Calreticulin Enhances Therapeutic Immune Responses of Dry Thermostat-Stressed Hepatocellular Carcinoma Cell Vaccine

Despite the promise of whole tumor cell vaccines in personalized cancer immunotherapy, their limited immunogenicity and lack of danger signals hinder their clinical efficacy. Herein, we introduce a time-efficient (within 2 h) dry thermostat (DT) device-based method to prepare ^CpGRTC-vax¹⁰⁰, a therapeutic vaccine generated by treating tumor cells through DT and further decorated with cholesterol-linked CpG-ODNs, to eliminate orthotopic hepatocellular carcinoma (HCC). The DT treatment preserves tumor cell morphology integrity and antigenic activity, promotes the translocation of calreticulin to the cell surface (approximately 13-fold), upregulates HSP70 (around 3.4-fold), and induces DNA breaks while immediately inactivating tumor cells within 5 min. This approach overcomes the limitations of existing whole cell vaccines by enhancing immunogenicity and exposing pathogen-associated and damage-associated molecular patterns. These changes led to efficient tumor antigen uptake, dendritic cell activation, and increased levels of CXCL-10, Gzms-B, IL-6, and IL-12 in tumors. Consequently, the T-cell infiltration into orthotopic HCC is enhanced. Mechanistic studies reveal that NK cells, CD4⁺T cells, and activated CD8⁺T cells are recruited, alongside with reduced Tregs within the tumor. Furthermore, ^CpGRTC-vax¹⁰⁰ activates patient-derived DC-T cells, demonstrating an efficient antitumor effect in patient-derived xenograft models through adoptive transfer of activated DC-T cells. Our findings highlight the potential of physical methods in enhancing tumor cell vaccine immunogenicity and their therapeutic promise for addressing the urgent needs of HCC treatment.