TNF-alpha plays pleiotropic role in innate immune activation and blood-brain barrier disruption driving brain metastasis

Tumor necrosis factor-alpha (TNFalpha) 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 TNFalpha 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 TNFalpha in EMT6 orthotopic models, where TNFalpha-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 TNFalpha-treated cells contains high concentrations of soluble TNFalpha 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, TNFalpha exerts a paradoxical influence: beneficial through HSP70-mediated innate immune activation, yet detrimental through TNFalpha-driven BBB disruption that facilitates brain metastatic outgrowth.To mechanistically separate these opposing functions in our ongoing studies, we are selectively depleting TNFalpha from EMT6-CM using immunoabsorption approaches while preserving HSP70-containing vesicles and soluble fractions. TNFalpha-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 TNFalpha-depleted, HSP70-enriched CM will reject EMT6-Luc tumors, demonstrating that HSP70-driven immunogenicity is sufficient for tumor protection in the absence of TNFalpha-induced BBB damage. Successful rejection will validate our model in which TNFalpha is necessary for BBB disruption but dispensable for HSP70-mediated anti-tumor immunity.Collectively, our findings reveal a previously unrecognized dichotomy in TNFalpha biology, with significant implications for cytokine-based immunotherapies and mechanisms underlying breast cancer brain metastasis. Understanding how to uncouple TNFalpha's immunostimulatory and pathological effects may guide development of safer and more effective immune-modulating strategies.