Disulfiram/Copper induces immunogenic cell death via NPL4 in hepatocellular carcinoma

Some chemotherapeutic agents have been found to enhance the antitumor immunity by inducing immunogenic cell death (ICD). The combination of disulfiram (DSF) and copper (Cu) has demonstrated anti-tumor effects in a range of malignancies including hepatocellular carcinoma (HCC). However, the potential of DSF/Cu as an ICD inducer and whether it could enhance the efficacy of immune checkpoint blockade in HCC remains unknown. Here, we showed that DSF/Cu-treated HCC cells exhibited characteristics of ICD in vitro, such as calreticulin (CRT) exposure, ATP secretion, high mobility group box 1 (HMGB1) release. DSF/Cu treated HCC cells elicited significant immune memory in a vaccination assay. DSF/Cu treatment promoted dendritic cell activation and maturation. The combination of DSF/Cu and CD47 blockade further facilitated DC maturation and subsequently enhanced CD8+ T cell cytotoxicity. Mechanically, DSF/Cu promoted the nuclear accumulation and aggregation of nuclear protein localization protein 4 (NPL4) to inhibit the ubiquitin-proteasome system, thus induced endoplasmic reticulum (ER) stress. Inhibition of NPL4 induced ICD-associated damage-associated molecular patterns. Collectively, our findings demonstrated that DSF/Cu induced ICD-mediated immune activation in HCC and enhanced the efficacy of CD47 blockade. HCCLM3 cells were treated with DSF/Cu in a concentration of 0.2μM for 24h or transfected with siRNA-NC or NPL4 siRNA-1 for 48h.