123 - Induction of Immunogenic Cell Death upon Combinatorial Therapy with Cold Physical Plasma and Chemotherapeutic Agents in Melanoma

Abstract

Introduction Chemotherapeutic agents remain as the essential regimen against malignant melanoma. Variety of combination therapies have been employed to sensitize tumor cells, circumvent drug resistance, and reduce systemic toxicity. However, there is still need for innovative combinational approaches to resolve these difficulties. In the current study, we investigated the potential synergism of cold physical plasma in conjunction with chemotherapeutic agents and identified mechanisms of synergistic immunogenic cell death (ICD) in tumor cells in vitro Methods In this study, we pretreated murine B16F10 and human SK-Mel 28 melanoma cells with increasing concentrations of Doxorubicin, Epirubicin, Oxaliplatin, Vorinostat and SAHA for 24h. This was followed by 30 secs exposure cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). After 6-hour incubation, IC50 was determined using alamar blue assay and compared with single treatment modalities. Cell culture supernatants were used to quantify markers of immunogenic cell death (CART, ATP, HMGB1, CXCL10) by ELISA, immunofluorescence and flow cytometry. Total cell counts, intracellular superoxide levels (DHE), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). Co culture and transwell migration assays was performed on THP1 monocytes and RAW 264.7 macrophages. For SK-Mel 28 tumor cells, 3D spheroid models were also investigated. Results Combination therapy of chemotherapeutic agents and cold physical plasma significantly decreased the IC50 values in melanoma cell lines in comparison to single treatment modalities in 2D and 3D culture conditions. The tumor cell killing was mediated by elevation of superoxide and loss of mitochondrial membrane potential. Co culturing SK-Mel 28 cells with THP1 monocytes or RAW macrophages with B16F10 following combination therapy led to pronounced tumor cell killing. ICD markers such as ATP, HMGB1, CART, and CXCL10 were induced in both single treatment conditions however were significantly elevated upon combination therapy demonstrating a synergistic effect. Transwell migration assay revealed migration of THP1 monocytes and RAW 264.7 macrophages following incubation with conditioned medium. In this study, we have demonstrated that cold physical plasma is a valuable tool to induce ICD in tumors pretreated with conventional chemotherapeutic agents.