Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150
Aim: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). Methods: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-l...
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Karlitepe, A. Kabadayi, H. Vatansever, S. Gurdal, M. Gunduz, C. Ercan, G. 2018-06-19T09:11:27Z 2018-06-19T09:11:27Z 2017 Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 / A. Karlitepe, H. Kabadayi, S. Vatansever, M. Gurdal, C. Gunduz, G. Ercan // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 212–218. — Бібліогр.: 32 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138541 Aim: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). Methods: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RTPCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR. Results: In miR150-transfected cells, the increased expression of NK cell-specific genes such as GZMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent following miRNA150 transfection. Conclusion: It was shown in vitro that NK-like cells could be obtained by differentiation from AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical studies in improvement of immunotherapeutic approaches for cancer therapy. This study was supported by grant No. 214S650 from the Turkish Scientific and Technological Research Institute (TUBITAK). en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 |
| spellingShingle |
Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 Karlitepe, A. Kabadayi, H. Vatansever, S. Gurdal, M. Gunduz, C. Ercan, G. Original contributions |
| title_short |
Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 |
| title_full |
Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 |
| title_fullStr |
Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 |
| title_full_unstemmed |
Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 |
| title_sort |
anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with mirna150 |
| author |
Karlitepe, A. Kabadayi, H. Vatansever, S. Gurdal, M. Gunduz, C. Ercan, G. |
| author_facet |
Karlitepe, A. Kabadayi, H. Vatansever, S. Gurdal, M. Gunduz, C. Ercan, G. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2017 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
Aim: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose tissue derived mesenchymal stem cells (AD-MSCs). Methods: NK-like cells were differentiated from AD-MSCs and activated by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RTPCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were determined by TUNEL and RT-PCR. Results: In miR150-transfected cells, the increased expression of NK cell-specific genes such as GZMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent following miRNA150 transfection. Conclusion: It was shown in vitro that NK-like cells could be obtained by differentiation from AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical studies in improvement of immunotherapeutic approaches for cancer therapy.
|
| issn |
1812-9269 |
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https://nasplib.isofts.kiev.ua/handle/123456789/138541 |
| citation_txt |
Anti-cancer efficiency of natural killer cells differentiated from human adipose tissue-derived mesenchymal stem cells and transfected with miRNA150 / A. Karlitepe, H. Kabadayi, S. Vatansever, M. Gurdal, C. Gunduz, G. Ercan // Experimental Oncology. — 2017 — Т. 39, № 3. — С. 212–218. — Бібліогр.: 32 назв. — англ. |
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2025-11-26T22:55:38Z |
| last_indexed |
2025-11-26T22:55:38Z |
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| fulltext |
212 Experimental Oncology 39, 212–218, 2017 (September)
ANTI-CANCER EFFICIENCY OF NATURAL KILLER CELLS
DIFFERENTIATED FROM HUMAN ADIPOSE TISSUE-DERIVED
MESENCHYMAL STEM CELLS AND TRANSFECTED
WITH miRNA150
A. Karlitepe1, H. Kabadayi2, S. Vatansever2, 3, M. Gurdal1, C. Gunduz4, G. Ercan1, 5, *
1Department of Medical Biochemistry, Ege University Medical School, Izmir 35100, Turkey
2Department of Histology and Embryology, Celal Bayar University Medical School, Manisa 45030, Turkey
3Experimental Health Sciences Research Center, Near East University, Mersin 33010, Turkey
4Department of Medical Biology, Ege University School of Medicine, Izmir 35100, Turkey
5Department of Stem Cell, Ege University Institute of Health Sciences, Izmir 35100, Turkey
Aim: The aim of this study is to investigate the effects of miR150 transfection on NK-like cells differentiated from adipose
tissue derived mesenchymal stem cells (AD-MSCs). Methods: NK-like cells were differentiated from AD-MSCs and activated
by miR150 transfection. Transfected/non-transfected NK-like cells were characterized by immunohistochemical and RT-
PCR analyzes. Apoptotic efficiency of the transfected/non-transfected NK-like cells on pancreatic cancer cells PANC1 were
determined by TUNEL and RT-PCR. Results: In miR150-transfected cells, the increased expression of NK cell-specific
genes such as GZMB, KIR2DL2, CD16, CD56, NKG2D, NKp46 and increased immunoreactivity of NK cell-specific surface
marker CD314 (NKG2D) were evident. TUNEL assays showed that NK-like cells with/without transfection induced apoptosis
in PANC1 cells in the same manner. The decrease in oncogene expression and the increase in the tumor suppressor gene
expression in PANC1 cells upon co-culture with NK-like cells differentiated from AD-MSCs were more prominent follow-
ing miRNA150 transfection. Conclusion: It was shown in vitro that NK-like cells could be obtained by differentiation from
AD-MSCs and their efficiency could be increased via miR150 transfection. The results are encouraging for further clinical
studies in improvement of immunotherapeutic approaches for cancer therapy.
