Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells
© Kucerova et al.; licensee BioMed Central Ltd. 2013
Received: 27 June 2013
Accepted: 3 November 2013
Published: 9 November 2013
Mesenchymal stromal cells (MSCs) represent heterogeneous cell population suitable for cell therapies in regenerative medicine. MSCs can also substantially affect tumor biology due to their ability to be recruited to the tumor stroma and interact with malignant cells via direct contacts and paracrine signaling. The aim of our study was to characterize molecular changes dictated by adipose tissue-derived mesenchymal stromal cells (AT-MSCs) and the effects on drug responses in human breast cancer cells SKBR3.
The tumor cells were either directly cocultured with AT-MSCs or exposed to MSCs-conditioned medium (MSC-CM). Changes in cell biology were evaluated by kinetic live cell imaging, fluorescent microscopy, scratch wound assay, expression analysis, cytokine secretion profiling, ATP-based viability and apoptosis assays. The efficiency of cytotoxic treatment in the presence of AT-MSCs or MSCs-CM was analyzed.
The AT-MSCs altered tumor cell morphology, induced epithelial-to-mesenchymal transition, increased mammosphere formation, cell confluence and migration of SKBR3. These features were attributed to molecular changes induced by MSCs-secreted cytokines and chemokines in breast cancer cells. AT-MSCs significantly inhibited the proliferation of SKBR3 cells in direct cocultures which was shown to be dependent on the SDF-1α/CXCR4 signaling axis. MSC-CM-exposed SKBR3 or SKBR3 in direct coculture with AT-MSCs exhibited increased chemosensitivity and induction of apoptosis in response to doxorubicin and 5-fluorouracil.
Our work further highlights the multi-level nature of tumor-stromal cell interplay and demonstrates the capability of AT-MSCs and MSC-secreted factors to alter the anti-tumor drug responses.
KeywordsAdipose tissue-derived mesenchymal stromal cells Human breast cancer Chemoresistance Proliferation Epithelial-to-mesenchymal transition Cytokine profile
Breast cancer still remains one of the most common malignancies in women with multiple risk factors . Any solid tumor derived from breast epithelial tissue is supported by tumor stroma – a non-malignant tumor compartment composed from multiple cell types and non-cellular components. The tumor microenvironment creates a complex signaling network which substantially affects tumor biology and therapeutic responsiveness [2, 3]. Adipose tissue is the most abundant stromal constituent in the breast and also a rich source of mesenchymal stromal cells (MSCs) which contribute to mammary carcinogenesis . As a fat grafting procedure is frequently used in breast reconstruction, breast contour deformity correction or even in breast augmentation, it also carries potential oncological risk of de novo breast cancer and/or its recurrence [5, 6].
The MSCs derived from the adipose tissue (AT-MSCs) share a number of key characteristics with the bone marrow-derived MSCs (BM-MSCs) [7–9]. MSCs from both sources were demonstrated to integrate into tumor-associated stroma and exhibit multiple regulatory functions in the tumor microenvironment [10–12]. Experimental data revealed the capability of BM-MSCs to differentiate into tumor-associated fibroblasts [13–15] and even create a cancer stem cell niche  when exposed to tumor-conditioned medium. The interaction of BM-MSCs and breast cancer cells was also shown to promote metastatic spread as a result of bidirectional paracrine signaling . Although the effect on proliferation of the tumor cells was not stimulatory in general, MSCs were shown to promote tumor cell migration, an epithelial-to-mesenchymal transition (EMT), mediate release from the hormone-dependence, and increase chemoresistance in breast cancer cells [18–23]. MSCs-secreted factors increased mammosphere formation and the exosomes from MSCs were sufficient to support the growth of tumor xenografts [24–26]. Taken together these data suggest that BM-MSCs promote breast cancer growth and/or metastatic spread. However, a suppression of the tumor growth by MSCs was reported for the tumor types other then breast; and the role of MSCs in tumor growth remains a matter of further investigations [12, 27–29]. Better understanding of the underlying mechanisms might lead to the therapeutic intervention with the aim to increase an antitumor response [30, 31]. MSCs themselves can be specifically engineered for the increased tumor-targeting and efficiency of the anti-tumor treatment . The introduction of specific transgene(s) into the AT-MSCs sensitized the breast cancer cells MDA-MB-231 to the chemotherapeutic drug 5FU for in vitro.
We have previously characterized the effect of AT-MSCs on the proliferation of breast cancer cells; and linked it to the cytokine secretion profile of AT-MSCs . In this study we have focused on the multiple alterations induced in human Her2-positive breast cancer cell line SKBR3 by the AT-MSCs. We have extended our investigation also on the effect of stromal cells on drug responses in the tumor cells. We have observed that the AT-MSCs induced an EMT, decreased proliferation, increased migration and other molecular changes in the SKBR3 cells. We have shown that the AT-MSCs could alter chemosensitivity of the tumor cells.
Human tumor cell line SKBR3 (ATCC® Number HTB-30™) was used for the study. Tumor cells were maintained in high-glucose (4.5 g/l) DMEM (PAA Laboratories GmbH) containing 10% FBS (Biochrom AG), 10.000 IU/ml penicillin (Biotica, Part. Lupca, Slovakia), 5 μg/ml streptomycin, 2 mM glutamine and 2.5 μg/ml amphotericin (PAA Laboratories GmbH).
