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Stem Cell Rev and Rep
DOI 10.1007/s12015-017-9772-y
Impact of Human Adipose Tissue-Derived Stem Cells
on Malignant Melanoma Cells in An In Vitro Co-culture Model
Fabian Preisner1 · Uwe Leimer1 · Stefanie Sandmann1 · Inka Zoernig2 ·
Guenter Germann1 · Eva Koellensperger1 © Springer Science+Business Media, LLC 2017
Abstract This study focuses on the interactions of human
adipose tissue-derived stem cells (ADSCs) and malignant
melanoma cells (MMCs) with regard to future cell-based
skin therapies. The aim was to identify potential oncological risks as ADSCs could unintentionally be sited within
the proximity of the tumor microenvironment of MMCs. An
indirect co-culture model was used to analyze interactions
between ADSCs and four different established melanoma
cell lines (G-361, SK-Mel-5, MeWo and A2058) as well as
two low-passage primary melanoma cell cultures (M1 and
M2). Doubling time, migration and invasion, angiogenesis,
quantitative real-time PCR of 229 tumor-associated genes
and multiplex protein assays of 20 chemokines and growth
factors and eight matrix metalloproteinases (MMPs) were
evaluated. Co-culture with ADSCs significantly increased
migration capacity of G-361, SK-Mel-5, A2058, MeWo and
M1 and invasion capacity of G-361, SK-Mel-5 and A2058
melanoma cells. Furthermore, conditioned media from all
ADSC-MMC-co-cultures induced tube formation in an
angiogenesis assay in vitro. Gene expression analysis of
ADSCs and MMCs, especially of low-passage melanoma
cell cultures, revealed an increased expression of various
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s12015-017-9772-y) contains
supplementary material, which is available to authorized users.
* Eva Koellensperger
Eva.Koellensperger@ethianum.de
1
ETHIANUM ‑ Clinic for Plastic, Aesthetic
and Reconstructive Surgery, Spine, Orthopedic
and Hand Surgery, Preventive Medicine, Voßstraße 6,
69115 Heidelberg, Germany
Department of Medical Oncology, National Center for Tumor
Diseases (NCT) Heidelberg, Heidelberg University Hospital,
Im Neuenheimer Feld 460, 60120 Heidelberg, Germany
2
genes with tumor-promoting activities, such as CXCL12,
PTGS2, IL-6, and HGF upon ADSC-MMC-co-culture. In
this context, a significant increase (up to 5,145-fold) in the
expression of numerous tumor-associated proteins could be
observed, e.g. several pro-angiogenic factors, such as VEGF,
IL-8, and CCL2, as well as different matrix metalloproteinases, especially MMP-2. In conclusion, the current report
clearly demonstrates that a bi-directional crosstalk between
ADSCs and melanoma cells can enhance different malignant
properties of melanoma cells in vitro.
Keywords Adipose tissue-derived stem cells (ADSCs) ·
Melanoma · Crosstalk · Tumor microenvironment · Skin
therapy · Regenerative medicine
Abbreviations
ADSCsAdipose tissue-derived stem cells
bFGFBasic fibroblast growth factor
CCLC-C motif-ligand
CDCluster of differentiation
COX-2Cyclooxygenase-2
CXCLC-X-C motif ligand
ECMExtracellular matrix
EMTEpithelial-mesenchymal-transition
EMMPRINExtracellular matrix metalloproteinase
inducer
FCSFetal calf serum
FNFibronectin
HGFHepatocyte growth factor
hMSCsHuman mesenchymal stem/stroma cells
HUVECHuman umbilical vein endothelial cells
ILInterleukin
MCAMMelanoma cell adhesion molecule
MMCsMalignant melanoma cells
MMPMatrix metalloproteinase
13
Vol.:(0123456789)
PMAPhorbol 12-myristate 13-acetate
SDStandard deviation
PTGS2Prostaglandin-endoperoxide synthase 2
TWTranswell
VEGFVascular endothelial growth factor.
Introduction
Human mesenchymal stem/stroma cells (hMSCs) consist
of a heterogeneous population of fibroblast-like progenitor
cells, capable of undergoing multilineage differentiation.
Due to their regenerative properties, they are considered a
useful tool for future cell-based therapies. Through paracrine
mechanisms, hMSCs are able to affect cells in their microenvironment, thus having immunomodulatory, pro-angiogenic,
anti-inflammatory, and anti-apoptotic effects [1]. Adipose
tissue-derived stem cells (ADSCs) are an abundant and readily available subset of hMSCs. In the context of stem cellbased therapies, ADSCs have been used in several clinical
trials, for example in the form of cell-assisted lipotransfer,
in order to enhance wound healing, tissue engineering, and
soft tissue augmentation after reconstructive surgery [2–4].
Interestingly, ADSCs also hold great promise for skin-based
therapies such as rejuvenation, scar remodeling and skin
repair [5, 6]. The mechanisms, through which the positive
effects are achieved, have not yet been fully elucidated and
are still under active investigation. The ADSCs secretome,
including a plethora of growth factors and cytokines, seems
to contribute to their therapeutic benefit. Recent reports suggest that ADSCs can stimulate collagen synthesis, reduce
UVB-induced apoptosis, protect dermal fibroblasts from
oxidative stress and cause a “whitening” effect based on
inhibition of melanin synthesis and tyrosinkinase activity
in melanocytes [7–11]. Also, hMSCs have been used successfully to limit scar formation in the context of cutaneous
wound healing [12].
However, ADSCs have also been shown to be actively
recruited to tumor sites and their inflammatory microenvironment, and thus, to contribute to tumor pathogenesis and
progression [13]. Additionally, ADSCs have been suggested
to be able to differentiate into tumor-promoting stromal cells,
also known as cancer-associated fibroblasts (CAFs) [14].
These cells are seen as an essential part within the tumorassociated stroma by influencing the neoplastic development
through secretion of various cytokines and proteases. As
a result of the reciprocal interactions between tumor and
stroma, the tumor cells’ motility, invasion and metastasis
were enhanced [15]. In fact, there are several studies demonstrating the tumor-promoting effects of hMSCs [16, 17].
