Molecular characterization of immunogenic cell death triggered by the high hydrostatic pressure in human tumor cells.

Abstract:

Background: Immunogenic cell death (ICD) is a form of cell death caused by certain chemicals or physical modalities: cytostatic agents such as anthracyclines, oxaliplatin and bortezomib, or radiotherapy and photodynamic therapy. Typical molecular events characteristic for ICD are surface exposure of calreticulin, HSP70, HSP90, release of intranuclear HMGB1 and the secretion of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells and analyzed the key components of apoptotic pathways. 

Methods: We treated wide spectrum of primary human tumor cells and human cancer cell lines (acute lymphoblastic leukemia, ovarian and prostate cancer) by HHP in the custom made device and analyzed the expression of ICD markers, signaling pathways triggered by the treatment and capacity of HHP treated tumor cells to induce tumor specific immune responses. 

Results: HHP induced rapid expression of HSP70, HSP90 and calreticulin on the cell surface of early apoptotic cells. Cells treated by HHP produced high levels of reactive oxygen species (ROS). Pretreatment of cells with ROS inhibitors, N-acetyl-L-cysteine or L-glutathione, inhibited ROS production and calreticulin expression, indicating the importance of ROS pathway in HHP-mediated ICD. The other key components of the ER stress mediated apoptotic pathway, such as phosphorylation of eIF2α, the activation of caspase-8 and caspase-8-mediated cleavage of the ER protein BAP31 were also detected. The interaction of dendritic cells (DCs) with HHP-treated tumor cells led to faster rate of phagocytosis, upregulation of CD83, CD86 and HLA-DR and release of IL-6, IL-12p70 and TNFα. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific T cells. DCs pulsed with HHP-treated tumor cells also induced the lowest number of regulatory T cells.

Conclusions: HHP is a reliable and potent inducer of ICD in human tumor cells. HHP activates the key components of ER stress mediated pathway. HHP treatment can be used for the induction of ICD in GMP settings in compliance with the regulatory requirement. This allows for the testing of HHP killed immunogenic tumor cells in cancer immunotherapy trials.

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Citation:
J Clin Oncol 32, 2014 (suppl; abstr e14008)