For mammosphere cultures, 4×104 EGFP-SKBR3 cells per well were plated in non-adherent 6-well plates (Ultra-low attachments plates, Corning, Amsterdam, NL) in serum free DMEM/F12 medium (GIBCO-Invitrogen BRL) supplemented with 10.000 IU/ml penicillin (Biotica, Part. Lupca, Slovakia), 5 μg/ml streptomycin, 2 mM glutamine, and 2.5 μg/ml amphotericin (Sigma, St. Louis, MO), 10 ng/ml bFGF (Miltenyi Biotec), 10 ng/ml EGF (Miltenyi Biotec), 4 μg/ml heparin (Sigma, St. Louis, MO), 2 μg/ml insulin (Sigma, St. Louis, MO) and B27 supplement (diluted 1:100, Gibco-Invitrogen BRL) and cultivated at 37°C in humidified atmosphere and 5% CO2 for 5 days. Specific inhibitors 1.63 μM LY294002 (Sigma, St. Louis, MO) or 0.5 μM SB203580 (Sigma, St. Louis, MO) were added to the MSCs-CM mammosphere medium as indicated.
AT-MSCs were isolated and characterized by immunophenotype and differentiation potential as previously described in  (Additional file 1). The AT-MSCs were expanded in low glucose (1.0 g/l) DMEM supplemented with 10% HyClone® AdvanceSTEM™ supplement (Thermo Scientific) and antibiotic/antimycotic mix (10.000 IU/ml penicillin, 5 μg/ml streptomycin, 2 mM glutamine, and 2.5 μg/ml amphotericin). Different isolates were used for the experiments (n = 4), each experiment was run at least twice with each isolate to draw the conclusions. Cells were maintained at 37°C in humidified atmosphere and 5% CO2.
Cell-free AT-MSCs conditioned medium (MSCs-CM) was collected from 80–90% confluent cultures after 24 hours of cultivation with fresh tumor cell culture medium or mammosphere culture medium, respectively, and filtered through 0.45 μm filters. Fresh MSCs-CM was always used for the experiments.
Stable transduction of SKBR3 to express enhanced green fluorescent protein (EGFP) was done by retrovirus gene transfer as described elsewhere . Transgene incorporation and EGFP expression was confirmed by PCR, reverse transcription coupled PCR and flow cytometric analysis performed on BD Canto II cytometer (Becton Dickinson, USA) equipped with FACS Diva program. FCS Express software was used for evaluation. The identity of SKBR3 and EGFP-SKBR3 cells was further confirmed by sustained expression of epithelial cell adhesion molecule (CD326, ≥98% positivity) verified by flow cytometry with specific antibody anti-EpCAM-PE (Miltenyi Biotec GmbH, Germany). Mouse IgG1-PE (Miltenyi Biotec GmbH, Germany) was used as negative isotype control.
Analysis of morphological changes in EGFP-SKBR3
Three ×105 EGFP-SKBR3 cells were mixed with 1.5×105 DiI-stained AT-MSCs and cocultured for 5–9 days. For a comparison, EGFP-SKBR3 cells alone were seeded and cell morphology was analyzed by fluorescent microscopy (Axiovert 200, Zeiss, Germany). Alternatively, quadruplicates of 4×104 tumor cells were seeded in MSC-CM or culture medium in 96-well plates. Phase-contrast images were taken in the IncuCyte ZOOM™ Kinetic Imaging System (Essen BioScience, UK). Cell confluence was evaluated by IncuCyte ZOOM™ 2013A software (Essen BioScience, UK) based on the confluence masks as recommended by manufacturer.
Fifty thousand EGFP-SKBR3 per well were plated in triplicates in ImageLock 96-well plates (Essen BioScience, UK) and let to adhere for 16 hrs. Confluent monolayers were wounded with wound making tool (Essen BioScience, UK), washed twice and supplemented with MSC-CM or culture medium. As indicated, medium was supplemented with receptor-tyrosine kinase inhibitors 150 nM Pazopanib, 250 nM Sorafenib or 200 nM Sunitinib (inhibitors kindly provided by National Cancer Institute, Bratislava). Images were taken every two hours for next 72 hrs in the IncuCyte ZOOM™ Kinetic Imaging System (Essen BioScience, UK). Cell migration was evaluated by IncuCyte ZOOM™ 2013A software (Essen BioScience, UK) based on the relative wound density measurements and expressed as means of three independent experiments run in triplicates ± SD.
Gene expression analysis
EGFP-SKBR3 tumor cells were cultured with or without MSC-CM for 6 days with everyday medium replenishment. Total RNA was isolated from 5×106 EGFP-SKBR3 cultured with or without MSC-CM. Cultured cells were collected by trypsinization, RNA isolated by NucleoSpin® RNA II (Macherey-Nagel) and treated with RNase-free DNase (Qiagen, Hilden, Germany). Total RNA was subjected to control PCR to confirm the absence of genomic DNA contamination. RNA was reverse transcribed with RevertAid™ H minus First Strand cDNA Synthesis Kit (Fermentas, Hanover, MD). 200 ng of cDNA was amplified in standard PCR performed in 20 μl 1x PCR master mix (Fermentas, Canada) with 0.5 μl respective specific primers (20 pmol/μl) and DNase free water (Fermentas, Canada) in DNA Engine Dyad™ Peltier Thermal Cycler (MJ Research, UK) with pre-set amplification profile and horizontal electrophoresis was used for detection of amplicons. Each reaction was run with appropriate no template controls and negative control (RNA template without reverse transriptase). Primer sequences were listed in Additional file 2.
Quantitative PCR was performed in 1 × ABsolute™ QPCR SYBR® Green Mix (ABgene, Surrey, UK), 0.16 μM primers and 200 ng of template cDNA on Bio-Rad CFX96™ and analyzed by Bio-Rad CFX Manager software version 1.6. Relative gene expression change was calculated according to ΔΔCt method. GAPDH and HPRT1 gene expression was taken as endogenous reference. Analysis was performed twice in triplicates and data expressed as means ± SD.
