Novel anthracyclines with enhanced immunogenic effects against drug resistant osteosarcoma cells

Doxorubicin (dox) is one of the first-line drug treatment in osteosarcoma. P-glycoprotein (Pgp) limits dox’s intracellular accumulation and efficacy in osteosarcoma. Part of the cytotoxic effects of dox are mediated by the induction of immunogenic cell death (ICD) that allows a durable eradication of the tumor by the host immune system. Pgp-overexpressing tumors, however, are also ICD-resistant. We recently synthesized two classes of synthetic doxs – nitric oxide (NO)-releasing dox and H2S-releasing dox – that were cytotoxic against different Pgp-expressing tumors. The aim of this work is to investigate if the lead compounds (termed Ndox and Sdox) were able to elicit ICD in Pgp-positive/dox-resistant osteosarcoma cells. Ndox and Sdox induced apoptosis in both sensitive and resistant cells, were localized within the endolasmic reticulum (ER), up-regulated ER stress-dependent cell death genes, promoted the translocation of calreticulin form ER to cell surface, induced the extracellular release of ATP and HMGB1, increased the phagocytosis of tumor cells by dendritic cells and the expansion of anti-tumor CD8+T-lymphocytes, in a NOand H2S-dependent manner, respectively. Expanded CD8+clones up-regulated immune-activating cytokines and down-regulated immune-suppressive cytokines. Dox induced the same events in sensitive cells, but not in Pgp-expressing/doxresistant cells. We suggest Ndox and Sdox as new multifunctional anthracyclines able to induce apoptosis of resistant osteosarcoma cells and contemporarily activate an anti-tumor immune response. These pro-drugs may have a future use in osteosarcoma patients with high Pgp expression, characterized by a poor outcome because of the lack of durable tumor eradication and the high frequency of relapse.