The human breast cancer MCF-7 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ atmosphere in a humidified incubator. Breast cancer tissues were obtained from female patients (n=12) aged 40–60 years (mean 52 years), who underwent surgical operation in the First People's Hospital of Lianyungang from April 2015 to July 2015 (Jiangsu, China), all patients provided written informed consent to participate in the study and all experiment protocols were approved by the Medical Ethics Committee of First People's Hospital of Lianyungang. The cell isolation procedure was typically undertaken within 4 h of tumor removal. In brief, fresh tissue specimens were collected, washed with PBS, cut into 1 mm³-sized pieces and put into culture dishes for 30 min at 37°C. Then, tissue samples were maintained in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin at 37°C with 5% CO₂. Medium was replaced every three days after the initial plating. When adherent fibroblast-like cells confluence reached ~80%, the cells were passaged into new flasks for further expansion. Cells at passage 3–4 were used for the evaluation of the experiments. Bone marrow-mesenchymal stem cells (BM-MSCs) from patients (n=12) aged 3–60 years (mean, 22 years) with suspected blood system diseases who were diagnosed with no hematological disease or other cancers at the First People's Hospital of Lianyungang from April 2015 to July 2015 (Jiangsu, China) were chosen as the MSC controls.

To investigate the surface antigen markers of different passages of BC-MSCs and BM-MSCs, flow cytometric analysis was performed using fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated antibodies: CD14 (cat. no. 561712; dilution, 1:50), CD34 (cat. no. 555822; dilution, 1:50) and CD45 (cat. no. 560976; dilution, 1:50) were FITC-conjugated, CD29 (cat. no. 561795; dilution, 1:50), CD44 (cat. no. 561858; dilution, 1:50), CD90 (cat. no. 561970; dilution, 1:50) and CD105 (cat. no. 562380; dilution, 1:50) were PE-conjugated. Mouse PE-IgG1 (cat. no. 349043; dilution, 1:50) and FITC-IgG1 (cat. no. 349041; dilution, 1:50) isotypic immunoglobulins were used as controls (BD Biosciences, San Jose, CA, USA). BC-MSCs and BM-MSCs cells (1.0×10⁶) were trypsinized, washed twice in PBS, incubated with monoclonal antibodies for 30 min on ice at 4°C and then washed with PBS. Labeled cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences).

To determine the ability of BC-MSCs to undergo osteogenic and adipogenic differentiation, BC-MSCs were seeded in 35-mm plates at 3×10⁴ cells/cm² and cultured in L-DMEM containing 15% FBS. The next day, the medium was changed to osteogenic medium or adipogenic medium (both from Cyagen Biosciences, Sunnyvale, CA, USA) in accordance with the culture protocol. After 2 weeks, the osteogenic differentiation of BC-MSCs was examined by alkaline phosphatase staining at room temperature for 15 min. At 3 weeks later, adipocytes were stained with Oil-Red-O at room temperature for 15 min to confirm the ability of adipogenic differentiation of BC-MSCs. A fluorescence-inverted microscope (Olympus Corporation, Tokyo, Japan) was used to observe the staining results at ×100 and ×400 magnification.

A total of (1.0×10⁶) BC-MSC cells were seeded in 35-mm plates and cultured in L-DMEM with 15% FBS. The following day, the media was removed and the cells were washed with PBS, re-incubated with fresh medium for 48 h. Next, BC-MSC-conditioned medium (BC-MSC-CM) and BM-MSC-conditioned medium (BM-MSC-CM) were collected, and centrifuged at room temperature to remove possible cell debris (1,000 × g for 10 min).

To investigate the effect of cisplatin, BC-MSC-CM combination cisplatin on the viability of MCF-7 cells, an MTT assay was performed. Briefly, MCF-7 cells were plated at a density of 1×10⁴ cells/well in a 96-well rounded bottom plate. After incubation for 12 h, cells were incubated with cisplatin for another 48 h at 37°C by a continuous induction from 2.5 to 80 µM (2.5, 5, 10, 20, 40 and 80 µM) in a stepwise increasing concentration manner in the presence or absence of BC-MSC-CM. Control medium was used as a control. All the cells were cultured for a further 48 h. Subsequently, MTT reagent was added into each well and the cells were incubated for an additional 4 h at 37°C. Following incubation and removal of the supernatant, 150 µl DMSO was added to dissolve the dye and the absorbance was measured at 490 nm with a microplate reader. IC₅₀ was defined as the drug concentration causing a 50% apoptosis relative to the negative control. In this experiment, the IC₅₀ was 20 µM. The experiment was repeated three times; six parallel samples were detected each time.

