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Background While microtubule-targeting agents (MTAs) traditionally exert anticancer effects through mitotic arrest, emerging evidence suggests that certain MTAs can induce immunogenic cell death (ICD) via alternative mechanisms, such as endoplasmic reticulum (ER) stress, leading to enhanced antitumor immunity.Methods We investigated BRG399, a novel brain-penetrant colchicine-binding site MTA, for its capacity to induce ICD across various cancer cell lines. We assessed the temporal induction of ICD markers calreticulin (CRT), ATP, HMGB1, and HSP70 at both transcriptional and protein levels. Cell viability and cell cycle analyses were conducted to determine the relationship between ICD induction and mitotic arrest. In vivo studies were performed in syngeneic murine models of colon cancer (MC38, CT26) to characterize immune cell modulation within the tumor microenvironment (TME) throughout the treatment course.Results BRG399 induced early transcriptional upregulation of ICD markers, with corresponding protein expression observed at 24–48 hours. Notably, these effects occurred at low nanomolar concentrations (30–100 nM), preceding any detectable G2/M cell cycle arrest, which was only evident at concentrations exceeding 100 nM after 24 hours. This early ICD induction suggests a mechanism independent of prolonged mitotic arrest . In vivo studies demonstrated that BRG399 treatment preserved tumor-associated immune cells and favorably modulated the immune landscape within the TME. This modulation aligns with established ICD mechanisms, wherein damage-associated molecular patterns (DAMPs) including CRT, ATP, and HMGB1 released from dying tumor cells act as critical signaling mediators to the immune system. Consequently, BRG399-induced ICD not only directly affects tumor cells but also reprograms the TME to support antitumor immune responses.Conclusions Our findings suggest BRG399 is an inducer of ICD at sub-cytotoxic concentrations that precede mitotic arrest. This distinctive early induction profile suggests that low doses of BRG399 may effectively stimulate antitumor immune surveillance while minimizing systemic toxicity, potentially enhancing immunotherapy efficacy. These characteristics position BRG399, for further investigation, as a candidate for rational combination strategies aimed at improving treatment outcomes across multiple cancer types.