Chitosan activates NLRP3 inflammasome and cGAS-STING to suppress cancer progression through hexokinase 2 dissociation and mitochondrial dysfunction

<b>Background:</b> Chitosan, a natural polysaccharide with known immunostimulatory potential, has shown promise in cancer therapy. However, its direct role in modulating antitumor immunity and the underlying mechanisms remain unclear. This study aimed to explore how unmodified chitosan influences tumor progression and immune responses through innate immune signaling pathways. <b>Methods:</b> Murine tumor models (CT26, B16-F10) were used to evaluate the antitumor effects of chitosan in vivo. Flow cytometry and histological analyses assessed changes in immune cell infiltration. Primary macrophages and gene knockout models were used to investigate the molecular mechanisms, including inflammasome activation, mitochondrial function, and hexokinase 2 (HK2) location, via ELISA, western blotting, mitochondrial assays. <b>Results:</b> Chitosan treatment suppressed tumor growth and metastasis, while promoting infiltration of neutrophils, monocytes, and activated T cells in the tumor microenvironment. Mechanistically, chitosan and its bioactive degradation product, N-acetylglucosamine (NAG), induced the dissociation of HK2 from mitochondria, triggering mitochondrial dysfunction, ROS overproduction, and mtDNA release. These signals jointly activated both the NLRP3 inflammasome and the cGAS-STING pathway. The antitumor effect of chitosan was attenuated in <i>Nlrp3</i> ^-/- and <i>Sting</i> ^-/- mice, confirming the essential roles of both pathways. <b>Conclusions:</b> Chitosan orchestrates dual activation of NLRP3 and cGAS-STING signaling via HK2 dissociation and mitochondrial dysfunction, reprogramming the tumor immune microenvironment and enhancing antitumor immunity. These findings support chitosan's potential as a multifunctional immunoadjuvant for improving immunotherapy in resistant cancers.