In brief, a total of 5.0×10⁴ MCF-7 cells were seeded into 6-well plates. After 48 h, cells were treated with 20 µM cisplatin diluted in complete medium or BC-MSC-CM. Control cells were treated with medium without the addition of cisplatin. After culturing for 48 h, cells were suspended in PBS and incubated with reagents from the Annexin V & Dead Cell kit and Count & Viability kit (both from Merck KGaA, Darmstadt, Germany) according to manufacturer's protocol. Data was processed using Muse^™ smart touch FACS (Merck KGaA) to generate dot plots. The values were exported to GraphPad Prism software (version 6.0; GraphPad Inc., La Jolla, CA, USA) for further analysis.

To investigate which component of the BC-MSC-CM decreased the level cisplatin-induced apoptosis and promote the viability of MCF-7 cells, the concentration of 6 cytokines and chemokines (IL-17A, IL-7, IL-6, VEGF, EGF, FGF) in BC-MSC-CM were quantified using a Luminex immunoassay. MILLIPLEXH human cytokine 96-well plate assays (cat. no. HCYTOMAG-60K, EMD Millipore, Billerica, MA, USA) were performed according to the manufacturer's protocol.

To evaluate the effect of IL-6 on cisplatin-induced apoptosis in MCF-7 cells, 10 µg/ml IL-6 neutralization antibody (R&D Systems, Inc., Minneapolis, MN, USA, MAB206.) was added to a total of 5.0×10⁴ MCF-7 cells cultured with BC-MSC-CM. Following incubation at 37°C for 48 h, cells were collected for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses.

Cells were treated as aforementioned and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.). Reverse transcription was performed using Superscript II reverse transcriptase, Oligo(dT) primer (Roche Diagnostics, Indianapolis, IN, USA) in a 40-µl reaction volume. qPCR was performed using the Veriti 96 Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions were as follows: 94°C for 30 sec, 60°C (primer) for 30 sec and 72°C for 30 sec, with a final extension at 72°C for 10 min, performed for 35 cycles. Primers of human IL-6 and β-actin were designed using the Primer 5.0 Software (Biosoft International, Palo Alto, CA, USA) as shown: IL-6 forward, 5′-GAGGAGACTTGCCTGGTGAA-3′ and reverse, 5′-GCGCAGAATGAGATGAGTTG-3′; β-actin forward, 5′-TGGACTTCGAGCAAGAGATG-3′ and reverse, 5′-GGATGTCCACGTCACACTTC-3′. Data was quantified by the 2^−ΔΔCq method (22).

Cells were treated as aforementioned, MCF-7 cells (1.0×10⁶) were lysed in radioimmunoprecipitation assay buffer containing 1 mM phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Nanjing, China). The protein concentrations were determined using a NanoDrop 2000 Micro-volume spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). A total of 200 µg protein in each lane was electrophoresed using 12% SDS-PAGE, and the gels were transferred onto a polyvinylidene fluoride membrane. Subsequently, membranes were blocked with 5% skim milk at room temperature for 1 h, and incubated with primary antibodies at 4°C overnight. Then the antibody-bound membranes were washed three times, each time for 10 min. The HRP conjugated goat anti-rabbit secondary antibody (cat. no. A0208; dilution, 1:1,000; Beyotime Institute of Biotechnology) incubated with membranes at 37°C for 1 h. After washed three times, the membranes were visualized by using LuminataTM Crescendo Western HRP substrate (EMD Millipore, USA). Primary antibodies were as follows: Anti-STAT3 (cat. no. MAB1799; dilution, 1:500), anti-phosphorylated (p)-STAT3 (cat. no. MAB4607; dilution, 1:500; R&D Systems, Minneapolis, MN, USA), anti-β-actin (cat. no. sc47778; dilution, 1:1,000) and anti-B-cell lymphoma-associated X (Bax) (cat. no. sc493; dilution, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

All values were expressed as the mean ± standard deviation. Statistically significant differences between groups were assessed by two-way analysis of variance or Student's t-test using GraphPad Prism software (version 6.0; GraphPad, Inc.). P<0.05 was considered to indicate a statistically significant difference.

