Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Nov 9:13:535.
doi: 10.1186/1471-2407-13-535.

Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

Lucia Kucerova  1 , Svetlana Skolekova, Miroslava Matuskova, Martin Bohac, Zuzana Kozovska
Affiliations

Altered features and increased chemosensitivity of human breast cancer cells mediated by adipose tissue-derived mesenchymal stromal cells

Lucia Kucerova et al. BMC Cancer. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
AT-MSCs induced epithelial–to-mesenchymal transition and increased mammosphere formation. A) SKBR3 cells were cultured in DMEM or MSC-CM for 6 days. Phase-contrast images revealed shift from epithelial to mesenchymal-like morphology in the majority of tumor cells (magnification 40x). B) AT-MSCs and EGFP-SKBR3 were directly cocultured for 6 days. Fluorescence microscopy confirmed morphological signs of an EMT in the tumor cells (magnification 100x). C) Markers of the EMT and pluripotency were up-regulated in the tumor cells cultured in MSC-CM. Quantitative RT-PCR confirmed significant increase in the expression of Nanog, Oct, Twist, Snail1, Snail2, αSMA and FAP in EGFP-SKBR3 cells exposed to MSC-CM in comparison to EGFP-SKBR maintained under the standard culture conditions. The data are expressed as means ± SD, *p < 0.05, #p < 0.001. D) Non-adherent culture conditions increased EGFP-SKBR3 mammosphere formation in the presence of MSC-CM, which could be abrogated by specific inhibitors of PI3K (1.63 μM LY294002) and p38 MAP kinases (0.5 μM SB203580) (magnification: light microscopy 40x, fluorescent microscopy 100x). Relative viability of EGFP-SKBR3 cells exposed to MSC-CM in the presence of inhibitors did not significantly differ from the viability in DMEM as evaluated by luminescent viability assay (right panel).
Figure 2
Figure 2
Increased migration of breast cancer cells due to the changes in a paracrine signaling and gene expression in MSC-CM-exposed SKBR3. A) Paracrine signaling in tumor and stromal cell cocultures. AT-MSCs were directly cocultured with EGFP-SKBR3 for 2 days and the cytokine levels were evaluated by human Bio-PlexTM 27-plex Cytokine Assay. Direct coculture of the tumor and stromal cells resulted in induction of IL-5, IL-7, IL-10, GM-CSF, IFN-γ and MIP-1a (‡). EGFP-SKBR3 or AT-MSCs alone did not produce detectable levels of these cytokines. Levels of cytokines IL-4, IL-9, eotaxin, IP-10, MCP-1 were significantly higher in comparison to the theoretically calculated additive value of EGFP-SKBR3 and AT-MSCs alone. Values were calculated as means of two independent experiments performed in duplicates, *p < 0.05, **p < 0.01. B) Expression analysis demonstrated the induction of VEGFR2 and c-Kit receptor expression in MSC-CM exposed EGFP-SKBR3 cells for 6 days. The expression of EGFR1, VEGFA, SCF and c-Met was detected in EGFP-SKBR3 cells. Induced expression of VEGFR2 and c-Kit was detected in MSC-CM cultured EGFP-SKBR3. Representative outcome is shown; the experiments were repeated at least three times with different MSCs isolates and similar outcome. C) EGFP-SKBR3 cells exhibited increased migration in the presence of MSC-CM as evaluated by live-cell imaging in a scratch wound assay. Confluent monolayers of EGFP-SKBR3 cells were wounded and the migration in the presence of MSC-CM or standard culture medium was observed for 72 hrs. Quantitative evaluation of a relative wound density demonstrated the capability of the secreted soluble factors by AT-MSCs to significantly increase the migration of tumor cells. Data are expressed as means of three independent measurements each run in quadruplicates ± SD. D) Increased migration of EGFP-SKBR3 in MSCs-CM could be significantly inhibited by 200 nM Sunitinib (VEGFR2, PDGFRβ and c-Kit inhibitor); and not by 150 nM Pazopanib (multi-target kinase inhibitor of VEGFR1, VEGFR2, VEGFR3, PDGFR, FGFR, c-Kit and c-Fms) or 250 nM Sorafenib (VEGFR-2, Raf-1 and B-Raf inhibitor).
Figure 3
Figure 3