Consequently, there have been concerns about the oncological safety of hMSCs in cell-based therapies, e.g. in the skin
[18].
13
Stem Cell Rev and Rep
The overall incidence of cutaneous melanoma continues to rise, especially in Caucasian populations [19, 20].
Considering the promising future for ADSCs in skin-based
therapies and regenerative medicine, it is of great importance to evaluate the oncological risk of possible interactions
between co-localized ADSCs and melanoma cells. Given
our focus on skin-based therapies, we investigated possible
interactions of ADSCs and melanoma cells, since it is conceivable that ADSCs could unintentionally be injected into
the proximity of a subclinical melanoma lesion. Therefore,
we co-cultured primary ADSCs with various established
melanoma cell lines and low-passage primary melanoma cell
cultures. Subsequently, we analyzed the conditioned media
for proteins and quantified changes in proliferation, migration, invasion, and gene expression of tumor-associated
genes and compared the results with those of the respective
mono-culture.
Methods
All chemicals, if not noted separately, were purchased from
Sigma–Aldrich, Munich, Germany.
Donor Specification
This study was conducted under the guidelines and with the
approval of the ethical committees of the University of Heidelberg and of the medical association of the local district
Baden-Wuerttemberg, Germany. After informed consent was
obtained, subcutaneous adipose tissue of six healthy women
with an age-range of 24–48 years (median age 37.5 years)
undergoing elective plastic surgery, was used for isolation
of ADSCs.
ADSCs Isolation, Culture and Characterization
ADSCs were isolated as previously described [15, 21]. Next,
cells were expanded, their multilineage differentiation potential was analyzed and the cells were examined for surface
marker expression using flow cytometry according to a previously described method [15].
ADSC‑MMC‑co‑Cultures
G-361-MMCs (#CRL-1424), A2058-MMCs (#CRL11,147), SK-Mel-5-MMCs (#HTB-70) and MeWo-MMCs
(#HTB-65) were purchased from American Type Culture
Collection (ATCC, Manassas, USA). Primary low-passage
melanoma cell cultures M1 (passage 16) and M2 (passage
Stem Cell Rev and Rep
were cultured up to 5 days in 4 ml individual co-culture
medium per well without medium change. Cell number was
determined daily after trypsinisation and trypan blue staining. MMC- and ADSC-mono-cultures – either in transwell
inserts or on 6-well culture plates – served as a control and
were treated like the respective co-culture. Supernatants of
day 4 were analyzed for proteins while the corresponding
cells were utilized for gene expression studies.
14) were kindly provided by Inka Zörnig (Department of
Medical Oncology, National Center for Tumor Diseases,
Heidelberg, Germany) (see Table 1).
For more comparable results, the use of a universal coculture medium would have been preferable. However, since
some melanoma cell lines have different nutritional requirements, an individual culture medium had to be designed for
each co-culture in which growth kinetics and morphology
of ADSCs and MMCs were least altered, when compared
to the use of officially recommended cell culture media.
ADSC-G361-co-cultures were performed in McCoy’s 5a
medium (ATCC, Manassas, USA) supplemented with 10%
fetal calf serum (FCS) (Biochrom), 10 ng/ml rhEGF and
10 ng/ml rhPDGF-BB (CellSystems, Troisdorf, Germany).
ADSC-SK-Mel-5- and ADSC-MeWo-co-cultures were carried out in Eagle’s Minimum Essential Medium (ATCC,
Manassas, USA) supplemented with 10% FCS, 10 ng/ml
rhEGF and 10 ng/ml rhPDGF-BB. ADSC-A2058-co-cultures were performed in an expansion medium consisting
of 60% Dulbecco’s modified Eagle’s medium (DMEM) high
glucose (4,5 g/l D-glucose) (Invitrogen, Life Technologies,
Darmstadt, Germany), 40% MCDB-201, 1% ITS (insulin
transferrin selenous acid) (Becton Dickinson, Heidelberg,
Germany), ­10− 8 M dexamethasone, 0.1 mM ascorbic acid2-phosphate, 2% FCS, 100 U/ml penicillin (Biochrom),
0.1 mg/ml streptomycin (Biochrom), 10 ng/ml rhEGF and
10 ng/ml rhPDGF-BB. ADSC-M1- and ADSC-M2-co-cultures were kept in RPMI 1640 (Gibco, Life Technologies,
Darmstadt, Germany) supplemented with 10% FCS, 1%
MEM non-essential amino acids (Gibco, Life Technologies, Darmstadt, Germany), 100 U/ml penicillin, 0.1 mg/ml
streptomycin, 10 ng/ml rhEGF and 10 ng/ml rhPDGF-BB.
All melanoma cell lines as well as ADSCs were cultured
in their individual co-culture medium for two passages
and then applied to the co-culture system. Co-culture of
melanoma cells and ADSCs was performed in a transwell
system. Therefore, 4 × 104 MMCs were seeded onto a polyester membrane transwell-clear insert (Corning, pore size
0.4 µm) while 4 × 104 ADSCs were seeded onto the bottom
of a 6-well cell culture plate with same cell density. Cells
Table 1 Melanoma cell
line donor information and
characteristics
Melanoma cell line
G-361*
SK-Mel-5*
A2058*
MeWo*
M1
M2
Determination of Cell Proliferation
Separate growth kinetics were evaluated during the exponential growth phase for both, mono- and co-cultured cells.
Therefore, cells of six wells per condition (ADSCs alone,
MMCs alone, and both cell types in co-culture) were harvested with trypsin/EDTA once every 24 h from day 1 to
day 5. The cells of each well were stained with trypan blue
and the viable cells were counted with a hemocytometer.
The doubling time DT was calculated by the formula: DT
[hours] = (log2 × T)/(logY − logX) with T = time in culture
[hours], Y = number of cells at the end of T, X = number of
cells at the beginning of T.
