Immunogenic cell death (ICD) is a form of apoptosis that kills susceptible populations of cancer cells while teaching the immune system to attack the remaining resistant cells. Chemotherapeutics which induce ICD elicit their immune response by inducing tumor cells to display or release damage-associated molecular patterns (DAMPs). There are two key biomarkers for ICD:  1) during the apoptotic process, tumor cells secrete ATP (extracellular ATP or eATP) and 2) during secondary necrosis, tumor cells release HMGB1 (high mobility group box 1). Because the dying tumor cells display these DAMP molecules, they stimulate the recruitment of dendritic cells (DCs) into the tumor bed and ultimately “teach” cytotoxic T-lymphocytes (CTLs) to respond to these tumor specific antigens. These primed CTLs will then kill additional tumor cells through a direct cytotoxic response. Therefore, therapeutics which provoke an ICD response offer a therapeutically desirable outcome for cancer therapy. Current assay methods such as ELISA assays and flow cytometry for measuring these ICD biomarkers are laborious and involve multiple transfer and wash steps. We have developed homogeneous (no wash), single addition assays for measuring the two principle ICD biomarkers. The eATP assay uses optimized luciferase detection chemistry that is applied directly to live cells to measure ATP release over 24 hours. The HMGB1 assay uses a complementary luciferase fragment-labeled antibody approach to measure protein concentration. We demonstrated the use of these two assays with model systems for inducing Immunogenic Cell Death with human and murine cell lines including U2OS, EL4, and U937 cells. The potency and response magnitude of ICD-inducing compounds including doxorubicin, idarubicin and mitoxantrone differs depending on the model cell line used. Further, the timing of extracellular ATP release and HMGB1 release differs between cell lines and inducers. Improved assays to measure two markers of Immunogenic Cell Death will expedite the discovery and development of inducers of Immunogenic Cell Death, including small molecule therapeutics, oncolytic viruses, radiation therapy and other novel therapeutics, by streamlining the workflow, increasing sample throughput and providing real time live cell data.

Citation Format: Richard L. Somberg, Kevin Kupcho, Andrew Niles, James Cali. Detection ofHMGB1 and extracellular ATP for the assessment of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1112.