All chemicals were purchased from Sigma-Aldrich if not stated otherwise. Human tumor cell lines MCF-7 (ATCC® HTB-22™), Sk-Br-3 (ATCC® Number HTB-30™), T47D (ATCC® HTB-133™), MDA-MB-231 (ATCC® HTB-26™) and MDA-MB-231 NucLight Red™ (Essen BioScience, Welwyn Garden City, UK) were used for the study. Mesenchymal stromal cells (MSC) were obtained from healthy individuals undergoing elective lipoaspiration, who provided an informed consent. No humans were involved in this research study, human material harvested from the healthy individuals after elective surgery was used as approved by the Ethics Committee of the University Hospital (Ruzinov, Ruzinovska 6, 826 06 Bratislava, Slovakia). MSC were isolated and characterized by the immunophenotype and differentiation potential as previously described in .
Stable transduction of MSC to express red fluorescent protein (RFP) was done by retrovirus gene transfer. MSC culture was transduced three times in three consecutive days with virus containing media supplemented with 1 μg/ml protamine sulphate. Cells were maintained in selective media containing appropriate concentration of G418 for 13 days, until the control (untransduced) MSC were dead. Virus-containing medium was collected from semi-confluent cultures of GP + env-AM-12/RFP cells incubated in fresh culture medium for 24 h, filtered through 0.45 μm filter and used either fresh or kept frozen at −80 °C until use. RFP expression was confirmed by flow cytometric analysis performed on BD Canto II Cytometer (Becton Dicinson, USA).
Tumor cells were maintained in high-glucose (4.5 g/l) DMEM (PAA Laboratories GmbH, Pasching, Austria) containing 10 % FBS (GIBCO® Invitrogen, Carlsbad, CA), 10.000 IU/ml penicillin (Biotica, Part. Lupca, Slovakia), 5 μg/ml streptomycin, 2.5 μg/ml amphotericin and 2 mM glutamine (PAA Laboratories GmbH). The MSC were expanded in low glucose (1.0 g/l) DMEM supplemented with 5 % HyClone® AdvanceSTEM™ supplement (Thermo Scientific, Waltham, MA, USA) plus 5 % FBS and antibiotic/antimycotic mix (10,000 IU/ml penicillin, 5 μg/ml streptomycin and 2.5 μg/ml amphotericin) and 2 mM glutamine. Cells were maintained at 37 °C in humidified atmosphere and 5 % CO2.
Cell-free MSC conditioned medium (CM) was collected from 2 × 105 cells plated on a 35 mm culture dish after 48 h of cultivation in high-glucose medium and filtered through 0.45 μm filters. Fresh CM was always used for the experiments.
Gene expression analysis
MSC were cultured with or without 1 μg/ml cisplatin overnight. Total RNA was isolated from 4 × 106 cells. Cultured cells were collected by trypsinization, RNA isolated by NucleoSpin® RNA II kit (Macherey-Nagel, Germany) 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, St. Leon-Rot, Germany). 200 ng of cDNA was amplified in standard PCR performed in 8 μl 1x Dream Taq PCR Master Mix (Thermo Scientific) with 0.3 μl respective specific primers (20 pmol/μl) and DNase free water (Fermentas) in BIORAD T100™ Thermal Cycler (MJ Research, UK) with pre-set amplification profile, and horizontal electrophoresis was used for detection of amplicons.
For quantitative PCR we used following protocol: activation step at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 5 s, 10 s annealing and polymerization at 58 °C and plate read for 5 s at 75 °C followed by final extension for 5 min at 72 °C and melt curve analysis. The PCR reaction mixture (16 μl) contained 1,5 μl cDNA, 0,3 μl respective specific primers (10 pmol/μl), water and Brilliant III QPCR SYBR® Green Mix (Agilent, Santa Clara CA). qPCR reaction ran on CFX96™ Real-Time PCR Detection System (BIO-RAD Laboratories, USA).
Drug resistance assay
For the evaluation of chemosensitivity of tumor cells, either 5 × 103 Sk-Br-3, 1, 5 × 102 MDA-MB-231 (resp. MDA-MB-231 NucLight Red™), 4 × 103 MCF-7 or 3 × 103 T47D cells were seeded in 96-well plates. On day 1, treatments were started with cisplatin (0.1–50 μg/ml) diluted in standard culture medium.
To test the effect of IL-6 and IL-8 on chemosensitivity, 1.5x102 MDA-MB-231 NucLight Red™ cells were seeded in 96-well plates. On day 1, treatments were started with cisplatin (0.5 μg/ml) diluted in standard culture medium with/without 50 ng/ml IL-6, IL-8, or both.
IncuCyte Zoom™ Kinetic Imaging System and/or luminescence assay were used for analysis of treatment effects.