In Vitro Analysis of Cell Migration and Invasive
Behavior
The migration and invasion capacity of ADSCs and MMCs
alone and in co-culture were evaluated by using the QCM
24-Well Colorimetric Cell Migration Assay (Merck Millipore) and QCM ECMatrix Cell Invasion Assay (ECM550,
Merck Millipore). All steps were conducted according to
the manufacturer’s instructions. Briefly, cells of each cell
type were seeded in their individual co-culture medium
either on the bottom of the supplied 24-well plate (9 × 103
cells per well) or onto the membrane of the transwell insert
(9 × 103 cells per insert). Cells were cultured separately for
24 h (ADSCs) or 72 h (MMCs) before co-culture conditions
(ADSCs on the well plate bottom, MMCs in the transwell
Clinical data
Age
Sex
Ethnicity
31
24
43
78
64
60
Male
Female
Male
Male
Male
Female
Caucasian
Caucasian
Caucasian
Caucasian
Caucasian
Caucasian
Primary
tumor site
Tissue origin
Skin
Skin
Skin
Skin
Skin
Skin
Primary tumor
Metastatic site: lymph node
Metastatic site: lymph node
Metastatic site: lymph node
Metastatic site: sigmoid colon
Metastatic site: pleural effusion
* according to ATCC
13
inserts and vice versa) were applied for another 24 h (migration) or 72 h (invasion). Both cell types alone in the inserts
were regarded as negative controls. After removal of nonmigrated / non-invasive cells and staining of the remaining cells, the evaluation was performed in an ELISA reader
(Sunrise-Basic, Tecan, Austria) by measurement of the
extracted dye at 560 nm.
Quantitative Real‑Time Polymerase Chain
Reaction (qPCR)
Gene expression analysis was performed for 229 different
genes in four main tumor-associated categories: chemokines,
apoptosis, molecular mechanisms of cancer, and metastasis.
RNA was isolated from ADSCs and MMCs by using the
Trizol plus Kit (Life Technologies, Carlsbad, USA). Next,
Quant-iT RNA-Assay (Life Technologies) was carried out
to calculate total RNA-concentration and 1 µg of RNA was
applied to cDNA synthesis by the High Capacity cDNA
Reverse Transcription Kit (Life Technologies). Gene expression analysis was performed on a Step One Plus Instrument
(Life Technologies) using TaqMan Real-Time PCR technology. Gene expression analysis was conducted by using predesigned TaqMan 96-well array plates, each containing 92
different genes of interest and four endogenous controls with
10 ng cDNA per well (Human Molecular Mechanisms of
cancer #4418806, Human Chemokines #4418729, Human
Cellular Apoptosis Pathway #4418762, Human Tumor
Metastasis #4418743, Life Technologies, Carlsbad, USA). In
order to detect possible changes associated with an epithelial
to mesenchymal transition (EMT), gene expression of Eand N-cadherin was evaluated using specific TaqMan gene
expression assays (Hs01023894 for E-cadherin, Hs00983056
for N-cadherin) with 10 ng of cDNA per sample. To confirm
the results of the TaqMan 96-well array plates, qPCR was
performed for various genes of interest using pre-designed
TaqMan Gene Expression Assays (Life Technologies, Darmstadt, Germany) (Supplementary Table S1). All qPCRs for
TaqMan array plate verification were carried out in triplicates and normalized to GUSB. Calculation of fold change
in gene expression was conducted using delta delta CT
(ΔΔCT) – relative quantitation method.
Human Cytokine Magnetic 30‑Plex Panel
To quantify the expression of 30 cytokines (bFGF, CCL2,
-3, -4, -5, CXCL-9, -10, EGF, Eotaxin, G-CSF, GM-CSF,
HGF, IFN-α, -γ, IL-1β, -1RA, -2, -2R, -4, -5, -6, -7, -8,
-10, -12, -13, -15, -17, TNF-α, and VEGF), seven different matrix metalloproteinases (MMP-1, -2, -3, -7, -8, -9,
-10) and extracellular matrix metalloproteinase inducer
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Stem Cell Rev and Rep
(EMMPRIN, CD147) simultaneously in samples of each
ADSCs mono-culture, MMC-mono-culture, and ADSCMMC-co-cultures, a human cytokine magnetic 30-plex
(LHC6003M and LHC6002, Life technologies, Carlsbad,
USA) and a human MMP magnetic Luminex Performance
Assay were conducted according to manufacturer’s instructions. Samples were analyzed with a Bio-Plex 200 instrument (Bio-Rad, USA). Standard curves were generated to
calculate the levels of cytokines and MMPs.
Analysis of Angiogenic Properties
An in vitro angiogenesis assay kit (ECM 625, Merck Millipore) was used to investigate the possible pro-angiogenic
effects of ADSCs and MMCs alone or in co-culture. Therefore, supernatants of each condition were collected at day
4 of cell culture and analyzed for induction of tube formation in human umbilical vein endothelial cells (HUVEC)
according to manufacturer’s instructions. First, wells of a
96-well plate were coated with an ECM Matrix solution, and
7.5 × 103 HUVEC cells were seeded onto the matrix in each
well. The different conditioned media from ADSC-mono-,
MMC-mono- or ADSC-MMC-co-cultures were added and
incubated for eight hours at 37 °C, 5% ­CO2. Tube formation was visualized with a light microscope after four and
eight hours of incubation. A positive control was induced
by Phorbol 12-myristate 13-acetate (PMA) (Abcam, Cambridge, UK, no. ab120297).
Statistical Analysis
The results of proliferation, migration and invasion experiments were evaluated using the Mann–Whitney U Test.
P-values of less than 0.05 were regarded as statistically
significant. Statistical analysis was performed using SPSS
v21.0 (IBM Corp., Armonk, NY, USA) and graphs were
constructed with GraphPad Prism v7.0b (GraphPad Software, San Diego, CA, USA).
