human tumor cell lines skbr3  (ATCC)

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-535

Figure Lengend Snippet: 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).

Article Snippet: Human tumor cell line SKBR3 (ATCC® Number HTB-30TM) was used for the study.

Techniques: Cell Culture, Fluorescence, Microscopy, Quantitative RT-PCR, Expressing, Comparison, Light Microscopy, Viability Assay

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-535

Figure Lengend Snippet: 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-Plex TM 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).

Article Snippet: Human tumor cell line SKBR3 (ATCC® Number HTB-30TM) was used for the study.

Techniques: Migration, Gene Expression, Cytokine Assay, Comparison, Expressing, Cell Culture, Live Cell Imaging, Scratch Wound Assay Assay

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-535

Figure Lengend Snippet: 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.

Article Snippet: Human tumor cell line SKBR3 (ATCC® Number HTB-30TM) was used for the study.

Techniques: Live Cell Imaging, Luminescence Assay, Comparison, Inhibition, Fluorescence, Cell Culture

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-535

Figure Lengend Snippet: 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 IC 50 (SKBR3) = 27 ng/ml DOX to IC 50 (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.

Article Snippet: Human tumor cell line SKBR3 (ATCC® Number HTB-30TM) was used for the study.

Techniques: Fluorescence, Comparison, Viability Assay, Activation Assay, Luminescence Assay, Derivative Assay

Journal: BMC Cancer

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

doi: 10.1186/1471-2407-13-535

Figure Lengend Snippet: 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. IC 50 shifted from IC 50 (SKBR3) = 70 ng/ml 5FU to IC 50 (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.

Article Snippet: Human tumor cell line SKBR3 (ATCC® Number HTB-30TM) was used for the study.

Techniques: Fluorescence, Activation Assay, Activity Assay, Derivative Assay