Key Words: pancreatic cancer, adipose tissue derived mesenchymal stem cells, natural killer cells, miRNA150.
Pancreatic cancer (PaCa) is currently the third
most frequent cause of cancer-related death in the
United States [1]. Pancreatic ductal adenocarcinoma
(PDAC), the major histological subtype comprising
90% of all PaCa, displays local invasion and metasta-
sis during early stages of the disease via developing
intrinsic resistance to most therapeutics, contributing
to its notoriously poor prognosis with 1–5% of 5-year
survival rates (~6 months of median survival duration
with currently available therapies) [2–4].
Natural killer (NK) cells constitute a significant com-
ponent of the innate immune system which contributes
particularly to the anti-tumor immune response [5, 6].
NK cells recognize virus-infected and transformed ma-
lignant cells and then kill these target cells without prior
sensitization [6, 7]. NK cells have various surface recep-
tors (NK cells are CD3-negative, CD16- and/or CD56-
positive) and cytotoxic granules. NK cells recognize
tumor cells and are thought to be good candidates for
adaptive immunotherapy against cancer [8]. Recently,
they gained attention for their spontaneous cytotoxic-
ity against a broad range of malignancies and for their
potential to become a “multi-purpose” anti-cancer
agent and studies are focused on isolating and expand-
ing NK cells abundantly [1, 6, 8]. NK cells exist in the
peripheral blood at a very low concentration and after
isolation there is a need to expand them in culture in order
to reach a sufficient concentration for clinical applica-
tions. However, prolonged culture leads to exhaustion
of NK cells and they become ineffective in killing target
cells and die within a few days in clinical applications.
Therefore, in recent years there have been great interests
in attempts to generate NK cells from more abundant cell
sources. Until today, NK cells have been differentiated
in vitro from different cell sources such as embryonic
stem cells (ESCs) or hematopoietic stem cells (HSCs) [6].
NK cells have been also directly isolated from umbilical
cord blood or peripheral blood cells [6, 9]. Mesenchymal
stem cells (MSCs) are multipotent stem cells with promis-
ing potential for cancer research [10–12]. Recently, these
cells draw attention because of their importance for tissue
microenvironment and their ability of suppressing immune
system [11]. They can be obtained from many tissues
such as bone marrow and adipose tissue, etc. and are
able to be proliferated or differentiated suitably in in vitro
conditions [13, 14]. Usually adipose tissue can be ob-
tained easily and abundantly by a surgical procedure,
which is not severely invasive and provides a rich source
of MSCs [15, 16]. In addition, adipose derived MSCs
(AD-MSCs) have less ethical problems when compared
with other sources.
MicroRNAs (miRNAs) are short (~22 nt) noncoding
regulatory RNAs that function as post-transcriptional
Submitted: August 4, 2017.