Results
Characterization of ADSCs
The isolated ADSCs were characterized according to the
ISCT and IFATS criteria for hMSCs by examination of distinct surface markers in flow cytometry as well as by evaluation of adipogenic and osteogenic differentiation potential
based on immunocytochemistry and qPCR analysis of lineage-specific genes or protein biomarkers [22, 23]. Isolated
ADSCs were positive for CD13, CD44, CD63, CD73, CD90,
Stem Cell Rev and Rep
CD105 and CD166, partially positive for CD29 and negative for CD31, CD34, CD45 and CD106 (Supplementary
Fig. S1).
After adipogenic differentiation for 14 days, ADSCs
showed markedly enhanced lipid storage in contrast to noninduced control cells. This finding was observed through
Oil Red O staining (Supplementary Fig. S2A). Perilipin
expression on the surfaces of the intracellular lipid droplets
was revealed by immunofluorescence analysis (Supplementary Fig. S2B). Furthermore, adipogenically differentiated
cells showed an up-regulation greater than 20 × 103-fold for
perilipin 1 (PLIN1) and a nearly fivefold up-regulation for
peroxisome proliferator-activated receptor gamma (PPARγ)
(Supplementary Fig. S2D). Moreover, osteogenically differentiated ADSCs showed significantly higher extracellular
calcium deposition than non-induced control-cells, analyzed
with Alizarin Red staining (Supplementary Fig. S2C). During and after osteogenic differentiation, ADSCs showed an
up-regulation of osteogenic markers runt-related transcription factor 2 (RUNX2) (sevenfold) and the tissue non-specific alkaline phosphatase (ALPL liver/ bone/ kidney) (up
to 24-fold) (Supplementary Fig. S2E).
Proliferation
Growth kinetics were analyzed during the exponential
growth phase (Supplementary Fig. S3A-E). When ADSCs
were cultured alone on a regular culture surface in the
six-well plate, the mean doubling time varied from 21 h
to 31 h depending on the selected culture medium. Also,
doubling times of MMCs differed greatly with low-passage primary melanoma cell cultures having the slowest proliferation rate. In most co-cultures, there were no
significant differences in proliferation compared to their
respective mono-cultures. However, proliferative activity of low-passage primary melanoma cell cultures M1
and M2 significantly increased during the exponential
growth phase when co-cultured with ADSCs (P = 0.037
for M1-MMCs and P < 0.01 for M2-MMCs). At the same
Table 2 Effect of ADSCMMC-co-culture on
proliferative activity
time, ADSCs doubling time significantly slowed down
in co-culture with M2 melanoma cells (P = 0.036). Also,
proliferative activity of ADSCs and G-361-MMCs significantly slowed down in co-culture (P = 0.01 for ADSCs
and G-361-MMCs) (Table 2).
Analysis of Gene Expression
After gene expression analysis was carried out for 229
different genes in four main tumor-associated categories
(chemokines, apoptosis, molecular mechanisms of cancer
and metastasis), genes whose expression increased (fold
change ≥ 2) or decreased (fold change ≤ 0.5) were once again
analyzed for validation purposes using specific TaqMan
Gene Expression Assays.
In this context, multiple changes in expression levels of
genes with tumor-promoting activities, such as CXCL12,
PTGS2, IL-6, IL-8, MMP-2 and HGF, could be detected in
both cell types. When comparing the reaction of the different
MMCs, low-passage primary melanoma cell cultures were
clearly most affected by the co-culture assay. Up-/downregulation of key genes in MMCs and ADSCs is illustrated in
Fig. 1a, b. For more details see Supplementary Table S2.
Multiplex Protein Analysis
After the co-culture of ADSCs and different melanoma cells,
the concentration of several proteins with pro-tumorigenic
activities was increased in the conditioned medium (Table 3
and Supplementary Fig. S4A-F). Amongst them, there were
multiple inflammatory and pro-angiogenic mediators, such
as CCL2, IL-6, IL-8 and VEGF. Also, protein secretion of
different pro-invasive molecules, such as MMP-2 and EMMPRIN, was enhanced under co-culture conditions. Changes
in protein secretion were again more obvious when ADSCs
were co-cultured with low-passage primary melanoma cell
cultures. Significant changes in the protein concentration
for bFGF, CCL5, CXCL-9, -10, Eotaxin, G-CSF, GM-CSF,
Co-culture
ADSCs
in monoculture
ADSCs in
co-culture with
MMCs
ADSC – G-361
ADSC – SK-Mel-5
ADSC – A2058
ADSC – MeWo
ADSC – M1
ADSC – M2
30.2 (3.6)
30.9 (4.5)
21.4 (1.3)
30.6 (4.4)
28.2 (2.8)
28 (3.5)
43.4 (10.5)
32.1 (3.1)
21.9 (1.4)
32.9 (3.5)
33 (4.6)
32.8 (3.3)
P-value MMCs in
monoculture
0.01
> 0.05
> 0.05
> 0.05
> 0.05
0.036
22.3 (2.2)
27.7 (3.4)
25.3 (3.8)
36.6 (4.2)
48.1 (5.2)
42.6 (3)
MMCs in coculture with
ADSCs
28.2 (3.7)
24.8 (2.4)
27.7 (2.5)
41.8 (4.9)
40.5 (3.9)
35.7 (3.7)
P-value
0.01
> 0.05
> 0.05
> 0.05
0.037
< 0.01
ADSC-MMC-co-cultures were setup for five days while doubling times were calculated during exponential
growth phase between day two and four. Proliferative activity is displayed as mean doubling time in hours.
Standard deviation is given in brackets (n = 6). Bold text indicates a statistically significant difference
13
Stem Cell Rev and Rep
Fig. 1 Up‑/downregulation of key genes in MMCs and ADSCs
after co‑culture Quantitative real-time PCR was performed on isolated cells from indirectly co-cultured (a) melanoma cells and (b)
ADSCs. Gene expression levels were compared to their respective
mono-cultures and expressed as relative gene expression. Standard
deviation is indicated as error bar (n = 3). Relative HGF-expression
in M1-MMCs (*) could not be calculated, since no expression was
detected in M1-mono-culture
IFN-α, IFN-γ, IL-1RA, -2, -2R, -4, -5, -7, -10, -12, -13, -15,
and − 17, and TNF-α could not be detected in any of the
co-cultures.
