Abstract A47: Immunogenic cancer cell death induced by high hydrostatic pressure and hyperthermia: Calreticulin exposure pathway

Abstract

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy, severe hyperthermia (sHT) or high hydrostatic pressure (HHP). HHP and sHT are able to trigger a specific antitumor immune response and induce the rapid translocation of calreticulin (CRT) to the plasma membrane surface (ecto-CRT). Ecto-CRT on dying tumor cells represents an “eat me” signal for dendritic cells as well as a major molecular determinant that makes the difference between immunogenic and non-immunogenic cell death. Nevertheless, the mechanisms implicated in CRT translocation after HHP treatment is unknown. Inducers of ICD trigger the ER stress response, Reactive Oxygen Species (ROS) generation and Ca2+ release. Kinetics of ecto-CRT was assessed in lung A549 and ovarian OV90 tumor cell lines. CRT localization in sHT- and HHP-treated tumor cells was tracked by endosomal staining (caveolin, clathrin, early and late endosomes) and visualized by confocal microscopy. For detection of CRT translocation pathway, the tumor cells were pretreated with selected cytoskeletal inhibitors, inhibitor of intracellular transport, ER stress activator or small interfering RNA followed by sHT and HHP treatment. The cells were analyzed by flow cytometry and confocal microscopy. Plasma membrane fraction and endosomes were isolated and analyzed by western blotting. Our results indicate that the ROS - PERK - eIF2α - caspase 2 - caspase 8 - caspase 8-mediated cleavage of the ER protein BAP31 is central for the perception of HHP-driven cell death as immunogenic. On the other hand our first results indicate that none of these processes are involve in exposure of CRT on cell surface in sHT treated tumor cells. We observed that sHT treatment does not lead to caspase 8 activation and Bap31 cleavage. However, we showed that translocation of CRT to the cell surface is dependent on endoplasmic reticulum-sessile kinase PERK and presence of Bax/Bak proteins. CRT colocalization with clathrin in tumor cells after HHP and particularly with early endosomes was detected. Surprisingly, we also did not observe colocalization with any endosomes in sHT treated tumor cells. Further, inhibition of microtubule-dependent transport with Nocodazole D results in decrease of CRT transport on cell surface in HHP treated tumor cells. Similar but less significant effect was seen when Brefeldin A was used to inhibit anterograde transport in both treatments. Inhibition of actin polymerization by Cytochalasin D did not significantly affect CRT translocation. In conclusion, PERK seems to play a central role in the exposure of CRT upon induction of ICD. However, up-stream and downstream signaling cascades differ depending the modality used to induce ICD, such as HHP or sHT. Citation Format: Sarka Vosahlikova, Irena Adkins, Linda Urbanova, Nada Hradilova, Lenka Sadilkova, Radek Spisek, Irena Moserova. Immunogenic cancer cell death induced by high hydrostatic pressure and hyperthermia: Calreticulin exposure pathway. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A47.