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Background: Chronic inflammation in the tumor microenvironment (TME) sustained by immune cells has a crucial role both in tumor initiation and progression. One of the central cytokines of inflammation, IL-1β, is produced as a biologically inactive precursor that requires proteolytic processing by caspase-1. Activation of caspase-1 is triggered by the formation of inflammasomes, multiprotein complexes that detect microbial and endogenous danger signals primarily via NOD-like receptors, such as NLRP3 and NLRC4. Biologically active IL-1β is believed to be released through membrane pores formed by gasdermin D during a lytic form of cell death called pyroptosis. Although IL-1β-mediated inflammation has been shown to have a detrimental role in tumor progression, the signaling pathway controlling IL-1β release in the TME and the exact effect of the cytokine on antitumor T-cell responses have not been fully elucidated. A better understanding of how IL-1β release is controlled in tumors will also pave the way towards the therapeutic utilization of small-molecule inhibitors available to target NOD-like receptors and caspase-1. Methods: First, we characterized the impact of IL-1β in the TME by assessing the immune cell composition and vasculature of Lewis lung carcinomas (LLC) and E0771 breast carcinomas in IL-1β-deficient mice using flow cytometry and histologic analysis. Next, we used mice deficient in different inflammasome components, including NLRP3, NLRC4 and caspase-1, to investigate the involvement of these proteins in controlling IL-1β release in LLC and E0771 tumors. Using immunoblots and small-molecule inhibitors, we further characterized the activation of alternative enzymatic pathways and their involvement in IL-1β release by tumor-associated myeloid cells. Finally, we examined the role of pyroptosis and necroptosis in IL-1β release using gasdermin D- and MLKL-deficient mice, respectively. Release of IL-1β was assessed using ELISA and immunoblots. Results: We found that IL-1β secretion was restricted to myeloid cells and promoted tumor progression in mouse models of lung and breast carcinoma. IL-1β deletion abrogated the tumor-induced mobilization of immunosuppressive neutrophils and normalized the tumor vasculature, thereby alleviating hypoxia. Consequently, proliferation of effector T-cells in the TME was enhanced, leading to higher CD4+ and CD8+ T-cell abundance in the absence of IL-1β. We observed that, although the NLRP3 inflammasome was active in tumor-infiltrating myeloid cells, NLRP3 and caspase-1 were not essential for the proteolytic maturation of pro-IL-1β and secretion of biologically active IL-1β in the TME. Inhibition or genetic deletion of caspase-8 reduced inflammasome-independent IL-1β release, indicating that caspase-8 provides an alternative pathway for proteolytic activation and secretion of IL-1β in tumor-infiltrating myeloid cells. Moreover, IL-1β release by tumor-infiltrating myeloid cells was independent of lytic cell death modalities including gasdermin D-mediated pyroptosis and MLKL-mediated necroptosis, suggesting an alternative release mechanism for the cytokine in the TME. Conclusions: Overall, our results demonstrate that tumor-infiltrating myeloid cells are able to release IL-1β independently of inflammasomes. We show that proteolytic maturation of IL-1β via caspase-8 in myeloid cells acts as an important driver of immune suppression in the TME through vascular destabilization, recruitment of immunosuppressive neutrophils and consequential inhibition of antitumor T-cell responses. We also show, that, unlike in autoinflammation, gasdermin D-mediated pyroptosis is not essential for the release of IL-1β in tumors. These results suggest that therapeutic inhibition of inflammasomes or pyroptosis will likely not be beneficial in certain tumor types due to the presence of an alternative caspase-8-mediated IL-1β release pathway in tumor-associated myeloid cells. Citation Format: Máté Kiss, Lieselotte Vande Walle, Helena Van Damme, Aleksandar Murgaski, Evangelia Bolli, Jiri Keirsse, Maria Solange Martins, Yvon Elkrim, Amelie Fossoul, Jens Serneels, Massimiliano Mazzone, Mohamed Lamkanfi, Jo A. Van Ginderachter, Damya Laoui. Inflammasome-independent IL-1β release by myeloid cells promotes vessel destabilization and immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A083.