Migration
13
Migration through the transwell pores could already be
observed when ADSCs and melanoma cells were cultured
Stem Cell Rev and Rep
Table 3 Changes in the protein expression levels of ADSCs and MMCs in co-culture compared to mono-culture
Protein
bFGF
CCL2
CCL3
CCL4
EMMPRIN
HGF
IL-1β
IL-6
IL-8
MMP-1
MMP-2
MMP-3
MMP-7
MMP-9
MMP-10
VEGF
ADSC–G-361
ADSC–SK-Mel-5
ADSC–A2058
ADSC–MeWo
ADSC–M1
ADSC –M2
ADSC
G-361
ADSC
SKMel-5
ADSC
A2058
ADSC
MeWo
ADSC
M1
ADSC
M2
-0.7
--9.3
0.5
-0.8
0.7
0.7
1.6
1.4
1.1
1.1
1.4
0.7
-n.a
--1.9
n.a
-74
9
433
65
817
n.a
2.2
29
n.a
-0.6
--8.5
0.4
-0.7
1.1
0.6
1.2
1.2
1.2
1
1.2
0.8
-38
--1.7
n.a
-3.9
1.4
6.7
347
257
n.a
1
8.7
7.5
1.2
0.9
--10
0.5
-0.9
1.4
1.7
8
1
-4.1
1
0.8
1.1
5.3
--3.3
446
-1.9
0.8
3.2
73
109
-1
8.7
10
1.1
0.7
-1.6
3.3
0.5
-1.2
1.6
0.4
1.3
0.5
-0.8
0.6
1.1
0.9
81
-2.1
1.8
n.a
-6.4
2.2
1281
1.9
227
-1.4
11
3.8
1.1
1.7
1.1
-17
1.6
1
1.4
1.7
1.1
2.8
1.3
2.2
1.1
1.3
1.5
1.3
5.3
2.5
-2.5
n.a
1.8
689
2.1
76
2664
313
n.a
1.3
17
8.9
1.3
2.6
1.4
-11
2.3
1.1
2.7
2.3
1.2
2.3
1.2
1.6
1.1
1.1
2.2
1.6
9.8
3.8
-2.6
n.a
2.4
1782
3.7
113
5145
269
n.a
1.2
16
19
Values are displayed as mean fold protein concentration in the conditioned medium in co-culture versus the respective mono-culture. Results
from 0 to 9.9 are shown with one decimal, results of 10 or higher are displayed without decimals. - -, not detected; n.a., not applicable (because
not detectable in mono- or co-culture)
alone. However, the migratory capacity of ADSCs significantly increased when co-cultured with G-361-MMCs
(P < 0.01), SK-Mel-5-MMCs (P < 0.01), MeWo-MMCs
(P = 0.045), M1-MMCs (P < 0.01) and M2-MMCs
(P < 0.01) while that of ADSCs co-cultured with A2058MMCs remained nearly unchanged (P > 0.05).
An incubation with ADSCs led to a significant increase in
the migratory capacity of G-361-MMCs (P < 0.01), SK-Mel5-MMCs (P < 0.01), A2058-MMCs (P = 0.037), MeWoMMCs (P < 0.01) and M1-MMCs (P < 0.01), whereas for
M2-MMCs only a moderate, but not significant increase
could be observed (P = 0.173) (Figs. 2 and 3).
Invasion
ADSCs and melanoma cell lines, especially G361-MMCs
and A2058-MMCs and to a lower extend SK-Mel-5-MMCs,
MeWo-MMCs, M1-MMCs and M2-MMCs showed invasive behavior by actively digesting the extracellular matrix
blocking the transwell pores and migrating to the lower surface of the transwell inserts’ floor. This was significantly
increased in ADSCs when co-cultured with G-361-MMCs
(P < 0.01), SK-Mel-5-MMCs (P < 0.01), A2058-MMCs
(P = 0.01), MeWo-MMCs (P = 0.02), M1-MMCs (P < 0.01)
and M2-MMCs (P < 0.01).
Co-culture with ADSCs on the bottom plate led to a significant increase of the invasion capacity of G-361-MMCs
(P = 0.01), SK-Mel-5-MMCs (P < 0.01) and A2058-MMCs
(P < 0.01) whereas no significant differences of the invasion
capacity of MeWo-MMCs (P = 0.3), M1-MMCs (P = 0.57)
and M2-MMCs (P = 0.52) could be observed (Figs. 2 and 3).
Angiogenesis
An incubation of HUVEC endothelial cells with conditioned media from ADSC- and MMC-mono-cultures and
their respective co-cultures led to formation of tubular networks, made visible through inspection under an inverted
light microscope after eight hours of incubation.
In this context, different phases of tube formation could
be detected, ranging from development of capillary tubes
with (ADSC-M1-MMCs and ADSC-M2-MMCs) or without
sprouting of new branches (ADSC-A2058-MMCs), forming of closed polygons (ADSC-G-361-MMCs and ADSCMeWo-MMCs) to the development of complex mesh like
structures (ADSC-SK-Mel-5-MMCs) (Fig. 4a–f).