Multiplex and SDF-1α secretion analysis
5×104 EGFP-SKBR3, 2.5×104 AT-MSCs alone, and 5×104 SKBR3 cells mixed with 2.5×104 AT-MSCs (ratio 2:1) were plated in the wells of 24-well plates and cultured in 2 ml of complete culture medium for two days. Cell-free supernatants were collected and subjected to human Bio-Plex™ 27-plex Cytokine Assay (Bio-Rad Laboratories Inc, Hercules, CA). Measurements were performed on Luminex 100 System (Luminex Corporation, Austin, TX) in duplicates with two different AT-MSCs isolates. Results were expressed as mean pg/ml of culture medium ± SD.
In order to confirm the SDF-1α secretion SDF1-α Quantikine Immunoassay (R&D Systems Inc.) was used. SDF-1α levels in cell free supernatants were determined on xMark™ Microplate Spectrophotometer (BIO-RAD).
The effect on tumor cell proliferation was evaluated as a relative fluorescence determined by green fluorescence readout (Ex. 485, Em. 520) on PolarStar OPTIMA reader (BMG Labtechnologies, Offenberg, Germany) in direct cocultures. Quadruplicates of 1×104 EGFP-SKBR3 cells were seeded in black-walled 96-well plates (Greiner Bio-One Intl. AG) with increasing numbers of AT-MSCs and cultured for 6 days. Green fluorescence was directly proportional to the number of viable tumor cells within the wells and the fluorescence value in the untreated cells was set to 100% by default. Experiments were evaluated as mean of quadruplicates ± SD.
In order to dissect the role of SDF-1α/CXCR4 axis in proliferation of EGFP-SKBR3 cells in cocultures with AT-MSCs, specific inhibitor of this signaling axis AMD 3100 (Sigma, St. Louis, MO) was used. Final concentration of 5 μg/ml AMD 3100 was added to EGFP-SKBR3 cells alone, cultured in MSC-CM or in coculture with AT-MSCs. The effect on proliferation was evaluated as a relative fluorescence as described above.
Relative cell viability was evaluated by CellTiter-Glo™ Luminescent Cell Viability Assay (Promega Corporation, Madison, WI) based on the ATP quantitation representative of metabolically active cells. Quadruplicates of 6×103 SKBR3 cells per well were seeded in 96-well plates overnight. Diluted MSCs-CM was added to the adherent tumor cells on the next day. Relative proliferation was determined on LUMIstar GALAXY reader (BMG Labtechnologies, Offenburg, Germany). Values were expressed as mean relative luminescence ± SD, when luminescence of control cells was taken as reference. Experiments were repeated at least twice with similar results and a representative result is shown.
Following drugs were used: 5-fluorouracil (5FU, Sigma, St. Lois, MO), doxorubicin (DOX, EBEWE Pharma, Austria) and cis-platin (EBEWE Pharma, Austria). For the evaluation of chemosensitivity, either 6×103 EGFP-SKBR3 cells alone or mixed with AT-MSCs (ratio 2:1) were seeded in 96-well plates. On day 0, treatments were started with doxorubicin (6.25 -100 ng/ml), 5FU (6.25-1000 ng/ml) or cis-platin (0.001-10 μg/ml). The chemosensitivity was determined by fluorescence measurements as described above 6 days later. Experiments were evaluated as means of three different experiments run in quadruplicates and the relative fluorescence in untreated cells was taken as 100% by default. Alternatively, 8×103 EGFP-SKBR3 were seeded in 96-well plates overnight and treated with the drugs diluted in MSCs-CM. Relative fluorescence and cell proliferation was determined as above.
Quadruplicates of 2×104 SKBR3 per well were seeded in 96-well white-walled plates (Corning Costar Life Sciences, Amsterdam, NL) overnight. Doxorubicin (100 ng/ml) or 5FU (100 μg/ml and 500 μg/ml) diluted in MSC-CM or culture media was added to the cells for the indicated period of time and a Caspase-3/7 activity was determined by the Caspase-Glo® 3/7 Assay (Promega Corporation, Madison, WI) on LUMIstar GALAXY reader (BMG Labtechnologies, Offenburg, Germany) at indicated timepoints. Values were determined as mean values of RLU ± SD.
Annexin V assay
In order to quantify a proportion of viable, apoptotic and necrotic cells in cocultures, adherent AT-MSCs were labeled with 5 μM carboxy-fluorescein diacetate, succinimidyl ester (CFDA-SE, Molecular Probes, Eugene, OR) in a serum-free DMEM for 15 min at 37°C. Medium was replaced for standard culture medium to incubate overnight. Next day, SKBR3 cells were mixed with CFDA-SE labeled AT-MSCs in a ratio 2:1 and plated onto 6-well plate (5×104 SKBR3, 5×104 AT-MSCs, or 5×104 SKBR3 with 2.5×104 AT-MSCs/well) for direct co-culture. Doxorubicin at final concentration 50 ng/ml was added to the respective wells one day later and cells were treated for 48 hrs. Apoptotic cells were stained with Phycoerythrin-labeled Annexin V (eBioscience, San Diego, CA); dead cells were detected with DAPI viability dye. Cells were analyzed using BD CantoII cytometer (Becton Dickinson, USA) equipped with FACSDiva program. FCS Express software was used for the evaluation.
