Download Plasma activation of immune system for cancer treatment

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
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A.J. Drexel Plasma Institute, 200 Federal Street Suite 500, US-08103 Camden, NJ, U.S.A.
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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.
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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
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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
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[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.
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