*Correspondence: E-mail: gulinnazalper@yahoo.com
Tel.: +90 232 390 43 55
Abbreviations used: AD-MSC — adipose derived mesenchymal
stem cell; ESC — embryonic stem cell; HSC — hematopoietic stem
cell; MSC — mesenchymal stem cell; NK — natural killer; PaCa —
pancreatic cancer; PDAC — pancreatic ductal adenocarcinoma.
Exp Oncol 2017
39, 3, 212–218
Experimental Oncology 39, 212–218, 2017 (September) 213
repressors of protein-coding target messenger RNAs
and regulate translational repression or degrada-
tion [17, 18]. miR150 is expressed in various immune
cells such as mature NK and innate NK T cells, B and
T cells and is up-regulated during cellular matura-
tion [19]. Particularly miR150 is responsible for the
development and activation of NK cells and regulates
the production of IFNγ by NK cells [19].
The aim of this study is to differentiate AD-MSCs
to NK-like cells by using miRNA transfection in ad-
dition to classical differentiation protocol and then
to investigate the effect of these cells on human PaCa
cells in vitro.
MATERIALS AND METHODS
Cell culture. AD-MSCs purchased from ATCC
were cultured in MesenPRO (Thermo Fisher Scientif-
ic, MA, USA) medium. Human pancreatic duct endo-
thelial cells (hTERT HPDE) (a kind gift of MD Anderson
Cancer Center, TX, USA) were cultured in keratinocyte
serum free medium (Thermo Fisher Scientific, MA,
USA) supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 10 ng/ml of endothelial
growth factor and 1% penicillin/streptomycin (P/S)
(BI, Cromwell, CT, USA). Human PaCa cell line
(Panc1) was obtained from MD Anderson Cancer
Center, TX, USA and cultured in DMEM medium
supplemented with 10% FBS, 2 mM L-glutamine
and 1% P/S. AD-MSC derived NK-like cells were
cultured in RPMI-1640 medium supplemented with
10% FBS, 12.5 ng/ml IL-2, 5 ng/ml IL-15 (Sigma,
USA) and 1% P/S.
Hematopoietic induction. AD-MSC were cul-
tured in Stemline Hematopoietic Stem Cell Expansion
Medium (Sigma, USA) supplemented with 1% bovine
serum albumin (BSA), 0.1 mM 2-mercaptoethanol,
200 μg/ml of transferrin, 40 mg/ml of low-density lipo-
protein (LDL), 10 mg/ml of insulin, 10 ng/ml of throm-
bopoietin (TPO), 50 ng/ml of stem cell factor (SCF),
50 ng/ml Flt3 ligand, 20 ng/ml of granulocyte-
macrophage-colony stimulating factor (GM-CSF),
20 ng/ml IL-3, and 20 ng/ml IL-6 for one week
(Sigma, USA). The incubation period lasted for one
week while refreshing with the medium which was not
supplemented with IL-6 and TPO on 3rd and 6th days.
NK cell differentiation. Hematopoietically in-
duced-AD-MSCs were cultured in RPMI-1640 medium
supplemented with 50 ng/ml SCF, 50 ng/ml Flt-3 li-
gand, 12.5 ng/ml IL-2, 20 ng/ml IL-7, 40 ng/ml IL-15,
2 mM L-glutamine, and 10% FBS. The incubation
period lasted for 4 weeks via refreshing the medium
every three days.
MiR150 transfection. At the end of the 4th week
of culturing, AD-MSC-NK cells were transfected
with miR150 (Sigma, USA) using a nanoparticle
(Xfect) (Takara Bio Inc., USA). In this procedure,
AD-MSC-NK cells were incubated with nanoparticle-
miRNA complex consisting of miRNA150 and trans-
fection polymer for 48 h at 37 °C according to Xfect
kit protocol.