Studies involving comparison between the two groups were analyzed by an unpaired Student's t-test in GraphPad Prism® software (LA Jolla, CA). The value of p < 0.05 was considered statistically significant.
AT-MSCs stimulate an EMT and mammosphere formation in the breast cancer cells SKBR3
Paracrine signaling and migration of SKBR3 cells is influenced by AT-MSCs
As it was previously suggested the MSC also affected the tumor cell migration . We could confirm significantly increased migration of MSC-CM-exposed SKBR3 in a wound healing assay as well (Figure 2C). The role of upregulated VEGFR2 or c-Kit signaling in the increased migration of MSC-CM-exposed SKBR3 was further examined by its pharmacological inhibition with multi-target kinase inhibitors Sunitinib (VEGFR2, PDGFRβ and c-Kit inhibitor), Sorafenib (VEGFR-2, Raf-1 and B-Raf inhibitor) and Pazopanib (multi-target kinase inhibitor of VEGFR1, VEGFR2, VEGFR3, PDGFR, FGFR, c-Kit and c-Fms). The migration of SKBR3 in MSC-CM was significantly decreased with 200 nM Sunitinib; and did not change in 150 nM Pazopanib or 250 nM Sorafenib (Figure 2D). These data reflect the differential properties of these inhibitors and a capability of sunitinib to revert MSC-CM-stimulated migration of SKBR3 cells. In accordance with these data, HGF/c-Met signaling was excluded to contribute to increased migration because the expression level of HGF and c-Met did not change and a specific inhibitor of this signaling axis SU11274 did not suppress MSC-CM stimulated SKBR3 migration (data not shown).
AT-MSCs inhibit proliferation of breast cancer cells SKBR3
SKBR3 chemosensitivity is altered in the presence of MSC-CM or AT-MSCs
MSCs represent multipotent cells valuable for regenerative therapies including augmentation of tissue regeneration in breast reconstruction after cancer-related surgery. Although recent results suggested that AT-MSCs might improve a long-term retention of the grafts, the risks of this cellular treatment still remain unresolved specifically in the context of a patient with cancer history [5, 6]. Tumors always encompass both malignant part and non-malignant cells of various cell lineages with complex mutual interactions between particular cell types [2, 40]. MSCs can contribute to the tumor microenvironment and play a role in mammary carcinogenesis . Our data showed that AT-MSCs did not increase the proliferation of the HER2-overexpressing, estrogen/progesterone receptor negative breast cancer cells SKBR3. However, AT-MSCs induced an EMT in tumor cells with increased tumor cell migration and mammosphere formation, potentially leading to increased aggressiveness and metastatic capability. MSCs derived from bone marrow were already described to affect breast cancer cell proliferation, migration, invasiveness, metastasis, morphology, chemoresistance and hormone responsiveness (reviewed in [11, 41]). According to our data the MSCs can alter tumor biology regardless of their tissue origin. Similarities in the MSCs secretome dictate the nature of the interaction with the other cell types . It has been shown that a gene expression profile of the MSCs derived from breast adipose tissue is comparable to the MSCs originating from abdominal adipose tissue resulting in comparable stimulation of proliferation in breast cancer cells MCF7 and MDA-MB-231 . Moreover, the MSCs from primary breast cancer tissues were also shown to exert stimulatory effect on MCF7 proliferation and tumor growth . Detailed study of migration properties of the tumor-cell exposed MSCs have unraveled increased migration of the MSCs isolated from breast adipose tissues in comparison to the migration of the MSCs derived from abdominal adipose tissue . Gene expression profile of these migratory MSCs was close to the profile of MSCs isolated from the tumor-adjacent breast adipose tissues . Thus the MSCs derived from abdominal adipose tissue with lower responsiveness to tumor-induced motility might be preferred exogenous cell source for fat grafting and breast augmentation to limit the effect on mammary carcinogenesis.
MSCs-secreted cytokines induced an EMT, increased expression of pluripotency genes and mammosphere formation in breast cancer cells (Figure 1C-D and ) which might suggest the capability of MSCs to increase the proportion of tumor initiating cells as a consequence of the EMT [35, 45]. MSC-CM induced expression of VEGFR2 concomitant with high VEGFA expression in SKBR3 cells could generate autocrine loop directly affecting a tumor cell survival and potentially more invasive phenotype . Based on these data, we hypothesized that SKBR3 cells in combination with AT-MSCs might have increased tumorigenicity. However, no increase in the tumor-forming capabilities was observed when AT-MSCs were coinjected with EGFP-SKBR3 cells in vivo. AT-MSCs could not support the xenotransplant growth in immunodeficient mice (data not shown). The EMT and upregulation of pluripotency genes induced by MSC-CM was not sufficient to promote tumor growth in low tumorigenic SKBR3 cells. Recently Karnoub's group demonstrated that the MSCs-mediated EMT was neither sufficient nor necessary for a generation of cancer stem cell phenotype, although it contributed to the increased metastasis in vivo. Future studies will be focused on the attempt to develop tumor xenotransplant model to test the MSCs-mediated alterations in the tumor behavior and its chemosensitivity in vivo.
Our data further support the dual role of MSCs in tumor cell proliferation. Previously we have reported increased proliferation of breast cancer cells T47D, MCF7 and MDA-MB-361 in response to AT-MSCs [19, 23] in contrast to antiproliferative action on SKBR3 cells (Figure 3). Our data correspond with the findings by Donnenberg et al., who did not show the capability of the AT-MSCs to increase the proliferation of dormant tumor cells . Several studies reported that the MSCs could actually inhibit tumor growth in vivo[29, 48–50] although in different tumor types (glioblastoma, leukemic, thyroid and colon cancer cells). More importantly, substantially altered composition of the chemokine secretome in tumor-stromal coculture indicated how an inflammatory component of the tumor might arise in vivo[51, 52]. IP-10 (chemokine CXCL10) is an important mediator in bidirectional MSCs/breast cancer signaling . Its increase in the normoxic conditions and different AT-MSCs/SKBR3 coculture model further extends its importance in stromal/breast cancer interactions.
