Abstract
Simple SummaryDendritic cells (DCs)-based anti-cancer vaccines displayed limited efficacy in clinical trials, mostly due to a lack of protocols for preparing immunogenic tumor antigens used in the vaccine. Here, a unique atmospheric pressure plasma jet was used to prepare a plasma-activated medium (PAM) which induced immunogenic cell death in tumor cells. This procedure increased the efficacy of tumor lysates in enhancing the immunogenicity of DCs according to their increased maturation, production of IL-12, and the capacity to induce cytotoxic CD8 T cells able to kill tumor cells. In contrast to the tumor lysates commonly used in DC vaccines, PAM-tumor lysates lacked the capacity to increase IL-10 production by DCs, and their potential to induce protumorogenic Th2 and regulatory T cells. Cumulatively, these results suggest that the novel method for preparing immunogenic tumor lysates with PAM could be suitable for improved DC-based immunotherapy of cancer patients.Autologous dendritic cells (DCs)-based vaccines are considered quite promising for cancer immunotherapy due to their exquisite potential to induce tumor antigen-specific cytotoxic T cells. However, a lack of efficient protocols for inducing immunogenic tumor antigens limits the efficacy of DC-based cancer vaccines. Here, we found that a plasma-activated medium (PAM) induces immunogenic cell death (ICD) in tumor cells but not in an immortalized L929 cell line or human peripheral blood mononuclear cells. PAM induced an accumulation of reactive oxygen species (ROS), autophagy, apoptosis, and necrosis in a concentration-dependent manner. The tumor lysates prepared after PAM treatment displayed increased immunogenicity in a model of human monocyte-derived DCs, compared to the lysates prepared by a standard freezing/thawing method. Mature DCs loaded with PAM lysates showed an increased maturation potential, as estimated by their increased expression of CD83, CD86, CD40, IL-12/IL-10 production, and attenuated PDL1 and ILT-4 expression, compared to the DCs treated with control tumor lysates. Moreover, in co-culture with allogeneic T cells, DCs loaded with PAM-lysates increased the proportion of cytotoxic IFN-γ+ granzyme A+ CD8+ T cells and IL-17A-producing T cells and preserved the Th1 response. In contrast, control tumor lysates-treated DCs increased the frequency of Th2 (CD4+IL-4+), CD4, and CD8 regulatory T cell subtypes, none of which was observed with DCs loaded with PAM-lysates. Cumulatively, these results suggest that the novel method for preparing immunogenic tumor lysates with PAM could be suitable for improved DC-based immunotherapy of cancer patients.
Highlights
Cold atmospheric plasma (CAP), called non-equilibrium atmospheric-pressure plasma (NEAPP), is a partially ionized gas generated under normal atmospheric pressure and ambient temperature [1]
Higher doses (50% and 100%) of plasma-activated medium (PAM) significantly reduced the metabolic activity of all cell types, but the effect was most prominent in cultures with the tumor A375 and Hep2 cells
According to the ISO standard on the cytotoxicity of medical devices, a 30% reduction in MTT is considered as non-cytotoxic [29], suggesting that the reduction of MTT in the cultures of L929 cells and Peripheral blood mononuclear cells (PBMCs) treated with 50% PAM could be considered as noncytotoxic as well
Summary
Cold atmospheric plasma (CAP), called non-equilibrium atmospheric-pressure plasma (NEAPP), is a partially ionized gas generated under normal atmospheric pressure and ambient temperature [1]. Specific compounds are created in the liquid phase which are responsible for the effects on cells (bacteria, plant cells, human cells, cancer cells, stem cells, etc.). The chemistry, both in a gas phase and liquid phase, depends on the plasma parameters (type of gas mixture, gas flow, concentration of electrons, temperature of electrons, deposited power etc.), and on the type of liquid medium that is being treated by plasma. PAM may be prepared by treating aqueous solutions, including cell culture media, with CAP In this process, RONS are transported from the gas phase into the liquid surface, dissolved into the medium, and undergo further reactions with dissolved molecules in the aqueous solution. Mixing of the gas-phase RONS with the medium is promoted (in this paper as well) by a strong flow of gas/plasma into the liquid whereby “bubbles” of plasma effluent are formed within the top layer of the liquid
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