Liposomal delivery of a disulfiram metabolite drives copper-mediated tumor immunity

Disulfiram, traditionally used as an alcohol-aversion drug, has demonstrated anticancer properties attributed to its metabolism into diethyldithiocarbamate (DDC), a potent copper-binding agent. Previously, we developed a liposomal formulation of copper diethyldithiocarbamate (Cu(DDC)2) by incorporating DDC into copper-containing liposomes. In this study, we present an improved formulation achieved by directly mixing DDC and copper in a suspension of empty liposomes, leveraging DDC's copper ionophore-like activity. This method minimizes Cu(DDC)2 precipitation outside the liposomes and enables efficient drug loading at defined molar ratios. The formulation effectively slowed the growth of subcutaneous MDA-MB-231 tumors in mice and showed increased efficacy in 4T1 tumors in immunocompetent mice compared to immunocompromised counterparts. In vitro, Cu(DDC)2-treated cancer cells exhibited upregulation of damage-associated molecular patterns (DAMPs), including ATP release, HMGB1 secretion, and calreticulin exposure. Transcriptomic analysis revealed increased expression of immune activation and copper transport genes, further supporting the potential for immunogenic cell death (ICD) induction. In a prophylactic tumor vaccination model, inoculation with Cu(DDC)2-treated CT26 cells delayed tumor growth and conferred protection in a subset of animals, indicating the induction of an adaptive immune response consistent with ICD. These findings align with previous reports of disulfiram-induced ICD and provide functional validation linking this activity to DDC's role as a copper ionophore. This formulation offers a robust and scalable platform for exploring the role of copper as an immune modulator and lays the groundwork for future optimization toward clinical application.