MSCs were also suggested to contribute to altered tumor drug resistance [21, 22, 54, 55]. Recently the study by Roodhart et al. demonstrated that cis-platin-preexposed MSCs mediated systemic resistance to cis-platin in tumor models including breast cancer cells MDA-MB-231 . However our experiments indicated that soluble factors present in the MSC-CM or the AT-MSCs concomitantly exposed to chemotherapeutic drug in direct coculture were not able to mediate chemoresistance (Figures 4 and 5). SKBR3 tumor cells in the presence of AT-MSCs had significantly increased sensitivity to chemotherapeutic drugs doxorubicin and 5FU that are frequently used for the breast cancer treatment. No significant difference in sensitivity to cis-platin (Figure 5C) or paclitaxel (data not shown) was detected when the AT-MSCs and tumor cells were exposed to the drug in cocultures. We believe that a concomitant exposure of stromal and tumor cells to the drug might actually increase the treatment efficiency. Contrastingly the exposure of (circulating) MSCs to the chemotherapy might induce secretion of mediators which subsequently contributed to increased tumor cell resistance [22, 55]. It remains to be further evaluated, which mechanisms are drug-specific, tumor cell type-specific or context specific. Taken together the mutual tumor/stromal interactions do not only determine the biological behavior of tumor as a complex organ, but also its response to the chemotherapeutic treatment. The effects of MSCs on tumor cells are multiple and depend on the state of the tumor cell (dormant vs. actively-proliferating), the properties of specific MSCs populations, and interactions with other cell types, such as tumor infiltrating immune cells origin . It is important to focus on the evaluation of interactions of MSCs with primary tumor cells to shed more light into the operating interactions and signaling pathways.
The aim of our study was to analyze biological effects of AT-MSCs on breast cancer cells SKBR3. We have demonstrated that AT-MSCs induced morphological changes, epithelial-to-mesenchymal transition, increased adherence, mammosphere formation, migration and decreased proliferation in SKBR3. These features and mechanisms of bidirectional signaling are shared by the MSCs originating from adipose tissue with the bone-marrow derived MSCs and considered to play an important role in the breast cancer pathogenesis. Our results indicated the capability of AT-MSCs and secreted soluble factors to increase the chemosensitivity of SKBR3 cells to doxorubicin and 5-fluorouracil. We concluded that the MSC-mediated influence on the drug resistance is dependent on the context of treatment, its timing and a cell type. Based on our observations, we concluded that the tumor and stromal cells interacted in a complex fashion that altered the properties of tumor cells and created dynamic interaction relevant for the tumor behavior and responses.
α-smooth muscle actin
Adipose tissue-derived mesenchymal stromal cells
Chemokine (C-C motif) ligand 5, RANTES
Stem cell factor receptor
Hepatocyte growth factor receptor
Chemokine (C-X-C) motif receptor 4, CXCL12 receptor
Epidermal growth factor
Enhanced green fluorescent protein
Fibroblast activating protein
Fibroblast growth factor
Granulocyte-colony stimulating factor
Granulocyte monocyte-colony stimulating factor
Hepatocyte growth factor
Hypoxanthine phosphoribosyltransferase 1
- IP-10 (CXCL10):
Chemokine (C-X-C motif) ligand 10
- MCP-1 (CCL2):
Monocyte chemoattractant protein-1, chemokine CCL2
- MIP-1a (CCL3):
Macrophage inflammatory protein-1alpha
Mesenchymal stromal cell-conditioned medium
Mesenchymal stromal cells
Platelet derived growth factor
- POU5F1 (Oct-3/4):
POU factor class 5 homeobeox 1
- RANTES (CCL5):
Regulated on Activation, Normal T-cell Expressed and Secreted, chemokine CCL5
Stem cell factor
Stromal cell-derived factor-1α, CXCL12
Tumor associated fibroblasts
Vascular endothelial growth factor
We thank L. Baranovicova for the help with MSC characterization, M. Dubrovcakova and V. Frivalska for the excellent technical assistance. This study was supported by the VEGA grants No.2/0088/11 (L.K.), 2/0130/13 (Z.K.) and 2/0171/13 (M.M.). This work was supported by the Slovak Research and Development Agency under the contract No.APVV-0230-11. The experiments on the IncuCyte ZOOM™ were enabled with the kind help and the financial support from the Cancer Research Foundation programmes WAC2003, RFL2009 and RFL2012.