Reverse transcription-polymerase chain reac-
tion (RT-PCR). Total RNA was isolated from miR150-
transfected and non-transfected AD-MSC-NK cells
and PANC1 cells before and after co-culturing using
RNeasy Mini Kit (Qiagen, Hilden, Germany). Ex-
pression of NK cells-specific genes such as GZMB,
KIR2DL2, CD16, CD56, NKG2D and NKp46 in NK-like
cells and cancer-related genes such as KRas, TP53,
CDKN2A, SMAD4, TGFBR2, ARID1A by RT-PCR
in PaCa cells was analyzed (Roche, LightCycler 480 In-
strument, Basel, Switzerland). The primers used are
given in Table.
Table. Primers used for RT-PCR
Gene Forward primer Reverse primer
GZMB 5'-GAAACGCTACTAACTA-
CAGG-3
5'-CCACTCAGCTAAGAGGT-3
KIR2DL2 5'-AGGGTTCTTCTTGCTGC-3' 5'-AGTGTCCTTAAACTTCCCT-3'
CD16 5'-CCCAAGCTTGGGGCC-
GCCACCATGTGGCAGCT-
GCTCCTCCCAACT-3'
5'-GGGATCCCCAGGTG-
GAAAGAATGATGAGAT-3'
CD56 5'-TTGTTTTTCCTGGGAACT-
GC-3'
5'-CCGGATCTGCAGGTAGTT-
GT-3'
NKG2D 5'-TTGACTACTGGACATCTTT-
GCTTTTG-3'
5'-GAATAATGAGTTTAG-
GAATACAGC-3'
NKp46 5�-GGCTGTGTCTGAGT-
CAGAG-3�
5�-GAGTTCATGTCCGGGAT-
GTAG-3'
KRAS 5-AAGGCCTGCTGAAAAT-
GACTG-3'
5'-CAAAGAATGGTCCTGCAC-
CAG-3'
TP53 5'-AACGGTACTCCGCCACC-3' 5'-CGTGTCACCGTCGTGGA-3'
CDKN2A 5�-AGAGGAGGGGCTGGCTG-
GTCA-3'
5�-CAGCGCCCGCACCTCCTC-
TA-3'
SMAD4 5'-TTCTAGGTGGCTGGTCG-
GAA-3'
5'-CAGGTGATACAACTC-
GTTCG-3'
TGFBR2 5'-TCCGGGAAGGCGCCGTC-
CGCT-3'
5'-CGACTGTCAAGCGCAGCG-
GAGAG-3'
ARID1A 5'-CTTCAACCTCAGT-
CAGCTCCCA-3’
5'- GGTCACCCACCT-
CATACTCCTTT-3'
Beta Actin 5'-GCACCACACCTTCTA-
CAATG-3'
5'-TGCTTGCTGATCCA-
CATCTG-3
Immunohistochemical analysis. The cells
were fixed with 4% paraformaldehyde for 30 min
and were washed two times with phosphate buffer
saline (PBS). They were then incubated 10 min with
0.1% Triton X-100 solution on ice for permeabiliza-
tion and 3% hydrogen peroxide (H2O2) was applied
for 5 min after washing with PBS. They were then
treated with blocking solution (Invitrogen, CA, USA)
for an hour at room temperature and were incubated
with primary antibody anti-hNKG2D (RD Systems,
MN, USA) overnight at 4 °C. The samples were
washed with PBS and incubated with biotinylated
rabbit anti-mouse second antibody (Invitrogen,
CA, USA) for 30 min. After washing with PBS
streptavidin-hydrogen peroxidase (Invitrogen, CA,
USA) was added for 30 min. In order to develop the
immunohistochemical reaction, diaminobenzidine
(DAB) (ScyTek, UT, USA) was applied for 5 min.
After washing with PBS, slides were stained with
Mayer’s hematoxylin and mounted with mounting
medium. The intensity of immunolabeling was evalu-
ated by the two investigators in different times with
light microscopy (BX40, Olympus, Tokyo, Japan).
