Abstract 4395: KP-1339 (IT-139) induces the hallmarks of immunogenic cell death in a colon cancer 3D model in vitro

Abstract

Abstract Chemotherapeutic drugs exert their activity by directly killing tumor cells through a variety of cellular targets eventually leading to apoptosis. Apoptosis is regarded as non-inflammatory and non-immunogenic, however, recent data confirm that the induction of damage-associated molecular patterns by dying cancer cells treated with certain chemotherapeutic agents, such as oxaliplatin, lead to their recognition by the immune system, and result in dendritic cell maturation and immune responses, a mechanism described as Immunogenic Cell Death (ICD). The main hallmarks of ICD are: Calreticulin (CRT) exposure on the cell membrane and release of high mobility group box-1 (HMGB-1) and ATP from dying cells. We have recently shown that the mode of action of KP-1339, a clinically investigated ruthenium-based metal complex, involves the enhancement of endoplasmic reticulum stress (ERS) and the induction of apoptosis-like events. We next investigated the ability of KP-1339 to trigger ICD. There is currently no reliable method to study ICD in vitro, and therefore we aimed at a more suitable model that better mimics the tumor microenvironment in comparison to 2D monolayer cultures. For this reason, ICD was assessed in human colorectal cancer HCT-116 3D multicellular spheroids in vitro. We demonstrate that spheroids treated with KP-1339 activate the PERK/Phospho-eIF-2alpha pathway of the ERS, a prerequisite for CRT exposure on the cell membrane. HMGB1 protein levels decrease considerably during a 72 hours drug treatment, as shown by Immunoblotting, suggesting the depletion of the protein. Additionally, treated spheroids released significantly more ATP than untreated controls. Immunofluorescence of cryosections of KP-1339 treated spheroids stained for CRT and HMGB1 confirmed the above observations and, clearly showed the translocation of CRT to the cell membrane. In conclusion, we have established a 3D tumor spheroid model as an alternative to the 2D monolayer cultures to study ICD in vitro. This model more closely resembles the tumor microenvironment and recapitulates relevant aspects of the in vivo situation. We successfully employed this model to demonstrate that the clinically investigated compound KP-1339 triggers the main hallmarks of ICD, which suggests enhanced induction anti-tumor immunity in colorectal cancer. Citation Format: Debora Wernitznig, Giorgia Del Favero, Konstantinos Kiakos, Nathalie Harrer, Herwig Machat, Doris Marko, Michael Jakupec, Wolfgang Sommergruber, Bernhard K. Keppler. KP-1339 (IT-139) induces the hallmarks of immunogenic cell death in a colon cancer 3D model in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4395.