Chemotherapy-derived DAMPs drive reprogramming of tumor-associated macrophages toward a pro-inflammatory phenotype in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a global health challenge, with treatment efficacy often compromised by the immunosuppressive tumor microenvironment (TME), particularly tumor-associated macrophages (TAMs). While chemotherapy induces immunogenic cell death (ICD) and the release of Damage-Associated Molecular Patterns (DAMPs), the specific impact of these molecules on macrophage plasticity and the underlying signaling mechanisms remain poorly understood. This study investigated the immunomodulatory effects of DAMPs released from HepG2 cells treated with standard chemotherapeutic agents, sorafenib and oxaliplatin. Macrophage polarization was assessed using murine RAW 264.7 and human monocyte-derived macrophages. The current study utilized flow cytometry, multiplex cytokine profiling, immunoblotting, and proteomic analysis to characterize phenotypic shifts and signaling pathways. Both Sorafenib and oxaliplatin induced significant cytotoxicity and the release of HMGB1 and S100A9, while sorafenib specifically triggered HSP90 release. Exposure to these chemotherapy-derived DAMPs effectively reprogrammed IL-4 stimulated M2 macrophages toward an M1-like phenotype, evidenced by downregulation of CD163/CD206, and upregulation of CD86. Mechanistically, this reprogramming was driven by a distinct, non-canonical signaling axis involving the upregulation of p-ERK and the NLRP3 inflammasome, occurring despite the downregulation of the classical NF-κB pathway. Proteomic analysis further revealed a metabolic shift toward cellular stress, ER stress and transcriptional reprogramming. Although the cytokine profile was predominantly pro-inflammatory (TNF-α, IL-1β), the concurrent secretion of IL-10 suggests a complex, mixed activation state. By overriding M2 immunosuppression via an ERK-NLRP3-dependent pathway, sorafenib-derived DAMPs act as an immunological primer to convert cold to hot tumors, providing a molecular rationale for chemo-immunotherapy combinations.