The immunoreactivities were considered as nega-
tive (-), weak (+), moderate (++) and strong (+++).
Co-culture. PANC1 cells were co-cultured with
AD-MSC-NK cells using polycarbonate membrane
214 Experimental Oncology 39, 212–218, 2017 (September)
(Sigma, MO, USA) in a ratio of 1:2 (NK:PANC1). After
incubation for 1 week, the number of dead cancer cells
was determined via TUNEL assay.
TUNEL assay. The in situ apoptosis detection
kit (ApopTag Plus Peroxidase In Situ Apoptosis De-
tection Kit, Millipore, MA, USA) was used. The cells
were fixed with 4% paraformaldehyde for 30 min
and were washed two times with PBS. They were
then incubated 10 min with 0.1% Triton X-100 solu-
tion on ice for permeabilization and endogenous
peroxidase activity was inhibited with 3% H2O2. The
cells were then incubated with equilibration buffer for
10–15 s and TdT-enzyme in a humidified atmosphere
at 37 ˚C for 60 min. They were subsequently put into
pre-warmed working strength stop/wash buffer
at room temperature for 10 min and incubated with
anti-streptavidin-peroxidase for 45 min. Each step
was separated by careful washing in PBS. Staining
was performed with DAB and counterstaining was
performed in Mayer’s hematoxylin.
Statistical analysis. Statistical analysis of the
data was performed using the Mann — Whitney U test
using the SPSS 16 (Statistical Package for the Social
Sciences) program. The statistical significance was
evaluated, considering p < 0.05 level as statistically
significant.
RESULTS
Hematopoietic induction and NK-like cell
differentiation. AD-MSCs in culture were ob-
served as fusiform cells adherent to the plastic
surface (Fig. 1, a). When cultured in hematopoietic
induction medium for a week, AD-MSCs detached
from plastic surface and became suspended after
forming sphere clusters (Fig. 1, b). Hematopoi-
etically induced-AD-MSCs were then cultured
in NK differentiation medium for 4 weeks in order
to obtain NK-like cells and these cells were called
AD-MSC-NK cells (Fig. 1, c).
Fig. 1. Characterization of AD-MSC: a — AD-MSC culture; b —
hematopoietic induction; c — AD-MSC-NK cell culture
Characterization of cultured cells was performed
immunohistochemically. Distributions of CD90 and
CD314 (NKG2D) were assessed following 4 weeks
of culturing of AD-MSCs and AD-MSC-NK cells (Fig. 2).
CD90 immunoreactivity was very weakly positive
(Fig. 2, e) while NKG2D (CD314) immunoreactivity was
strongly positive in AD-MSC-NK cells (Fig. 2, f) at the
30th day of culture. In control slides, negative staining
for both CD90 and CD314 was detected (Fig. 2, a, b).
Following miR150 transfection of NK cells differenti-
ated from AD-MSC, NKG2D immunoreactivity was found
to be strongly positive for miR150-AD-MSC-NK cells
in comparison to non-transfected AD-MSC-NK cells
(Fig. 3).
Fig. 3. Immunoreactivity of CD314 (NKG2D) in AD-MSC-NK cells
non-transfected (a) and miR150-transfected (b)
Expression of NK cell specific genes such as GZMB,
KIR2DL2, CD16, CD56, NKG2D and NKp46 signifi-
cantly increased in AD-MSC-NK cells (p < 0.05) when
Fig. 2. Characterization of AD-MSC (a, c, e) and AD-MSC-NK
(b, d, f) cells. Control staining (a, b), CD90 (c, d), CD314 (NK-
G2D) (e, f)
Experimental Oncology 39, 212–218, 2017 (September) 215
compared to undifferentiated AD-MSC as the control
group (Fig. 4) and following miR150 transfection,
the expression of these genes was found to be even
greater in the miR150-transfected AD-MSC-NK cells
(miR150-AD-MSC-NK) than the non-transfected
AD-MSC-NK cells (AD-MSC-NK) (Fig. 5).