Discussion
Over the last few years, adipose tissue-derived stem cells
have repeatedly been described as a promising tool in future
regenerative medicine especially due to their paracrine
effects and immunomodulatory properties [24]. With regard
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Stem Cell Rev and Rep
Fig. 2 Changes in migratory and invasive capacity of ADSCs
and different melanoma cell lines under co‑culture conditions
Migratory (left diagrams) and invasive capacity (right diagrams) of
ADSCs and different melanoma cell lines alone and in co-culture
were measured and are displayed as level of optical density at 560 nm
with standard deviation indicated as error bars (n = 6): (a) ADSCG-361-co-culture, (b) ADSC-SK-Mel-5-co-culture, (c) ADSCA2058-co-culture, (d) ADSC-MeWo-co-culture, (e) ADSC-M1-coculture, (f) ADSC-M2-co-culture. Figure keys are displayed at the
bottom of the figure
to cell-based skin therapies, ADSCs have been reported to
increase collagen synthesis in dermal fibroblasts, reduce
UVB-induced apoptosis and cause a “whitening” effect
based on inhibition of melanin synthesis and tyrosinkinase
activity in melanocytes [7, 9–11, 25]. Furthermore, the use
of ADSCs has been successfully investigated in the context
of experimental wound healing, inflammatory skin disorders
and the promotion of skin flap survival [5, 26, 27].
13
Stem Cell Rev and Rep
Fig. 3 Qualitative assessment of changes in migration and invasion of ADSCs and different melanoma cell lines Representative light microscopic images showing stained ADSCs or melanoma
cells on the lower surface of the microporous transwell insert after
migration or invasion under different conditions and removal of
non-migrating / non-invading cells: (a) ADSC-G-361-co-culture,
(b) ADSC-SK-Mel-5-co-culture, (c) ADSC-A2058-co-culture, (d)
ADSC-MeWo-co-culture, (e) ADSC-M1-co-culture, (f) ADSCM2-co-culture. Migration and invasion capacity was quantified by
extracting the dye and measuring optical density at 560 nm, displayed
in Fig. 2
In vivo, however, there is always a risk of malignant cells
being located in the vicinity of the transplanted ADSCs.
ADSCs have been thought to influence the neoplastic cells
directly via paracrine communication or indirectly via modulation of the surrounding non-neoplastic compartment.
Multiple studies have addressed this concern suggesting that
ADSCs can exert cancer-promoting effects through several
mechanisms such as promotion of angiogenesis [28], differentiation into cancer-associated fibroblasts [29], stimulation of epithelial-mesenchymal transition (EMT) [30] and
promotion of metastatic spread [17, 31].
Numerous in vitro and in vivo studies focusing on the
complexity of ADSC-mediated influence on tumor growth
have been conducted so far, including ADSC-melanoma
experiments, partially leading to conflicting results. This
was clearly reflected by a study from Kucerova et al., in
which the interplay between different tumor cell lines and
ADSCs led to a ADSC-mediated tumor-favoring effect on
A375 melanoma cells, but not on 8MGBA glioblastoma
cells [16].
In the present study we demonstrated that paracrine crosstalk between ADSCs and different types of melanoma cells
takes place and leads to various changes in gene expression
and secreted protein levels as well as remarkable promotion
of migration and invasion in both cell types. Moreover, a
cocktail of different cytokines and growth factors within the
conditioned medium of ADSC-MMC-co-cultures stimulated
neoangionesis, an essential process for tumor growth.
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Stem Cell Rev and Rep
Fig. 4 Induction of angiogenesis Light microscopic evaluation of
tube formation in an angiogenesis assay with incubation of HUVEC
cells with conditioned media (cm) from different ADSC mono-cultures (cmADSC), different melanoma cell mono-cultures (cm [name
of melanoma cell line]) and from different ADSC-MMC-co-cultures
(cmADSC + [name of melanoma cell line]) in (a) ADSC-G361-,
(b) ADSC-SK-Mel-5-, (c) ADSC-A2058-, (d) ADSC-MeWo-, (e)
ADSC-M1-, and (f) ADSC-M2-experiments
The novelty in our report lies in the fact that we not only
analyzed the impact of ADSCs on established melanoma cell
lines but also on low-passage primary melanoma cell cultures, since most of the previous experiments on ADSC-melanoma interaction were performed using murine B16 melanoma cells or the well known high-passage A375 melanoma
cell line [16, 32–34]. The aim of our study was by no mean
to question this previously obtained data, since established
cell lines have repeatedly been proven to be valuable tools in
advancing science and allow for a better comparison among
different reports. However, there are also limitations which
must be considered. In this context, it is crucial to remember,
that the co-culture assay itself only represents a simplistic
model of the in vivo situation. Thus, it is of great importance
to maintain high cell line quality and to minimize possible
effects of long term cell culture, which could lead to questionable scientific data. Multiple studies have demonstrated
passage dependent effects on a wide range of cell types,
including cancer cell lines, affecting their growth rate, cellular response to stimuli, morphology as well as protein- and
gene expression [35–39]. Mouriaux et al. performed geneand protein expression analysis on different uveal melanoma
cell lines at various passages and found that cell passaging
severely altered the expression of tumor characteristic genes
[40]. Besides, repeated passaging may lead to a selection
of variable subsets of cancer cells which are more likely to
differ from their original tumor in vivo in terms of genotype
and phenotype. In other words, low-passage cancer cells are
thought to more closely resemble the cancer cells in vivo
and thus produce more reliable results. Still, the fact that
passage-dependent effects may have already occurred to a
certain extent in low-passage melanoma cell lines can be
regarded as a limitation of the study. Therefore, it remains
unclear if passage 14 and 16 is less enough to rule out possible effects of long-term cell culture.
Nevertheless, co-culturing of ADSCs and low-passage
primary melanoma cell cultures indeed induced multiple
changes in gene expression and protein synthesis in both
cell types, whereas co-cultures of ADSCs and high-passage
melanoma cell lines mainly had an impact on ADSCs. With
regard to a possible tumor-favoring effect, we could show
that the gene expression and/or protein synthesis of different proteins with tumor-promoting activity was increased in
ADSCs and MMCs.
First and foremost we observed an enhanced hepatocyte
growth factor (HGF) secretion and strong up-regulation of
HGF gene expression in M2-MMCs upon co-culture with
ADSCs. HGF is a multifunctional cytokine, which - upon
binding to its receptor c-Met - activates numerous downstream pathways involving angiogenesis, tumor growth,
migration and metastasis [41–43]. Besides, HGF has shown
to activate melanoma proliferation through MAPK-signaling
[44]. This latter effect could at least in part be responsible
for increased proliferation of low-passage primary melanoma cell cultures since increased HGF expression was not
observed in co-culture with established melanoma cell lines.
