22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Uniform nanosecond pulsed DBD treatment of macrophages for stimulation of
anti-cancer effects
A.G. Lin, G. Fridman, D. Dobrynin, A. Fridman and V. Miller
A.J. Drexel Plasma Institute, 200 Federal Street, NJ-08103 Camden, NJ, U.S.A.
Abstract: Macrophages are immune cells that can be activated for anti-cancer effects. We
explored the potential of uniform nanosecond DBD to stimulate this effect in macrophages
in vitro. Our preliminary results showed that the media from plasma treated THP-1
differentiated macrophages reduced A549 lung cancer growth, and arrested them in the
G0/G1 phase.
Keywords: Non-thermal plasma, macrophages, cancer, pro-inflammatory stimulation
1. Introduction
Non-thermal plasmas are currently being developed as
an adjuvant therapy for cancer [1, 2]. In vitro treatment
of cell lines selectively kills cancer cells while normal
cells remain viable [2]. Local application of plasma to
tumours in vivo has led to reduced tumour size and
increased life expectancy of treated animals [2]. Most
studies are focused on the direct influence plasma has on
tumour. However, the body’s immune system also plays
a vital role in the control of cancer and must be
investigated to address all aspects of tumour therapy [3].
Macrophages are one of the key immune cells involved in
the initiation of protective immunological responses.
Macrophages originate from bone marrow progenitor
cells. These cells proliferate and are released into the
bloodstream where they develop into monocytes.
Monocytes circulate in the blood before taking residence
in various tissue where they differentiate into tissue
specific macrophages [4]. Depending on the local
environment the macrophages reside in, they can be
activated to display different phenotypes [5].
Macrophages that are classically activated are M1
macrophages and considered pro-inflammatory. They can
attack tumours through the release of factors and
cytotoxic substances such as tumour necrosis factor-α and
reactive oxygen and nitrogen species [6].
M2
macrophages, on the other hand, are activated on the
alternative pathway and release anti-inflammatory
cytokines; these macrophages play a role in wound
healing and tissue repair [5].
Tumour associated
macrophages (TAMs) are believed to belong to this class
of macrophages, and they are known to contribute to
angiogenesis and growth of the tumour [7].
Reactive oxygen species (ROS) are known to be
important molecules for pro-inflammatory immune
response ranging from killing pathogens to immune cell
migration. These effects may be produced through direct
interaction with cell membranes or through stimulation of
secondary pathways [8]. Recently, it was shown that
ROS are need for the activation of M1 macrophages [9].
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Since it is well known that plasma can affect the redox
state of cells [10], we hypothesize that plasma can
activate macrophages to display the M1 phenotypes
through delivered ROS. Stimulated macrophages in turn
may release pro-inflammatory cytokines that are toxic to
cancer cells. In the past, our lab has shown that certain
regimes of plasma can stimulate macrophage function in a
migration assay [11]. Here we investigated the ability of
uniform nanosecond pulsed dielectric barrier discharge
(nspDBD) to polarize THP-1 M0 macrophages into M1
phenotypes and its efficacy in promoting anti-cancer
activity.
2. Materials and Methods
The THP-1 human monocyte cells were differentiated
into macrophages with phorbol myristate acetate (PMA)
in 24-well plates and treated with nspDBD at different
doses. Cells were incubated for 48 hours to allow for the
production and secretion of pro-inflammatory cytokines
into the media. This “conditioned media” (CM) was
collected and transferred onto A549 human lung cancer
cells growing in separate 24-well plates. After 24 and 48
hours, a Trypan Blue exclusion test was performed to
determine cell viability and growth. 24 hours after CM
treatment, A549s cells were fixed in ethanol and stained
with Propidum Iodide for cell cycle analysis . Data were
collected with image cytometry via the Nexcelom
Cellometer and further gating and analysis was performed
on the FCS flow cytometry software. A schematic of the
experimental setup is shown below in Fig. 1.
3. Results
Trypan Blue exclusion test of A549 lung cancer cells 24
hours after treatment with CM from plasma stimulated
macrophages showed a decrease in live cell counts
(Fig. 2). At 48 hours, while all cells continued to
proliferate, the proliferation rate of CM treated cells
treated was less than 40% of those grown in just RPMI
with 10% fetal bovine serum (FBS). Cell cycle analysis
performed 24 hours post treatment revealed that CM
1
stimulated macrophages can inhibit cancer proliferation.
Co-culturing of plasma treated macrophages with cancer
cell lines is underway. Ongoing work also includes
examining changes in gene expression in macrophages
following plasma treatment with RT-PCR. The CM will
also be further studied with ELISA analysis to determine
the specific cytokines the macrophages are secreting into
the media following treatment. Macrophage polarization
will be confirmed by the expression of cell surface
markers specific to M1 and M2 macrophages. This
information will help us further elucidate the mechanism
of the anti-tumour effect of non-thermal plasma.
Figure 1. Experiment design for the study.
treatment arrested the A549 lung cancer cells in the
G0/G1 phase (Table 1). Our results indicate that CM
from plasma stimulated macrophages can inhibit A549
lung cancer cell growth. These results further suggest that
uniform nspDBD may be employed for the stimulation of
pro-inflammatory functions of macrophages.
5. Acknowledgments
This work is funded internally and partially by NIH
R01 EB 013011-01 (Freeman).
6. References
[1] M. Vandamme et al. Plasma Process Polym., 7, 3-4
(2010)
[2] M. Keidar et al. Br. J. Cancer, 105, 9 (2011)
[3] D. Hanahan et al. Cell, 144, 5 (2011)
[4] C. Lewis et al. Cancer Res., 66, 2 (2006)
[5] K. Spiller et al. Biomaterials, 35, 15 (2014)
[6] A. Hedbrant et al. Int. J. Oncol., 46, 1 (2015)
[7] A. Mantovani et al. Trends Immunol., 23, 11 (2002)
[8] A. Varin and S. Gordon Immunol. Res., 26, 1
(2002)
[9] L. Padgett et al. J. Immunol., 192 (2014)
[10] Terry Paper
[11] V. Miller et al. Plasma Process Polym., 11, 12
(2014)
Figure 2. Live A549 cell counts 24hr and 48hr post CM
treatment was measured. A student’s t-test was used to
determine significance between the treated groups and the
control group cultured in RPMI+10% FBS (n = 3,
* p<0.05, + p<0.01).
Table 1.
treatment.
A549 cell cycle analysis 24hr post CM
Apoptotic
G0/G1
S
G2/M
Cancer Media
0.00
62.40±4.4
14.10±1.3
23.50±3.0
CM: 0Hz
0.00
64.79±0.7
12.99±0.3
22.22±0.4
CM: 15Hz
0.00
67.80±6.1
12.26±2.2
19.94±4.0
CM: 30HZ
0.09
69.64±2.8*
12.01±2.5
18.26±2.8*
4. Ongoing Work
Our results indicate that conditioned media from plasma
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