mRNA expression
0.00
1.00
2.00
3.00
4.00
5.00
*
*
*
*
*
*
6.00
7.00
8.00
9.00
Re
la
tiv
e
fo
ld
Genes
GZMB KIR2DL2 CD16 CD56 NK2D NKP46
AD-MSC control
NK
Fig. 4. Variations in gene expressions of GZMB,
KIR2DL2, CD16, CD56, NKG2D and NKp46 genes in AD-
MSC-NK cells in comparison to AD-MSC as the control
group (Mean ± Standard Deviation, Mann — Whitney
U test was applied; *p < 0.05 (n = 3 for each group)
mRNA expression
*
*
*
* *
*
NK control
NK (miR150)
0.00
1.00
2.00
3.00
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la
tiv
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fo
ld
Genes
GZMB KIR2DL2 CD16 CD56 NK2D NKP46
Fig. 5. Variations in gene expressions of GZMB, KIR2DL2,
CD16, CD56, NKG2D and NKp46 genes in miR150-trans-
fected AD-MSC-NK cells in comparison to non-transfected
AD-MSC-NK cells as the control group (Mean ± Standard Deviation,
Mann — Whitney U test was applied; *p < 0.05 (n = 3 for each group)
Induction of apoptosis in PANC1 cells after
co-culturing with AD-MSC-NK cells. The TUNEL
me thod based on detection of DNA strand breaks
to identify apoptotic cells was used. According to our
TUNEL assay results, AD-MSC-NK and miR150-AD-
MSC-NK were both able to kill PANC1 cancer cells
(Fig. 6). TUNEL-positive cells were not detected
in the control staining of PANC1 cells, whereas 11%
of TUNEL-positive cells were observed in PANC1 cells
after co-culturing with NK cells (Fig. 6, b). After
miR150 transfection of NK cells, the percentage
of TUNEL-positive cells was 8.8% in miR150-NK-
PANC1 co-culture (Fig. 6, d) but only 3.0% in miR150-
NK-hTERT co-culture (Fig. 6, f). Only 4% of TUNEL-
positive cells were found in PANC1-AD-MSC co-
culture (Fig. 6, h).
After PANC1 cel ls were co-cultured with
AD-MSC-NK cells, the expression of KRAS oncogene
significantly decreased (p < 0.05), while the expression
of tumor suppressor genes such as P53, CDKN2A,
TGFBR2, and ARID1A significantly increased (p < 0.05)
in comparison with that in PANC1 cells before co-
culturing (Fig. 7). After miR150 transfection, expres-
sion of tumor suppressor genes further increased
while the oncogene expression was further reduced
in PANC1 cells co-cultured with the miR150-trans-
fected AD-MSC-NK cells in comparison to co-culture
of PANC1 cells with non-transfected AD-MSC-NK cells
(Fig. 8).
Fig. 6. TUNEL assay: a — control (PANC1); b — PANC1-NK co-cul-
ture; c — control (PANC1); d — PANC1-miR150-transfected NK co-
culture; e — control (hTERT-HPDE); f — hTERT-HPDE-miR150-trans-
fected NK co-culture; g — control (PANC1); h — PANC1-AD-MSC
co-culture
DISCUSSION
PaCa, which is frequently seen in the elderly,
is an aggressive progressing cancer with 5-year
216 Experimental Oncology 39, 212–218, 2017 (September)
survival rate being less than 5%. In recent years,
the incidence of the disease has been increasing
steadily and it is in the first place among cancer related
deaths [20, 21]. Among the studies of PaCa, cell-
based immunotherapeutic approaches are the most
interesting ones. NK cell-based immunotherapeutic
approaches in PaCa have been investigated in phase
studies in which NK cells isolated from the donors
or the patient were used [22].