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Stem Cell Rev and Rep
Furthermore, IL-6 gene expression was up-regulated in
ADSCs and low-passage primary melanoma cell cultures
and protein synthesis was increased under co-culture conditions. IL-6 is a multifunctional cytokine which signals
through STAT3, which in turn is a regulator for proliferation, anti-apoptosis and angiogenesis [45]. Fittingly, IL-6
has been reported to promote proliferation in various tumor
types in vivo [46–49] and with special regard to melanoma,
IL-6 is known to convert from a paracrine growth inhibitor
to an autocrine stimulator during melanoma progression [50,
51]. Moreover, an elevated serum concentration of IL-6 has
been linked to cancer progression in different tumor types
[52–55] and has been identified as an independent prognostic biomarker of poor survival in patients with metastatic
melanoma [56].
In addition, gene expression and/or protein synthesis of
key vasculogenic factor VEGF and other pro-angiogenic
factors, such as CCL2 and IL-8, were up-regulated in some
ADSC-MMC-co-cultures, especially in the presence of lowpassage primary melanoma cell cultures. These molecules
have all been shown to exhibit potent angiogenic activity
by causing increased migration, proliferation and survival
of endothelial cells [57–65]. Along with that, Wu et al.
showed that overexpression of IL-8 in melanoma cells both
increased microvessel density and significantly enhanced
tumor growth and lung metastasis in vivo [64]. By supplying the tumor with nutrients and oxygen, the growth of new
blood vessels is a crucial step in tumorgenesis [66]. Fittingly,
we found increased angiogenic potential in every ADSCMMC-co-culture compared to their respective mono-culture
in vitro. Since the expression of multiple pro-angiogenic
factors was increased, it appears reasonable to think that a
mixture of mediators contributed to an increased tube formation rather than a single key factor.
It has been previously demonstrated that the presence of
pro-angiogenic factors, such as IL-8, can induce secretion
of matrix metalloproteinases (MMPs), especially of MMP-2
and MMP-9 [65, 67]. This leads to both facilitated angiogenesis and degradation of extracellular matrix and thus invasive tumor growth. Indeed, bi-directional crosstalk between
ADSCs and melanoma cells led to a strong increase in protein secretion of matrix metalloproteases, mainly of MMP-2.
In some co-cultures protein concentration of MMP-9 was
also slightly elevated compared to mono-culture. Plus, we
detected a moderate up-regulation of MMP-2 and MMP-9
gene expression in some ADSCs and MMCs. Accordingly,
paracrine interaction between ADSCs and different melanoma cells could in most cases significantly increase migration and/or invasion of both cell types in vitro, although
increased MMP-expression did not correlate with the extent
of increased migration/invasion. In contrast to most established melanoma cell lines, invasion of low-passage primary
cell cultures M1 and M2 was not enhanced under co-culture
conditions, although a strong increase in the gene expression
and protein secretion of different MMPs could be observed
in the co-culture assay. At this point, however, it is important to remember that MMP-activity is strongly regulated at
several levels [68]. For example, it is conceivable that tissue
inhibitors of metalloproteinases (TIMPs), which can inhibit
local MMP-activity, had an influence on cell invasion [69].
Besides that, it has been reported that tumorigenic properties of tumor cell lines can increase with cell passaging
[40]. Thus, passage-related effects should also be considered. Nevertheless, elevated expression of matrix degrading enzymes has already been linked to poor prognosis and
disease progression in various types of cancer [70–73]. Also,
in situ hybridization experiments have proven that in most
cancer tissues MMPs are expressed by stromal, rather than
cancer cells [74].
Regarding the extracellular matrix, protein concentration
of EMMPRIN (CD147) was also elevated in the conditioned
medium of ADSC-MMC-co-cultures. Increased expression
of EMMPRIN was noted in various human carcinomas
including malignant melanoma and was found to be associated with tumor invasiveness, proliferation and VEGF-production and to correlate with tumor progression by inducing
MMP-expression in stromal fibroblasts [75–78].
During tumor progression, a process termed epithelialmesenchymal transition (EMT) leads to the acquisition of a
more mesenchymal-like phenotype in various human cancer cells, including malignant melanoma [79]. This includes
detachment from surrounding cell-cell-adhesions, downregulation of E-cadherin and up-regulation of N-cadherin
[80]. After ADSC-MMC-co-culture, a mild to moderate
down-regulation of E-cadherin could be detected in some of
the melanoma cell lines, suggesting ongoing functional and
morphological changes pointing towards EMT. However, no
significant up-regulation of N-cadherin could be detected
upon co-culture (data not shown).
There is growing evidence that chronic inflammation
drives carcinogenesis [81]. In this regard, it is important
to see that co-culturing also led to a mild to moderate upregulation of different C-C motif ligand-chemokines (CCL)
and C-X-C motif ligand-chemokines (CXCL), such as
CCL2, CCL7, CCL20, CCL28, CXCL6, CXCL12, CXCL13,
CXCL14 as well as of the PTGS2 gene, which encodes for
COX-2. Within the tumor microenvironment some of these
molecules serve as chemoattractant for various immune cell
subsets, such as tumor-associated macrophages (TAMs). It
has previously been shown that CCL2 expression correlates with density of tumor-associated macrophages at the
tumor site, which in turn is associated with enhanced tumor
vascularization and increased degradation of extracellular
matrix, thus tumor progression and poor outcome [82–85].
In addition, strong up-regulation of CXCL12 gene expression could be detected in low-passage primary melanoma
13
cell cultures after ADSC-MMC-co-culture. Dysregulation of
the CXCL12/CXCR4 axis has been implicated in tumor progression regarding various types of human cancers, including malignant melanoma [86, 87]. However, it is important
to consider that post-transcriptional modifications of many
chemokines occur, affecting their biological function [88].
