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22nd International Symposium on Plasma Chemistry July 5-10, 2015; Antwerp, Belgium Plasma activation of the immune system for cancer treatment V. Miller1, A. Lin2, N. Sang2, S. Chen2, S. Lin3 and A. Fridman1 1 3 A.J. Drexel Plasma Institute, 200 Federal Street Suite 500, US-08103 Camden, NJ, U.S.A. 2 Department of Biology, Drexel University, Philadelphia, PA, U.S.A. Department of Radiation Oncology, Fujian Provincial Cancer Hospital, Fuzhou, Fujian Province, P.R. China Abstract: Treatment of cancer, as a disease, is a major challenge facing the medical community. This paper will address a novel approach whereby non-thermal atmospheric pressure plasma is used to: (i) stimulate the function of immune cells directly so that a more natural elimination of cancers results and also by (ii) cause immunogenic death of cancerous cells. This will result in control of metastatic tumours as well as in prevention of relapse. Keywords: macrophage, cancer, immunogenic cell death, DBD plasma 1. Introduction Resistance to cell death is a hallmark of cancer cells [1]. They also have the ability to evade surveillance by the immune system [2]. Hence, restoration of immunogenicity of cancer cells to allow for recognition by the immune system followed by their elimination can be a viable therapeutic approach for treatment of cancers. Non-equilibrium atmospheric pressure plasma (NEAPP) offers a unique therapeutic solution. It kills cancer cells directly [3] and stimulates function of immune cells in vitro [4]. Non-thermal plasma has also been used to treat tumour-bearing animals with mixed results [5, 6]. While tumours shrink in size, cure has not yet been achieved. Recently, the induction of immunogenic cancer cell-death has been identified as a possible pathway for cancer therapy [7]. NEAPP treatment regimens may be optimized to avoid immunologically silent or immunosuppressive cell death and induce immunogenic cell death. This would achieve two outcomes: decrease in cancer cell burden through direct elimination and an immunological stimulation to further increase cancer cell killing. Hence both cell death and immunological stimulation may be achieved directly as well as through physiological mechanisms. Macrophages are one of the key cells involved in control of tumours [8]. They function by phagocytosis, antigen presentation, production of cytokines and recruitment of T and B cells [9]. We investigated the ability of plasma to stimulate macrophages to kill cancer cells. We also examined if plasma treatment of cancer cells resulted in an immunogenic cell death. It was determined that nspDBD stimulates macrophages to kill A549 lung carcinoma cells. Low dose treatment of A549 cells enhanced the killing activity of THP1 cells, suggesting that plasma may be modulated to cause tumour cell death in an immunogenic way. IN-02 2. Materials and Methods THP-1 human monocyte cells were allowed to differentiate into macrophages using phorbol myristate acetate (PMA) in 24-well plates overnight. They were then treated for 10 seconds with nspDBD (29.5 kV, 15 Hz or 30 Hz) at a distance of 1 mm. A549 lung carcinoma cell line was grown to confluency, overnight in the inserts of transwell chambers. Immediately following plasma treatment of THP1 cells, inserts containing the A549 cells were placed above them. They were allowed to coculture without direct contact between the two cell types for 48 hours. The only communication between cells was via soluble mediators. At the end of 48 hours, A549 cells were collected from the transwell inserts by trypsinization, stained with propidium iodide and examined using an image cytometer (Nexcelom Cellometer). Data was analysed using the FCS flow cytometry software. All cell counts were normalized to A549 cells growing in RPMI with 10% FBS. Cells were also observed under a Nikon Eclipse TE2000 and images were captured with a SPOT camera. In other experiments, A549 cells were treated with plasma and M0 THP1s were placed in transwells. Viability of A549s was determined 48 hours after coculture as above. 3. Results Fig. 1 shows the results of co-culture of A549 cells with plasma treated THP1 cells. PMA treatment of THP1 cells matures them into M0 macrophages. These cells are “uncommitted” and may be further differentiated in vitro into M1 phenotype by stimulation with interferon–gamma and lipopolysaccharide (LPS) or into M2 macrophage subtypes by growing with interleukin-4. A549 cells grown in RPMI without any THPs in the lower well maintained their cell numbers and viability. They did not increase in numbers because they were already confluent. A549 cells co-cultured with M0 macrophages without any exposure to nspDBD yielded only 35% viable cells. In the presence of M0 THP1 stimulated at 15 Hz, there was a 1 69% loss of viable cells. Following treatment at 30 Hz, there was an 85% loss of viable cells. Since there was no direct contact between the two cell types, it is hypothesized that the effect was upregulated via soluble mediators produced by the THP1 cells as a direct result of plasma treatment. Fig. 1. Viable A549 lung cancer cells 48 hours after incubation with NEAPP activated THP-1 macrophages. Following plasma treatment of PMA matured THP1 cells, A549 cells were co-cultured in transwell chambers for 48 hours. Viability of A549 cells was determined by examining cells in an image cytometer following propidium iodide staining. When M0 THP1 cells were co-cultured with nspDBD treated A549 cells, an enhancement of tumour cell killing was observed over that seen with plasma treatment alone (Fig. 2). The loss of viable cells was not as dramatic as in the case of plasma treatment of THP1s. 4. Discussion An enhanced killing of A549 cells by THP1 macrophages was observed when either one of the two cell types was exposed to plasma. This suggests that plasma can activate macrophages directly. It also implies that plasma treated cancer cells are able to “communicate” with macrophages to influence their function. This effect was not as pronounced because the two cell types were not in direct contact. The goal of successful anticancer therapy should be production of immunogenic cancer cell death. This would cause the induction of both innate and adaptive immune responses, resulting in eradication of cancer cells. 5. References [1] M.V. Blagosklonny. "Prospective strategies to enforce selectively cell death in cancer cells". Oncogene, 23, 2967-2975 (2004) [2] R. Kim, M. Emi and K. Tanabe. "Cancer immunoediting from immune surveillance to immune escape". Immunology, 121, 1-14 (2007) [3] M. Keidar, et al. "Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy". Brit. J. Cancer, 105, 1295-1301 (2011) [4] V. Miller, et al. "Plasma Stimulation of Migration of Macrophages". Plasma Process. Polymers, 11, 1193-1197 (2014) [5] L. Brulle, et al. "Effects of a Non Thermal Plasma Treatment Alone or in Combination with Gemcitabine in a MIA PaCa2-luc Orthotopic Pancreatic Carcinoma Model". Plos One, 7 (2012) [6] M. Vandamme, et al. "Antitumor Effect of Plasma Treatment on U87 Glioma Xenografts: Preliminary Results". Plasma Process. Polymers, 7, 264-273 (2010) [7] A. Tesniere, et al. "Immunogenic cancer cell death: a key-lock paradigm". Curr. Opinion Immunol., 20, 504-511 (2008) [8] J. MacMicking, Q.W. Xie and C. Nathan. "Nitric oxide and macrophage function". Ann. Rev. Immunol., 15, 323-350 (1997) [9] C. Lamagna, M. Aurrand-Lions and B.A. Imhof. "Dual role of macrophages in tumor growth and angiogenesis". J. Leukocyte Biol., 80, 705-713 (2006) Fig. 2. Viable A549 lung cancer cells 48 hours after incubation THP-1 macrophages. Following plasma treatment of A549 cells, PMA matured THP1 cells were co-cultured in transwell chambers for 48 hours. Viability of A549 cells was determined by examining cells in an image cytometer following propidium iodide staining. Results from other tumour cell lines and macrophage combinations will also be presented. 2 IN-02