- Nelson HD, Zakher B, Cantor A, Fu R, Griffin J, O'Meara ES, Buist DS, Kerlikowske K, van Ravesteyn NT, Trentham-Dietz A, et al: Risk factors for breast cancer for women aged 40 to 49 years: a systematic review and meta-analysis. Ann Intern Med. 2012, 156: 635-648. 10.7326/0003-4819-156-9-201205010-00006.View ArticlePubMedPubMed CentralGoogle Scholar
- Egeblad M, Nakasone ES, Werb Z: Tumors as organs: complex tissues that interface with the entire organism. Dev Cell. 2010, 18: 884-901. 10.1016/j.devcel.2010.05.012.View ArticlePubMedPubMed CentralGoogle Scholar
- Hanahan D, Coussens LM: Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 2012, 21: 309-322. 10.1016/j.ccr.2012.02.022.View ArticlePubMedGoogle Scholar
- Zhao M, Dumur CI, Holt SE, Beckman MJ, Elmore LW: Multipotent adipose stromal cells and breast cancer development: Think globally, act locally. Mol Carcinog. 2010, 49: 923-927. 10.1002/mc.20675.View ArticlePubMedPubMed CentralGoogle Scholar
- Donnenberg VS, Zimmerlin L, Rubin JP, Donnenberg AD: Regenerative therapy after cancer: what are the risks?. Tissue Eng Part B Rev. 2010, 16: 567-575. 10.1089/ten.teb.2010.0352.View ArticlePubMedPubMed CentralGoogle Scholar
- Zimmerlin L, Donnenberg AD, Rubin JP, Basse P, Landreneau RJ, Donnenberg VS: Regenerative therapy and cancer: in vitro and in vivo studies of the interaction between adipose-derived stem cells and breast cancer cells from clinical isolates. Tissue Eng Part A. 2011, 17: 93-106.View ArticlePubMedGoogle Scholar
- Schaffler A, Buchler C: Concise review: adipose tissue-derived stromal cells–basic and clinical implications for novel cell-based therapies. Stem Cells. 2007, 25: 818-827. 10.1634/stemcells.2006-0589.View ArticlePubMedGoogle Scholar
- Ciavarella S, Dominici M, Dammacco F, Silvestris F: Mesenchymal stem cells: a new promise in anticancer therapy. Stem Cells Dev. 2011, 20: 1-10. 10.1089/scd.2010.0223.View ArticlePubMedGoogle Scholar
- Strioga M, Viswanathan S, Darinskas A, Slaby O, Michalek J: Same or not the same? Comparison of adipose tissue-derived versus bone marrow-derived mesenchymal stem and stromal cells. Stem Cells Dev. 2012, 21: 2724-2752. 10.1089/scd.2011.0722.View ArticlePubMedGoogle Scholar
- Kidd S, Spaeth E, Watson K, Burks J, Lu H, Klopp A, Andreeff M, Marini FC: Origins of the tumor microenvironment: quantitative assessment of adipose-derived and bone marrow-derived stroma. PLoS One. 2012, 7: e30563-10.1371/journal.pone.0030563.View ArticlePubMedPubMed CentralGoogle Scholar
- El-Haibi CP, Karnoub AE: Mesenchymal stem cells in the pathogenesis and therapy of breast cancer. J Mammary Gland Biol Neoplasia. 2010, 15: 399-409. 10.1007/s10911-010-9196-7.View ArticlePubMedGoogle Scholar
- Kucerova L, Skolekova S: Tumor microenvironment and the role of mesenchymal stromal cells. Neoplasma. 2013, 60: 1-10.View ArticlePubMedGoogle Scholar
- Mishra PJ, Humeniuk R, Medina DJ, Alexe G, Mesirov JP, Ganesan S, Glod JW, Banerjee D: Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res. 2008, 68: 4331-4339. 10.1158/0008-5472.CAN-08-0943.View ArticlePubMedPubMed CentralGoogle Scholar
- Hall B, Dembinski J, Sasser AK, Studeny M, Andreeff M, Marini F: Mesenchymal stem cells in cancer: tumor-associated fibroblasts and cell-based delivery vehicles. Int J Hematol. 2007, 86: 8-16. 10.1532/IJH97.06230.View ArticlePubMedGoogle Scholar
- Spaeth EL, Dembinski JL, Sasser AK, Watson K, Klopp A, Hall B, Andreeff M, Marini F: Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression. PLoS One. 2009, 4: e4992-10.1371/journal.pone.0004992.View ArticlePubMedPubMed CentralGoogle Scholar
- Li HJ, Reinhardt F, Herschman HR, Weinberg RA: Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E2 signaling. Cancer Discov. 2012, 2: 840-855. 10.1158/2159-8290.CD-12-0101.View ArticlePubMedGoogle Scholar
- Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL, Polyak K, Tubo R, Weinberg RA: Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007, 449: 557-563. 10.1038/nature06188.View ArticlePubMedGoogle Scholar
- Martin FT, Dwyer RM, Kelly J, Khan S, Murphy JM, Curran C, Miller N, Hennessy E, Dockery P, Barry FP, et al: Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: stimulation of epithelial to mesenchymal transition (EMT). Breast Cancer Res Treat. 2010, 124: 317-326. 10.1007/s10549-010-0734-1.View ArticlePubMedGoogle Scholar
- Rhodes LV, Antoon JW, Muir SE, Elliott S, Beckman BS, Burow ME: Effects of human mesenchymal stem cells on ER-positive human breast carcinoma cells mediated through ER-SDF-1/CXCR4 crosstalk. Mol Cancer. 2010, 9: 295-10.1186/1476-4598-9-295.View ArticlePubMedPubMed CentralGoogle Scholar
