1. Isolation of CD133+ HPCs
The Human Ethics Committee of the First Affiliated Hospital, College of Medicine at Xi’an Jiaotong University approved the experimental protocol(2017-041). Written informed consent was signed the parents of individual newborns. Fresh and healthy human UCB samples (about 60–100 ml) were collected from five human newborns in our hospital. The CD133 + HPCs were isolated by immunomagnetic beads and Macs column (Mitenyi Biotec, Germany). Briefly, the mononuclear cells in the UCB samples were isolated by density gradient centrifugation using human lymphocyte separation solution. After being washed, the collected mononuclear cells (1 × 108 cells/sample) in 5 µg/ml BSA and 2 mmol/ml EDTA buffer were blocked with anti-CD16/anti-CD32 and stained with anti-human CD133 immunomagnetic beads, followed by loaded in the Macs column. During the column was washed, the flow-through CD133- cells were collected and the bound CD133 + HPCs were eluted. The CD133 + and CD133- cells were stained with PE-anti-CD133 and FITC-anti-CD34 (MBS850595, MyBioSource, California, USA). The percentages of CD133 + CD34 + cells were analyzed by flow cytometry, the remaining cells were designated CD133- human umbilical cord blood cells (HUCBCs). The isolated CD133 + CD34 + HPCs were routine-cultured in IMDM medium containing 10% of fetal bovine serum (FBS, MyBioSource).
2. Culture of breast cancer cells
We purchased human breast cancer MCF-7 and MDA-MB-231 cells from Shanghai Cell Bank of Chinese Academy of Sciences and characterized them by STR. We regularly cultured them in 10% FBS DMEM.
3. Cell proliferation assay
The effect of CD133 + HPCs on the proliferation of breast cancer cells was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays[20]. We co-cultured MCF-7 or MDA-MB-231 cells (2 × 103 cells/well) with or without (blank control), CD133 + HPCs and CD133- HUCBCs at a ratio of 20:1 in triplicate in 96-well plates for 24–120 h. We quantified the viability of individual wells by MTT.
4. Transwell invasion assay
We examined the influence of CD133 + HPCs on breast cancer cell invasion by transwell invasion assay[21].Briefly, We cultured MCF-7 or MDA-MB-231 cells (1 × 105 cells/well) in FBS-free medium in the upper chambers that had been coated with Matrigel (BD356234,BD Sciences༌New Jersey, USA) and CD133 + HPCs or CD133- HUCBCs (5 × 103 cells/well) in the lower chamber of 24-well Transwell plates (8 µm pore size, BD353097༌BD Sciences) in 10% FBS medium for 24 h. The breast cancer cells in the upper chamber and medium in the lower chamber served as the blank control. We photoimaged and counted the invaded cells in a blinded manner.
5. Cell apoptosis assay
We tested the role of CD133 + HPCs in spontaneous apoptosis of MCF-7 or MDA-MB-231 cells by flow cytometry[22].The cancer cells were cultured alone (blank control), or together with CD133 + HPCs or CD133- HUCBCs at 20:1 were cultured in the upper and lower chambers of Transwell chamber (0.4 µm pore size), respectively, for 72 h. The cancer cells (3 × 105 cells/tube) were tested for their apoptosis by flow cytometry after staining with Annexin V-FITC and Propidium iodide (PI, MyBioSource). The apoptotic FITC + and FITC + PI + cells were quantified.
6. Western blotting
After co-cultured with CD133 + HPCs or CD133- HUCBCs or cultured alone for 72 h, the MCF-7 and MDA-MB-231 cells were harvested and lyzed in 250 µl RIPA buffer on ice for 10 minutes, centrifuged, and the cell lysates were collected for Western blot[23]. We analyzed the cell lysates (50 µg/lane) using 10% gels, and specific antibodies including rabbit anti-E-cadherin (1:2000), anti-Vimentin (1:500), anti-N-cadherin (1:500), anti-β-actin (1:2000). We quantified the relative levels of interested proteins to the control β-actin using ImageJ software.
7. Animal experiment
The protocol was authorized by the Animal Care and Use Committee of Xi’an Jiaotong University(license number:SCXK 2017-003). Female BALB/c nude mice at 6 weeks of age were obtained from Silaike Laboratory Animal, Shanghai, China.We implanted each mouse subcutaneously with 1 × 107 MCF-7, MDA-MB-231 cells alone, or together with 5 × 105 CD133 + HPCs or CD133- HUCBCs (n = 8 per group, randomly). We monitored the tumor dynamic growth every 2 days up to 30 days post inoculation by measuring the tumor volumes (volume = length × width2 × 0.5).Each group of eight mice is housed in the separated individual standard cleaned cages under automatically controlled air condition system with temperature (22 ± 2 °C), humidity (about 60%), and lighting (12:12-h light–dark cycle).Diet and sterilized water are provided in the experiments.At the end of the experiment mice were euthanized by inhalation of CO2 followed by cervical dislocation.
8. Statistical analysis
Data are expressed as mean ± SD. We statistically analyzed the significance among groups by one way ANOVA and between groups by Student’s T test using the SPSS 22.0 software. A P-value of < 0.05 was defined as a statistical significance.