Use of drug-killed cancer cells: A method to assess the therapeutic effectiveness of immunogenic cell death.

Cancer immunotherapy has revolutionized cancer treatment by harnessing the immune system's potential to combat cancer. Among the various strategies in this field, the use of killed tumor cells (KC) induced by immunogenic cell death (ICD) inducers has gained attraction. This approach involves the treatment of cancer cells in vitro, followed by the subcutaneous injection of these killed cells into tumor-bearing mice. ICD induction triggers the exposure and release of damage-associated molecular patterns (DAMPs) and neoantigens, activating both innate and adaptive immune responses against cancer. Vaccination assays with immunocompetent mice and syngeneic cancer cells are considered the gold standard for identifying ICD inductors, as they effectively demonstrate the immunized host's capacity to achieve tumor rejection, typically showing more than 50% of protection. Despite significant progress in understanding ICD mechanisms, translating these findings into clinical practice faces challenges. Controversially, some reports indicate ICD induction with <50% protection in prophylactic vaccination. This variability in ICD interpretation can lead to "false positives" or overestimations of the immunogenicity of cell death induced by antitumor treatments, potentially complicating its clinical translation. Thus, rigorous adherence to the gold standard is necessary, and complementary experiments to assess the immunogenicity of cell death are advantageous. Here, we present a protocol to confirm the immunogenicity and therapeutic effectiveness of cell death induced by an ICD-inducer and evaluate its ability to reduce tumor burden in an established syngeneic mouse model.