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Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca(2+) homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca(2+) concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca(2+) depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca(2+), targeted expression of the ligand-binding domain of the IP(3) receptor and inhibition of the sarco-endoplasmic reticulum Ca(2+)-ATPase pump reduced endoplasmic reticulum Ca(2+) load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca(2+) levels control the exposure of CRT.

The conventional treatment of cancer relies upon radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Nonetheless, there are circumstances in which conventional anti-cancer therapy can induce a modality of cellular demise that elicits innate and cognate immune responses, which in turn mediate part of the anti-tumor effect. Although different chemotherapeutic agents may kill tumor cells through an apparently homogeneous apoptotic pathway, they differ in their capacity to stimulate immunogenic cell death. We discovered that the pre-apoptotic translocation of intracellular calreticulin (endo-CRT) to the plasma membrane surface (ecto-CRT) is critical for the recognition and engulfment of dying tumor cells by dendritic cells. Thus, anthracyclines and gamma-irradiation that induce ecto-CRT cause immunogenic cell death, while other pro-apoptotic agents (such as mitomycin C and etoposide) induce neither ecto-CRT nor immunogenic cell death. Depletion of CRT abolishes the immunogenicity of cell death elicited by anthracyclines, while exogenous supply of CRT or enforcement of CRT exposure by pharmacological agents that favor CRT translocation can enhance the immunogenicity of cell death. For optimal anti-tumor vaccination and immunogenic chemotherapy, the same cells have to expose ecto-CRT and to succumb to apoptosis; if these events affect different cells, no anti-tumor immune response is elicited. These results may have far reaching implications for tumor immunology because (i) ecto-CRT exposure by tumor cells allows for the prediction of therapeutic outcome and because (ii) the re-establishment of ecto-CRT may ameliorate the efficacy of chemotherapy.

In contrast to prior belief, tumor cell apoptosis is not necessarily silent but can be immunogenic. By tracing how anthracyclines and gamma-irradiation trigger immunogenic cell deaths, we found that they were causally connected to the exposure of calreticulin on the tumor cell surface, before apoptosis in the tumor cell itself occurred. Furthermore, we showed that calreticulin exposure was necessary and sufficient to increase proimmunogenic killing by other chemotherapies. Our findings suggest that calreticulin could serve as a biomarker to predict therapy-associated immune responses, and that tactics to expose calreticulin might improve the clinical efficacy of many cancer therapies.

Some chemotherapeutic agents can elicit apoptotic cancer cell death, thereby activating an anticancer immune response that influences therapeutic outcome. We previously reported that anthracyclins are particularly efficient in inducing immunogenic cell death, correlating with the pre-apoptotic exposure of calreticulin (CRT) on the plasma membrane surface of anthracyclin-treated tumor cells. Here, we investigated the role of cellular Ca2+ homeostasis on CRT exposure. A neuroblastoma cell line (SH-SY5Y) failed to expose CRT in response to anthracyclin treatment. This defect in CRT exposure could be overcome by the overexpression of Reticulon-1C, a manipulation that led to a decrease in the Ca2+ concentration within the endoplasmic reticulum lumen. The combination of Reticulon-1C expression and anthracyclin treatment yielded more pronounced endoplasmic reticulum Ca2+ depletion than either of the two manipulations alone. Chelation of intracellular (and endoplasmic reticulum) Ca2+, targeted expression of the ligand-binding domain of the IP3 receptor and inhibition of the sarco-endoplasmic reticulum Ca2+-ATPase pump reduced endoplasmic reticulum Ca2+ load and promoted pre-apoptotic CRT exposure on the cell surface, in SH-SY5Y and HeLa cells. These results provide evidence that endoplasmic reticulum Ca2+ levels control the exposure of CRT.Cell Death and Differentiation (2008) 15, 274–282; doi:10.1038/sj.cdd.4402275; published online 23 November 2007 [ABSTRACT FROM AUTHOR]