- Rhodes LV, Muir SE, Elliott S, Guillot LM, Antoon JW, Penfornis P, Tilghman SL, Salvo VA, Fonseca JP, Lacey MR, et al: Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence. Breast Cancer Res Treat. 2010, 121: 293-300. 10.1007/s10549-009-0458-2.View ArticlePubMedGoogle Scholar
- Greco SJ, Patel SA, Bryan M, Pliner LF, Banerjee D, Rameshwar P: AMD3100-mediated production of interleukin-1 from mesenchymal stem cells is key to chemosensitivity of breast cancer cells. Am J Cancer Res. 2011, 1: 701-715.PubMedPubMed CentralGoogle Scholar
- Roodhart JM, Daenen LG, Stigter EC, Prins HJ, Gerrits J, Houthuijzen JM, Gerritsen MG, Schipper HS, Backer MJ, van Amersfoort M, et al: Mesenchymal stem cells induce resistance to chemotherapy through the release of platinum-induced fatty acids. Cancer Cell. 2011, 20: 370-383. 10.1016/j.ccr.2011.08.010.View ArticlePubMedGoogle Scholar
- Kucerova L, Kovacovicova M, Polak S, Bohac M, Fedeles J, Palencar D, Matuskova M: Interaction of human adipose tissue-derived mesenchymal stromal cells with breast cancer cells. Neoplasma. 2011, 58: 361-370. 10.4149/neo_2011_05_361.View ArticlePubMedGoogle Scholar
- Klopp AH, Lacerda L, Gupta A, Debeb BG, Solley T, Li L, Spaeth E, Xu W, Zhang X, Lewis MT, et al: Mesenchymal stem cells promote mammosphere formation and decrease E-cadherin in normal and malignant breast cells. PLoS One. 2010, 5: e12180-10.1371/journal.pone.0012180.View ArticlePubMedPubMed CentralGoogle Scholar
- Sullivan NJ, Sasser AK, Axel AE, Vesuna F, Raman V, Ramirez N, Oberyszyn TM, Hall BM: Interleukin-6 induces an epithelial-mesenchymal transition phenotype in human breast cancer cells. Oncogene. 2009, 28: 2940-2947. 10.1038/onc.2009.180.View ArticlePubMedGoogle Scholar
- Zhu W, Huang L, Li Y, Zhang X, Gu J, Yan Y, Xu X, Wang M, Qian H, Xu W: Exosomes derived from human bone marrow mesenchymal stem cells promote tumor growth in vivo. Cancer Lett. 2012, 315: 28-37. 10.1016/j.canlet.2011.10.002.View ArticlePubMedGoogle Scholar
- Kidd S, Spaeth E, Klopp A, Andreeff M, Hall B, Marini FC: The (in) auspicious role of mesenchymal stromal cells in cancer: be it friend or foe. Cytotherapy. 2008, 10: 657-667. 10.1080/14653240802486517.View ArticlePubMedGoogle Scholar
- Klopp AH, Gupta A, Spaeth E, Andreeff M, Marini F: Concise review: dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth?. Stem Cells. 2011, 29: 11-19. 10.1002/stem.559.View ArticlePubMedGoogle Scholar
- Kucerova L, Matuskova M, Hlubinova K, Altanerova V, Altaner C: Tumor cell behaviour modulation by mesenchymal stromal cells. Mol Cancer. 2010, 9: 129-10.1186/1476-4598-9-129.View ArticlePubMedPubMed CentralGoogle Scholar
- Anton K, Glod J: Targeting the tumor stroma in cancer therapy. Curr Pharm Biotechnol. 2009, 10: 185-191. 10.2174/138920109787315088.View ArticlePubMedGoogle Scholar
- Hiscox S, Barrett-Lee P, Nicholson RI: Therapeutic targeting of tumor-stroma interactions. Expert Opin Ther Targets. 2011, 15: 609-621.View ArticlePubMedGoogle Scholar
- Cihova M, Altanerova V, Altaner C: Stem cell based cancer gene therapy. Mol Pharm. 2011, 8: 1480-1487. 10.1021/mp200151a.View ArticlePubMedGoogle Scholar
- Kucerova L, Poturnajova M, Tyciakova S, Matuskova M: Increased proliferation and chemosensitivity of human mesenchymal stromal cells expressing fusion yeast cytosine deaminase. Stem Cell Res. 2012, 8: 247-258. 10.1016/j.scr.2011.11.006.View ArticlePubMedGoogle Scholar
- Kucerova L, Altanerova V, Matuskova M, Tyciakova S, Altaner C: Adipose tissue-derived human mesenchymal stem cells mediated prodrug cancer gene therapy. Cancer Res. 2007, 67: 6304-6313. 10.1158/0008-5472.CAN-06-4024.View ArticlePubMedGoogle Scholar
- Ansieau S: EMT in breast cancer stem cell generation. Cancer Lett. 2013, 338: 63-68. 10.1016/j.canlet.2012.05.014.View ArticlePubMedGoogle Scholar
- Ungefroren H, Sebens S, Seidl D, Lehnert H, Hass R: Interaction of tumor cells with the microenvironment. Cell Commun Signal. 2011, 9: 18-10.1186/1478-811X-9-18.View ArticlePubMedPubMed CentralGoogle Scholar
- Moharita AL, Taborga M, Corcoran KE, Bryan M, Patel PS, Rameshwar P: SDF-1alpha regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis. Blood. 2006, 108: 3245-3252. 10.1182/blood-2006-01-017459.View ArticlePubMedGoogle Scholar
- Cabioglu N, Summy J, Miller C, Parikh NU, Sahin AA, Tuzlali S, Pumiglia K, Gallick GE, Price JE: CXCL-12/stromal cell-derived factor-1alpha transactivates HER2-neu in breast cancer cells by a novel pathway involving Src kinase activation. Cancer Res. 2005, 65: 6493-6497. 10.1158/0008-5472.CAN-04-1303.View ArticlePubMedGoogle Scholar