In our study, the expression of NK cell-specific
genes such as GZMB, KIR2DL2, CD16, CD56,
NKG2D, and NKp46 and immunoreactivity of NK cell-
specific surface marker CD314 (NKG2D) were evalu-
ated in NK-like cells before and after performing
nanoparticle mediated miR150 transfection in AD-
MSC-NK cells. Both miR150-transfected and non-
transfected NK cells were co-cultured with PaCa
cells (PANC1) in order to evaluate their efficiency
in killing cancer cells and our results showed that
miR150-transfected NK cells and non-transfected
NK cells have similar apoptotic activities. However,
RT-PCR results indicated that the decrease in gene
expressions of oncogenes and the increase in the
expression of tumor suppressor genes of PaCa
were more prominent in co-culturing PaCa cells with
miR150-transfected NK cells in comparison to that
in co-culturing PaCa cells with non-transfected
NK cells.
The results of the immunohistochemical analysis
showed that CD314/NKG2D staining was strongly posi-
tive in miR150-NK and AD-MSC-NK cells compared
to AD-MSCs. RT-PCR analysis indicated that GZMB,
KIR2DL2, CD16, CD56, NKG2D, and NKp46 genes
expressed in NK cells were found to be more potent
in the miR150-transfected cells.
According to TUNEL results, both miR150-trans-
fected AD-MSC-NK and non-transfected AD-MSC-
NK-like cells were similarly effective in inducing apop-
totic death of PaCa cells. While cells gone to apoptosis
can be analyzed by TUNEL assay, the pre-apoptotic
cells cannot be analyzed by this assay so we per-
formed Annexin V in our continuing study and deter-
mined 30% of pre-apoptotic cells in Annexin V assay
(data not given).
It should also be noted that there is another
pathway leading to cell death named pyroptosis,
which is a proinflammatory apoptosis pathway.
It will also be valuable to evaluate the pyroptosis
pathway in PANC1-NK cells co-culturing via adding
caspase-1 analysis to the procedures already used
in our study. IL-18, an inflammatory cytokine as-
sociated with NK-like cells, is active in the pathway
of pyroptosis, via activating caspase-1, which is not
effective in the known apoptosis pathway but is ef-
fective in pyroptosis [23].
MSCs are derived from tissues that contain
a rich source for the surrounding connective tis-
sue. And thus AD-MSCs are promising immuno-
modulator stem cells which can be obtained easily
and abundantly from several tissues, in particular
by liposuction and lipectomy procedures [24, 25],
and AD-MSCs do not carry any legal problems
as it is an issue for ESC-based studies. Moreover,
the cells are easily obtained by getting ethical per-
mission of donors who are subjected to lipectomy
procedures voluntarily [26]. Allogeneic NK cells
can be easily obtained via differentiation protocol
following hematopoietic induction from AD-MSCs
and also the efficiency of NK cells can be increased
via miRNA transfection. With their high potential
as a new immunotherapeutic source, such NK cells
will then be available to be used for clinical pur-
poses in the future.