After 10–14 days of primary culture of tissue samples, a small population of fibroblastic cells was observed. When the confluence of adherent fibroblast-like cells reached ~80%, the cells were passaged into new flasks, and spindle-shaped morphologies were observed on BC-MSCs and BM-MSCs (Fig. 1A). Flow cytometric analysis demonstrated that BC-MSCs and BM-MSCs expressed high levels of CD29, CD44, CD90, and CD105, but were negative for CD14, CD34 and CD45 (Fig. 1B and C). In addition, in order to evaluate the pluripotent differentiation potential of BC-MSCs, cells were cultured in osteoblastic-induction medium for two weeks and numerous Alkaline phosphatase staining-positive cells were observed. In addition, a number of BC-MSCs were positive for Oil-Red-O intracellular staining following three weeks of incubation indicating adipogenic induction. Control cells cultured in the complete medium were negative for Oil-Red-O staining (Fig. 1D).

To evaluate the effects of BC-MSCs in the cisplatin-induced MCF-7 cell response, cells were seeded in 96-well plates or 6-well plates for different experiments. As presented in Fig. 2A, the results of an MTT assay revealed that MCF-7 cells exposed to cisplatin in the presence of BC-MSC-CM exhibited significantly higher growth rates compared with those treated with cisplatin alone (P<0.01) and cisplatin plus BM-MSC-CM-treated cells (P<0.05). FACS analysis revealed that the proportion of annexin-V-bound MCF-7 cells cultured with control medium increased significantly in response to cisplatin treatment compared with those cultured with BC-MSC-CM (P<0.01; Fig. 2B). The anti-apoptosis effect of BC-MSC-CM was significantly increased compared with that in the BM-MSC-CM group (P <0.05; Fig. 2B). Co-treatment with BC-MSC-CM and cisplatin revealed that the vitality of MCF-7 cells increased significantly when compared with cells treated with cisplatin alone (Fig. 2C; P<0.01). Furthermore, BC-MSCs had a significantly higher potential to decrease MCF-7 cell apoptosis and promote cell proliferation compared with BM-MSCs (P<0.05; Fig. 2). These results suggested that BC-MSCs weakened the antitumor effect of cisplatin more effectively than BM-MSCs.

BM-MSCs exhibit marked tropism for tumor sites and have the ability to transition to cancer-associated stromal cells (23). In other words, cancer-associated stromal cells may originate from BM-MSCs. Detecting the different expression of paracrine factors between BC-MSCs and BM-MSCs may reveal which component of the BC-MSC-CM enhanced the survival of MCF-7 cells. The expression of important cytokines was therefore assessed using a Luminex immunoassay, which demonstrated that IL-6 secretion was significantly higher in the BC-MSC-CM group compared with that in the BM-MSC-CM group (P<0.01; Fig. 3A). RT-qPCR confirmed that the level of IL-6 mRNA was significantly increased in MCF-7 cells treated with cisplatin and BC-MSC-CM compared with the control and cisplatin group. IL-6 neutralizing antibody was added to cisplatin+BC-MSC-CM, IL-6 mRNA expression significantly decreased when compared with the cisplatin+BC-MSC-CM group (P<0.01; Fig. 3B). IL-6 secreted by BC-MSCs may therefore have a pivotal role in mediating the enhanced proliferation of MCF-7 cells.

To investigate whether the effect of BC-MSCs secreted IL-6 promoted MCF-7 cell survival from cisplatin, IL-6 neutralization antibody was added to BC-MSC-CM. The resultant depletion of IL-6 recovered cisplatin-induced apoptosis (Fig. 4A). Furthermore, MCF-7 cell viability evidently decreased following IL-6 neutralization antibody treatment (Fig. 4B). To assess whether the change in response of MCF-7 cells to cisplatin treatment caused by BC-MSCs was mediated through activation of the IL-6/STAT3 signaling pathway, western blot analysis was performed (Fig. 4C). Data indicated that in the presence of BC-MSC-CM, levels of p-STAT3 were raised markedly in MCF-7 cells, which could be reversed by incubation with IL-6-neutralizing antibody. The expression of the apoptosis-associated protein Bax was reduced in MCF-7 cells on incubation with BC-MSC-CM compared with cisplatin alone treated cells. The aforementioned data indicated that IL-6 is crucial in the BC-MSC-mediated reduction in apoptosis induced by cisplatin in breast cancer cells.