- Serrati S, Margheri F, Fibbi G, Di Cara G, Minafra L, Pucci-Minafra I, Liotta F, Annunziato F, Pucci M, Del Rosso M: Endothelial cells and normal breast epithelial cells enhance invasion of breast carcinoma cells by CXCR-4-dependent up-regulation of urokinase-type plasminogen activator receptor (uPAR, CD87) expression. J Pathol. 2008, 214: 545-554. 10.1002/path.2309.View ArticlePubMedGoogle Scholar
- Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell. 2011, 144: 646-674. 10.1016/j.cell.2011.02.013.View ArticlePubMedGoogle Scholar
- Casiraghi F, Remuzzi G, Abbate M, Perico N: Multipotent Mesenchymal Stromal Cell Therapy and Risk of Malignancies. Stem Cell Rev. 2012, -Google Scholar
- Kim J, Escalante LE, Dollar BA, Hanson SE, Hematti P: Comparison of Breast and Abdominal Adipose Tissue Mesenchymal Stromal/Stem Cells in Support of Proliferation of Breast Cancer Cells. Cancer Invest. 2013, -Google Scholar
- Yan XL, Fu CJ, Chen L, Qin JH, Zeng Q, Yuan HF, Nan X, Chen HX, Zhou JN, Lin YL, et al: Mesenchymal stem cells from primary breast cancer tissue promote cancer proliferation and enhance mammosphere formation partially via EGF/EGFR/Akt pathway. Breast Cancer Res Treat. 2012, 132: 153-164. 10.1007/s10549-011-1577-0.View ArticlePubMedGoogle Scholar
- Senst C, Nazari-Shafti T, Kruger S, Honer Zu Bentrup K, Dupin CL, Chaffin AE, Srivastav SK, Worner PM, Abdel-Mageed AB, Alt EU, Izadpanah R: Prospective dual role of mesenchymal stem cells in breast tumor microenvironment. Breast Cancer Res Treat. 2013, 137: 69-79. 10.1007/s10549-012-2321-0.View ArticlePubMedGoogle Scholar
- Sarrio D, Franklin CK, Mackay A, Reis-Filho JS, Isacke CM: Epithelial and mesenchymal subpopulations within normal basal breast cell lines exhibit distinct stem cell/progenitor properties. Stem Cells. 2012, 30: 292-303. 10.1002/stem.791.View ArticlePubMedGoogle Scholar
- Weigand M, Hantel P, Kreienberg R, Waltenberger J: Autocrine vascular endothelial growth factor signalling in breast cancer. Evidence from cell lines and primary breast cancer cultures in vitro. Angiogenesis. 2005, 8: 197-204. 10.1007/s10456-005-9010-0.View ArticlePubMedGoogle Scholar
- El-Haibi CP, Bell GW, Zhang J, Collmann AY, Wood D, Scherber CM, Csizmadia E, Mariani O, Zhu C, Campagne A, et al: Critical role for lysyl oxidase in mesenchymal stem cell-driven breast cancer malignancy. Proc Natl Acad Sci U S A. 2012, 109: 17460-17465. 10.1073/pnas.1206653109.View ArticlePubMedPubMed CentralGoogle Scholar
- Kucerova L, Matuskova M, Hlubinova K, Bohovic R, Feketeova L, Janega P, Babal P, Poturnajova M: Bystander cytotoxicity in human medullary thyroid carcinoma cells mediated by fusion yeast cytosine deaminase and 5-fluorocytosine. Cancer Lett. 2011, 311: 101-112. 10.1016/j.canlet.2011.07.014.View ArticlePubMedGoogle Scholar
- Zhu Y, Sun Z, Han Q, Liao L, Wang J, Bian C, Li J, Yan X, Liu Y, Shao C, Zhao RC: Human mesenchymal stem cells inhibit cancer cell proliferation by secreting DKK-1. Leukemia. 2009, 23: 925-933. 10.1038/leu.2008.384.View ArticlePubMedGoogle Scholar
- Lu YR, Yuan Y, Wang XJ, Wei LL, Chen YN, Cong C, Li SF, Long D, Tan WD, Mao YQ, et al: The growth inhibitory effect of mesenchymal stem cells on tumor cells in vitro and in vivo. Cancer Biol Ther. 2008, 7: 245-251. 10.4161/cbt.7.2.5296.View ArticlePubMedGoogle Scholar
- Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F: Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther. 2008, 15: 730-738. 10.1038/gt.2008.39.View ArticlePubMedGoogle Scholar
- Mantovani A, Allavena P, Sica A, Balkwill F: Cancer-related inflammation. Nature. 2008, 454: 436-444. 10.1038/nature07205.View ArticlePubMedGoogle Scholar
- Chaturvedi P, Gilkes DM, Wong CC, Luo W, Zhang H, Wei H, Takano N, Schito L, Levchenko A, Semenza GL: Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. J Clin Invest. 2013, 123: 189-205.View ArticlePubMedGoogle Scholar
- Dittmer A, Fuchs A, Oerlecke I, Leyh B, Kaiser S, Martens JW, Lutzkendorf J, Muller L, Dittmer J: Mesenchymal stem cells and carcinoma-associated fibroblasts sensitize breast cancer cells in 3D cultures to kinase inhibitors. Int J Oncol. 2011, 39: 689-696.PubMedGoogle Scholar
- Houthuijzen JM, Daenen LG, Roodhart JM, Voest EE: The role of mesenchymal stem cells in anti-cancer drug resistance and tumour progression. Br J Cancer. 2012, 106: 1901-1906. 10.1038/bjc.2012.201.View ArticlePubMedPubMed CentralGoogle Scholar
- Zimmerlin L, Park TS, Zambidis ET, Donnenberg VS, Donnenberg AD: Mesenchymal stem cell secretome and regenerative therapy after cancer. Biochimie. 2013, -Google Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/535/prepub
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