There are many miRNAs that are expressed
in human and mouse NK cells [27]. miR150, which
regulates the function of NK cells is highly exo-
genously expressed in resting NK cells in human and
PANC control
PANC co-culture
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Re
la
tiv
e
fo
ld
10.00
Genes
KRAS P53 CDKN2A SMAD4 TGFBR2 ARID1A
mRNA expression
* *
*
*
*
Fig. 7. Variations in gene expressions of KRAS, P53, CDKN2A,
TGFBR2, and ARID1A genes in PANC1 cells after co-cultured
with AD-MSC-NK cells in comparison to the PANC1 cells before
co-culturing as the control group (Mean ± Standard Deviation,
Mann — Whitney U test was applied; *p < 0.05 statistical signifi-
cance (n = 3 for each group)
Fig. 8. Variations in gene expressions of KRAS, P53, CDKN2A,
TGFBR2, and ARID1A genes in PANC1 cells after co-cultured
with miR150-transfected AD-MSC-NK cells in comparison
to the PANC1 cells after co-culturing with the non-transfected
AD-MSC-NK cells as the control group (Mean ± Standard Devia-
tion, Mann — Whitney U test was applied; *p < 0.05 statistical
significance (n = 3 for each group)
PANC control
PANC co-culture
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Re
la
tiv
e
fo
ld
Genes
KRAS P53 CDKN2A SMAD4 TGFBR2 ARID1A
mRNA expression
*
*
*
*
*
*
Experimental Oncology 39, 212–218, 2017 (September) 217
mice [28]. In our study, NK-like cells differentiated
from AD-MSCs were transfected with miR150 using
nanoparticles for the first time. RT-PCR analyzes
showed that in both non-transfected NK cells and
miR150-transfected NK cells oncogene KRAS ex-
pression decreased, while expression of tumor sup-
pressor genes (P53, CDKN2A, TGFBR2, SMAD4 and
ARID1A increased).
In a recent study, Joshi and colleagues investi-
gated the effect of miRNAs responsible for regulating
activation and function of NK cells on melanoma can-
cer stem cells. In their study, NK cells isolated from
the peripheral blood and then transfected with anti-
miR155 or anti-miR146 both showed antitumorigenic
effect on melanoma cells and increased production
of antitumorigenic factors released from NK cells such
as INFγ and granzyme B [29]. In ano ther study, Bezman
and colleagues demonstrated that mice with a targeted
deletion of miR150 have an impaired, cell lineage — in-
trinsic defect in their ability to generate mature NK cells
while miR150 transgene promotes the development
of NK cells, with more mature phenotype and also more
responsive to activation indicating that miR150 transfec-
tion enhances the function of NK cells [19]. In accor-
dance with these findings, in our study, it was shown that
miR150 increased the expression of NK cell-specific
genes as it was shown in Fig. 3 and 5.
The efficiency of NK cells derived from HCSs ob-
tained from fresh or frozen cord blood samples was
investigated in cancer cell lines using three different
culture systems developed by Spanholtz et al. Ac-
cording to their data, the most effective NK cells were
obtained from frozen cord blood HSCs and these cells
can be used in cancer treatment [30]. A study by Lu-
evano and his team supported these results. It has
been reported that NK cells obtained from frozen cord
blood HSCs were more effective [9].
Since the activity of NK cells in cancer patients
diminishes or disappears with time, cytokines which
have potential in activating NK cells such as IL-2,
IL-12, IL-15, IL-18, IL-21 are systemically adminis-
tered to the patients in order to induce endogenous
NK cells [31]. However, in these treatment approaches
high doses of these cytokines are administered to the
patients to strengthen and enumerate NK cells, and
it is known that this high dose cytokine treatment has
systemic toxic effects [32]. In our study, while obtain-
ing NK-like cells from AD-MSCs which has potential
for allogeneic use, we used cytokines at concentra-
tions such as 50 ng/ml SCF, 50 ng/ml Flt-3 ligand,
12.5 ng/ml IL-2, 20 ng/ml IL-7, 40 ng/ml IL-15 for
4 weeks in vitro during the differentiation protocol.
In our study, when performing differentiation protocol
in vitro we used IL-2 and IL-15 at the nanogram levels
which would not lead to systemic toxicity in the future
even we will continue to use cytokines at low levels
during in vivo studies.
Since PaCa is one of the most aggressive cancers,
our results are promising in improving immunothera-
peutic approaches to cure malignancies. The results
of this study are encouraging for further clinical studies
in improvement of immunotherapeutic approaches
for treatment.
ACKNOWLEDgEMENT
This study was supported by grant No. 214S650 from
the Turkish Scientific and Technological Research In-
stitute (TUBITAK).
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