaya. TNF-alpha plays pleiotropic role in innate immune activation and blood-brain barrier disruption driving brain metastasis 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 184.
Celiker et al. (Fri,) studied this question.