Screening of novel immunogenic cell death inducers within the NCI mechanistic diversity set

Immunogenic cell death (IC D) inducers can be defined as agents that exert cytotoxic effects while stimulating an immune response against dead cell-associated antigens. When initiated by anthracyclines, IC D is accompanied by stereotyped molecular changes, including the pre-apoptotic exposure of calreticulin (CR T) on the cell surface, the lysosomal secretion of ATP during the blebbing phase of apoptosis, and the release of high mobility group box 1 (HMGB1) from dead cells. By means of genetically engineered human osteosarcoma U2OS cells, we screened the 879 anticancer compounds of the National Cancer Institute (NCI ) Mechanistic Diversity Set for their ability to promote all these hallmarks of IC D in vitro. In line with previous findings from our group, several cardiac glycosides exhibit a robust propensity to elicit the major manifestations of IC D in cultured neoplastic cells. This screen pointed to septacidin, an antibiotic produced by Streptomyces fibriatus, as a novel putative inducer of IC D. In low-throughput validation experiments, septacidin promoted CR T exposure, ATP secretion and HG MB1 release from both U2OS cells and murine fibrosarcoma MCA 205 cells. Moreover, septacidin-killed MCA 205 cells protected immunocompetent mice against a re-challenge with live cancer cells of the same type. Finally, the antineoplastic effects of septacidin on established murine tumors were entirely dependent on T lymphocytes. Altogether, these results underscore the suitability of the high-throughput screening system described here for the identification of novel IC D inducers.