Immunogenic Potential Of Cold Atmospheric Plasma For The Treatment Of Pancreatic Cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) forms the third leading cause of cancer related deaths in western countries. PDAC is a tumor with a fibroblastic stroma compartment that consists of pancreatic stellate cells (PSC) which play a complex role in supporting carcinogenesis, immunosuppression and therapy resistance. Therefore, the 5-year survival rate for PDAC patients remains below a disappointing 8%, stressing the need for new and more effective treatments [1]. Recently, cold atmospheric plasma (CAP) has emerged as a potent treatment option for cancer [2]. Although, CAP is being investigated for several years [3], the involvement of the immune system after CAP treatment remains poorly understood.

The immunogenic cell death (ICD) concept describes that the killing of cancer cells leads to direct activation of the immune system. Cancer cells dying in such an immunogenic fashion release so-called ‘danger-associated molecular patterns’ that are able to induce a specific antitumoral immune response. ICD can be elicited by several physical means such as irradiation and photodynamic therapy, providing a rationale for the induction of ICD after CAP treatment [4]. The aim of this study is to investigate the induction of a specific antitumoral immune response after CAP treatment in PDAC, in vitro.

Phosphate-buffered saline (PBS) was treated with CAP, generated by the kINPenIND®, and subsequently added to the monocultures of both pancreatic cancer cell (PCC) lines and PSC lines. To evaluate the four most important hallmarks of ICD, being membrane exposure of calreticulin, secretion of ATP and release of HMGB1 and type I interferon, the treatment parameters were optimized (i.e. treatment time, gas flow and gap distance) to obtain 50% cell death. The cellular difference in sensitivity between these two cell types for CAP treatment was assessed through cytotoxic analysis. After attaining the optimal treatment parameters, we investigated the translocation of calreticulin onto the cell surface with flow cytometry. ATP secretion was investigated with a bioluminescence assay, while ELISA was used to monitor the release of HMGB1 and interferon type I in the extracellular compartment.

Our data report a cytotoxic and immunogenic effect of CAP treatment in vitro on both PCC and PSC cell lines. These results warrant further in vivo validation to refine the involvement of the immune system after CAP treatment.