AT-MSCs and MSC-CM can inhibit the proliferation of SKBR3 breast cancer cells. A) MSC-CM-exposed EGFP-SKBR3 cells show significantly increased relative confluence as determined by the kinetic live-cell imaging. Data were pooled from the three independent experiments and expressed as means ± SD. B) Relative proliferation of the SKBR3 cells in serially diluted MSC-CM was determined by the viability luminescence-based assay after 6 days. MSC-CM supplemented culture medium gradually decreased the cell proliferation in comparison to the standard culture conditions. Proliferation was significantly inhibited in each MSC-CM dilution in comparison to standard culture medium (p < 0.05). C) The inhibition of proliferation was determined for the three different AT-MSCs isolates tested as above, *p < 0.05. D) Direct coculture of the EGFP-SKBR3 with AT-MSCs confirmed the inhibition of tumor cell proliferation based on the decrease in relative green fluorescence corresponding to the signal from the viable tumor cells. Relative proliferation was significantly lower in comparison to the proliferation of EGFP-SKBR3 alone when ≥ 1,000 AT-MSCs were admixed to the 10,000 EGFP-SKBR3 cells (p < 0.05). E) Immunoassay confirmed the production of SDF-1α in the AT-MSCs and cocultures of AT-MSCs and EGFP-SKBR3. F) EGFP-SKBR3 cells were cultured in the presence of AT-MSCs CM or AT-MSCs with or without 5 μg/ml AMD3100 - specific inhibitor of SDF1α/CXCR4 signaling. Relative tumor cell viability was significantly lower in the presence of AT-MSCs and the inhibitory effect was abrogated in the presence of the AMD3100. Each experiment was performed at least three times in quadruplicates with similar results and one representative outcome is shown. Data were expressed as means ± SD. *p < 0.05.
Figure 4
Figure 4
Increased chemosensitivity of SKBR3 in MSC-CM or AT-MSCs cocultures. A) Doxorubicin decreased relative fluorescence of the EGFP-SKBR3 in the presence of MSC secreted factors, indicative of increased chemosensitivity of EGFP-SKBR3 cells in the MSC-CM to doxorubicin concentrations of 12.5 ng/ml and 25 ng/ml, *p < 0.05. B) Relative viability of MSC-CM-exposed tumor cells is significantly lower in the presence of 25 ng/ml doxorubicin in comparison to doxorubicin treatment in culture medium, *p < 0.05. C) Direct comparison of the doxorubicin sensitivity revealed shift in IC50(SKBR3) = 27 ng/ml DOX to IC50(SKBR3 in MSCs-CM) = 13 ng/ml DOX, as determined by luminescent viability assay, *p < 0.05. D) Cytotoxic treatment with doxorubicin induced significantly higher Caspase-3/7 activation in the SKBR3/MSC-CM cultures as determined by a Casp-3/7 luminescence assay. Each experiment was performed three times with four different MSCs isolates and one representative evaluation is shown. Data are expressed as means ± SD, *p < 0.05. E) Flow cytometric analysis of the directly cocultured cells unraveled significantly increased proportion of apoptotic and necrotic tumor cells as determined by the Annexin V and/or DAPI positivity in cocultures. Tumor cells were mixed with AT-MSCs (2:1) and treated with 50 ng/ml doxorubicin for 48 hrs. Representative data derived from one experiment were shown, *p < 0.05.
Figure 5
Figure 5
AT-MSCs affect chemoresistance to 5FU in the direct cocultures with SKBR3 cells. A) AT-MSCs were directly cocultured with EGFP-SKBR3 in the presence or absence of 6.25 ng/ml - 1 μg/ml 5FU for 6 days. Relative viability of EGFP-SKBR3 was determined by fluorescence measurements. The presence of the AT-MSCs did not interfere with the fluorescence signal. AT-MSCs significantly decreased the resistance to 12.5 ng/ml and 50 ng/ml 5FU. IC50 shifted from IC50(SKBR3) = 70 ng/ml 5FU to IC50(SKBR3 in MSCs-CM) = 35 ng/ml 5FU, *p < 0.05. B) AT-MSCs significantly increased Caspase3/7 activation in SKBR3 cells in response to 5FU treatment for 48 hrs. No Caspase 3/7 activity was induced in AT-MSCs cell under these conditions due to their inherent chemoresistant nature, *p < 0.05. C) AT-MSCs did not significantly affect sensitivity of tumor cells to cis-platin treatment. Data were derived from the three independent experiments performed in quadruplicates. Values were expressed as means ± SD, p > 0.05.
See this image and copyright information in PMC

References

    1. 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. doi: 10.7326/0003-4819-156-9-201205010-00006. - DOI - PMC - PubMed
    1. Egeblad M, Nakasone ES, Werb Z. Tumors as organs: complex tissues that interface with the entire organism. Dev Cell. 2010;18:884–901. doi: 10.1016/j.devcel.2010.05.012. - DOI - PMC - PubMed
    1. Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 2012;21:309–322. doi: 10.1016/j.ccr.2012.02.022. - DOI - PubMed
    1. 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. doi: 10.1002/mc.20675. - DOI - PMC - PubMed
    1. 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. doi: 10.1089/ten.teb.2010.0352. - DOI - PMC - PubMed

Publication types

Substances