Sapogenin-based nanoscale assemblies induce immunogenic cell death, remodel the tumor microenvironment, and drive systemic antitumor immunity in melanoma

Immunogenic cell death (ICD) inducers represent a promising strategy for in situ vaccination (ISV) by enabling tumors to serve as endogenous sources of antigens and activating antitumor immunity. We investigate the immunotherapeutic potential of sapogenin-based nanoscale assemblies (S-NAs), focusing on AG-08 and its derivatives that self-assemble into cytotoxic supramolecular structures, inducing regulated necrosis and endoplasmic reticulum stress, hallmarks of ICD relevant to ISV. We demonstrated that S-NAs trigger hallmark features of ICD in melanoma cell lines, including surface exposure and release of danger-associated molecular patterns. S-NAs-mediated tumor cell death activated dendritic cells (DCs) and suppressed B16F10 growth. Their bioactivity was found to critically depend on formulation stability. In protein-free saline solutions, S-NAs underwent agglomeration and lost cytotoxicity, whereas stabilization with 1% human serum albumin (HSA) preserved nanoscale dispersion and biological activity. In a syngeneic B16F10 melanoma model, intratumoral administration of AG-08 and CG-05 NAs suppressed primary tumor growth. Notably, local AG-08 NAs treatment elicited a systemic immune response, characterized by enhanced activation of DCs, natural killer cells (NK), and CD8 + T cells. AG-08 NAs also remodeled the tumor microenvironment by reducing macrophages, myeloid-derived suppressor cells, and regulatory T cells, while increasing CD4 + T cell infiltration and DCs activation. Importantly, local treatment with AG-08 NAs suppressed pulmonary metastases and enhanced immune cell infiltration at metastatic sites, while exhibiting a safer profile as confirmed by biochemical and histological analyses. Collectively, these findings demonstrate S-NAs as a safe and effective small-molecule platform for ICD-driven ISV against metastatic cancers.