ADSC-MMC-co-culture also led to an increased expression of proto-oncogenes, especially of c-FOS and c-JUN.
FOS and JUN are both considered as major components
of the activator protein-1 (AP-1) complex which has been
attributed an important role in melanoma development and
progression [89, 90]. However, underlying regulatory mechanisms in vivo are complex and complicate the interpretation
of in vitro data.
ADSCs for co-culture experiments were obtained
exclusively from female donors. Therefore, potential gender-related differences of ADSCs regarding their cellular
characteristics also have to be addressed. It has been demonstrated, for example, that donor gender can have an impact
on the differentiation potential, proliferation capacity and
paracrine activity of ADSCs [91]. The latter aspect should
especially taken into account when focusing on paracrine
cell–cell interactions, as female ADSCs have shown to have
a higher secretion capacity of HGF and VEGF and hence
may produce slightly different results when used in co-culture experiments.
Furthermore, the melanoma cells’ tissue origin has to be
considered as another possible influencing factor when interpreting the data. Except for the G-361 melanoma cell line,
which is derived from a primary melanoma lesion of the
skin, every melanoma cell line in our experiments is derived
from a metastatic melanoma lesion. Plenty of research exists
on the diverse genetic alterations which occur during melanoma formation and progression. In this context, a range
of somatic mutations in dominant melanoma oncogenes
are considered to occur early during tumor development,
such as NRAS and BRAF, whereas other mutations are
more commonly seen in late-stage melanomas, for example
mutations of CDK2NA, PTEN and TP53 [92]. During later
stages in tumor development, tumor cells must acquire a
metastatic phenotype in order to spread to distant organs.
This involves changes in matrix degradation capabilities as
well as alterations in cell surface and gene expression [93,
94]. In this regard, drastic differences in gene expression
patterns between primary and nodally metastatic melanomas
have been shown previously [95]. As a consequence, cell
lines derived from metastatic sites are likely to differ from
primary cancer cells in terms of their genotype and phenotype and thus may show a different paracrine behavior when
confronted with ADSCs.
Conflicting data exist regarding the impact of ADSCs on
the proliferation of melanoma cells. Lee et al. previously
described a time- and dose-dependent anti-proliferative
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Stem Cell Rev and Rep
effect of ADSCs conditioned medium on B16 melanoma
cells, mediated through cell cycle modulation followed by
melanoma cell G1-arrest [32]. Also, an inhibitory effect
of ADSCs conditioned medium on the growth of A375
melanoma cells was reported by Ahn et al., suggesting that
ADSCs could function as a novel therapeutic agent in melanoma therapy [33]. In line with these results, our experimental data show that the growth of ADSCs and G-361
melanoma cells significantly slowed down in co-culture
compared to their respective mono-culture. However, qPCR
analysis did not reveal any major changes in the gene expression of cell cycle-modulating genes (data not shown). In
most of the co-cultures the proliferative activity was not
altered significantly, whereas proliferation of low-passage
primary melanoma cell cultures M1 and M2 significantly
increased during their exponential growth phase, when cocultured with ADSCs. Latter results go in accordance with
the previously mentioned study from Kucerova et al. who
demonstrated that co-culturing ADSCs with A375 melanoma cells leads to an increase of melanoma cell proliferation, thereby promoting tumor growth in vivo [16]. However,
in vitro results always have to be interpreted in the right
context, since the interplay between ADSCs/MMCs and the
non-malignant stromal compartment is not respected in the
co-culture assay. Thus, increased cell proliferation in vitro
does not necessarily have to lead to an enhanced tumor
growth in vivo. Also, co-culturing conditions were found to
have a remarkable influence on melanoma and mesenchymal
stem cell proliferation and hence may be in part responsible
for the adverse conclusions [96].
Taken together, the present study is first to prove that
ADSCs can enhance various malignant properties of melanoma cells in vitro, thereby also using low-passage primary melanoma cell cultures. Also, with regard to previously published data on the interaction between ADSCs and
squamous cell carcinoma cells [15], our results already add
more insight into potential oncological risks of ADSC-based
therapies with focus on the skin.
Conclusion
Plenty of evidence exists that ADSCs interact with cancer
cells in a paracrine fashion, which can eventually lead to
tumor progression and enhanced metastasis. Since ADSCs
have gained tremendous attention in the context of skin
repair and regeneration, it is vital to investigate how they
communicate with tissue-specific tumor cells. In this regard,
our study provides data supporting various potentially
tumor-promoting effects by reciprocal interactions of colocalized ADSCs and melanoma cells in vitro, affecting different malignant properties such as migration, invasion and
metastasis. Changes in gene expression and protein synthesis
Stem Cell Rev and Rep
were more apparent when ADSCs were co-cultured with
low-passage primary melanoma cell cultures, suggesting
that possible passage-related effects may have had an influence on melanoma cell behavior. For that reason, key in vitro
experiments focusing on ADSC-tumor-interaction should
always include primary cells or low-passage cell lines in
order to strengthen the scientific findings, if possible. With
special regard to melanoma cells, this can be achieved best
through the isolation of primary melanoma cells from metastatic tissue, since fresh specimens from patients’ primary
tumor samples are needed not only for pathologic analysis
but also to identify the molecular signature of the tumor
for a possible melanoma targeted therapy. Altogether, the
complexity of a potential ADSC-mediated impact on tumor
growth and metastasis reflected in this study, highlights
the necessity to fully elaborate possible oncological risks
of ADSC-based therapies to achieve a successful transfer
from bench to bedside.
Acknowledgements We would like to thank Claudia Ziegelmeier and
Iris Kaiser for technical support with the multiplex analysis and Prof.
Dr. Holger Sültmann (Division of Cancer Genome Research, NCT and
German Cancer Research Center, Heidelberg, Germany) for providing
access to the Bio-Plex 200 System.
Compliance with Ethical Standards Disclosure of interest The authors have no commercial, proprietary,
or financial interest in the products or companies described in this
article.
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