Kinetic measurement of Caspase-3/7 activity
To measure caspase-3/7 activity corresponding to the induction of apoptosis in cells cultivated in the presence of cisplatin, 7.5 × 103 MSC were seeded in 96-well plates and treated with 1 and 10 μg/ml cisplatin. CellPlayer 96-Well Kinetic Caspase-3/7 reagent (Essen BioScience) was used at a final concentration of 5 μM in growth medium and added directly to cells in 96-well plates. Caspase-3/7 reagent is non- fluorescent substrate which crosses the cell membrane where it is cleaved by activated caspase-3/7 resulting in the release of the DNA dye and green fluorescent staining of nuclear DNA. Kinetic activation of caspase-3/7 was monitored using IncuCyte Zoom™ Kinetic Imaging System and quantified using the IncuCyte™ FLR object counting algorithm.
Senescence β-Galactosidase Staining
MSC were examined also for the presence of senescent cells with the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology). Three × 105 MSC were seeded per well in low glucose DMEM in 6-well plate, and the next day treated with/without 1 μg/ml cisplatin in standard culture medium for 48 h. The β-Galactosidase activity was detected at pH 6 by light microscopy; the blue color development indicated β-Gal-positive senescent cells.
ALDH activity was measured in MDA-MB-231 and MCF-7 cells cultivated in standard medium, CM or pretreated CM (pr.CM) after reaching confluence (after 4–5 days). Four hundred thousand cells were seeded on a 35 mm culture dish in standard medium, which was replaced by fresh 5 ml of standard medium, CM or pr.CM the next day. Flow cytometry ALDEFLUOR® Assay (StemCell Technologies, Vancouver, BC) was used to assess ALDH activity. Control cells were exposed to diethylaminobenzaldehyde (DEAB) prior measurement. Two hundred fifty thousand cells were centrifuged for 5 min at 250 x g, the supernatant was removed and the cells were suspended in 500 μl of ALDEFLUOR Assay buffer.
Measurement was performed using BD FACSCanto™ II Flow cytometer (Becton Dickinson, USA) equipped with FacsDiva program. Data were analysed with FCS Express program.
Sk-Br-3 cells were cultivated in standard CM or pr.CM for 5 days. CD24-PE, CD44-APC and EpCAM-FITC antibody (Miltenyi Biotec GmbH, Germany) were used at a 1:50 dilution and incubated for 15 min with 250.000 tumor cells per sample. Triple staining was used for analysis of CD24−/CD44+/EpCAM+ population on BD FACSCanto™ II Flow cytometer (Becton Dickinson, USA).
Analysis of phosphorylation profiles of kinases and their protein substrates, as well as analysis of expression of apoptosis-related proteins was done by the Human Phospho-Kinase Array (R&D Systems, Minneapolis, MN) and Human Apoptosis Array Kit (R&D Systems). For both, untreated and overnight 1 μg/ml cisplatin pretreated MSC were solubilized at 1 × 107 cells/ml in lysis buffer at 2–8 °C for 30 min and proceeded according to manufacturer’s protocol. ImageJ software (NIH, Bethesda, MD) was used for the quantitative evaluation; pixel density was determined and calculated.
Cell supernatant of untreated MSC and pretreated MSC as above was analyzed by Human Cytokine Array Kit (R&D Systems) used to simultaneously detect the relative levels of 36 different cytokines, chemokines, and acute phase proteins according manufacturers protocol.
Gene expression array
For evaluation of the effect of direct co-culture of tumor cells with MSC (untreated or pretreated with 1 μg/ml cisplatin), 200.000 of MCF-7 were cultivated with 200.000 of RFP-MSC for 5 days and then sorted on BD Influx (BD Biosciences, USA) based on RFP positivity. Excitation laser was 561 nm and emission filter 585/29. RNA from MCF-7 cells were then isolated by Agilent Total RNA Isolation Mini Kit (Agilent Technologies, USA). RNA was reverse transcribed with RT2 Profiler PCR Array and expression of 84 human breast cancer related genes was analyzed.
In vivo experiments
Six week old athymic nude mice (Balb/c-nu/nu) were used in accordance with the institutional guidelines under the approved protocols. Five x106 MDA-MB-231 cells were injected subcutaneously in 100 μl serum free DMEM (PAA Laboratories GmbH). Animals were subsequently divided into following groups: control group (n = 4), cisplatin i.p. alone (n = 5), i.v. 2.5 × 105 MSC with i.p. cisplatin (n = 6), i.v. 2.5 x105 MSC alone (n = 4). Animals were treated with 3 mg/kg cisplatin with/without MSC every 12, 19 and 26 day.
Animals were regularly inspected for the tumor growth and the tumor volume was calculated according to the formula volume = length x width2/2. Animals were sacrificed, when the tumors exceeded 1 cm3 in accordance with the ethical guidelines.
Project was performed in the approved animal facility (licence number SK PC 14011) as approved by the institutional ethic committee and by the national competence authority (State Veterinary and Food Administration of the Slovak Republic, registration number Ro 3108/14-221) in compliance with the Directive 2010/63/EU of the European Parliament and the European Council and the Regulation 377/2012 on the protection of animals used for scientific purposes.
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.