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 . 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 . 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 . Additionally, ADSCs have been suggested to be able to differentiate into tumor-promoting stromal cells, also known as cancer-associated fibroblasts (CAFs) . 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 . 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 . 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 . 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 13 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 . With regard 13 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 , differentiation into cancer-associated fibroblasts , stimulation of epithelial-mesenchymal transition (EMT)  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 . 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. 13 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 . 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 . 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. 13 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 . 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 . 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 . By supplying the tumor with nutrients and oxygen, the growth of new blood vessels is a crucial step in tumorgenesis . 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 . For example, it is conceivable that tissue inhibitors of metalloproteinases (TIMPs), which can inhibit local MMP-activity, had an influence on cell invasion . Besides that, it has been reported that tumorigenic properties of tumor cell lines can increase with cell passaging . 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 . 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 . This includes detachment from surrounding cell-cell-adhesions, downregulation of E-cadherin and up-regulation of N-cadherin . 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 . 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 . 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 . 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 . 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 . 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 13 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 . 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 . 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 . 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 . 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 , 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. 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