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 Magnetic Activated Cell Sorting (MACS) column (Mitenyi Biotec, Germany) according to the manufacturer’s instructions. Briefly, the mononuclear cells in the UCB samples were isolated by density gradient centrifugation at 2,000 rpm for 25 mins using human lymphocyte separation solution. After being washed, the collected mononuclear cells (1×108 cells/sample) in 5 μg/ml BSA (Merck, Germany) and 2 mmol/ml EDTA buffer (Promega, USA) were blocked with anti-CD16/anti-CD32 (Promega, USA) and stained with anti-human CD133 immunomagnetic beads, followed by loading into the Macs column. During column washing, 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 (MBS851590, MyBioSource, California, USA) 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 (Novagen, USA) medium containing 10% of fetal bovine serum (FBS, MyBioSource), and semi suspended in a 37oC / 5% CO2 incubator. Cell growth was monitored daily.
2. Culture of breast cancer cells
MCF-7 and MDA-MB-231 breast cancer cell lines were purchased from Shanghai cell bank of the Chinese Academy of Sciences. The cells were cultured with DMEM (MCLAB, USA) containing 10% newborn bovine serum (MCLAB, USA) under the conditions of 37oC and 5% CO2. 0.25% trypsin (MCLAB, USA) was used to digest and passage. Fresh medium was replaced every 2-3 days.
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 [24]. MCF-7 and MDA-MB-231 breast cancer cells in logarithmic growth period were inoculated in 96 well plates, 2×103 breast cancer cells for each group. After 12 hours of culture, CD133+ HPCs and CD133- HPCs were added to the experimental group and the negative control group in a 20:1 proportion of breast cancer cells to HPCs. 24 hours later, each well was dosed with 200 µL serum-free medium DMEM and 20 µl 5 mg/ml MTT (MCLAB, USA). After 4 hours, 150 µL of DMSO (MCLAB, USA) was added to each well and incubated at room temperature for 10 minutes, oscillated with a micro-oscillator for 15 minutes, and optical density value was recorded at 570 nm with a flow cytometer instrument. All measurements were performed in triplicate.
4. Transwell invasion assay
We examined the influence of CD133+ HPCs on breast cancer cell invasion by Transwell invasion assay [25]. Briefly, the upper and lower chambers of a Transwell insert are separated by polycarbonate microporous membrane (8μm pore size), which is coated with Matrigel (Thermo Scientific, USA) on the upper chamber surface and dried at room temperature. 1x105 breast cancer cells were added to the upper chamber. CD133+ HPCs and CD133- HPCs were added to the lower chamber of the experimental group and the negative control group. The cell number ratio of breast cancer cells to HPCs was 20:1. After culturing at 37 oC for 24 h, the non-invasive cells were wiped off with a cotton swab. The filter membrane was fixed with 4% paraformaldehyde solution (Thermo Scientific, USA) for 30 minutes, and 0.01% crystal violet dye solution was added for a 20 minute incubation. The migrating cells were photoimaged and counted under the microscope. All measurements were performed in triplicate, and the experiments were repeated three times independently.
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 [26]. The cancer cells were cultured alone (blank control), or together with CD133+ HPCs or CD133- HUCBCs at a proportion of 20:1 in the upper and lower chambers of the Transwell insert (0.4 μm pore size), respectively, for 72 h. The cancer cells (3x105 cells/tube) were tested for apoptosis by flow cytometry after staining with 5 µL Annexin V-FITC (Promega, USA) and 10 µL Propidium iodide (PI, MyBioSource). The apoptotic FITC+ and FITC+PI+ cells were quantified and the experiment was repeated three times.
6. Western blotting
The Transwell (0.4 µm) indirect co-culture method and groups were described as above. The MCF-7 and MDA-MB-231 breast cancer cells were digested with 0.25% trypsin, and 250 µL RIPA lysate was added, incubated on ice for 10 minutes, and centrifuged at 4 oC for 20 minutes. Following centrifugation, the supernatant was collected and the protein concentration of each group of samples was detected on the spectrophotometer. A 10% separation gel and 5% concentrated gel were prepared, a 20 μg protein was mixed with 2×SDS sample buffer in a 1:1 ratio. Samples were added to the gel followed by electrophoresis (concentrating gel 80V; separating gel 100V). The gel interlayer was removed and the target protein and β-actin were isolated according to molecular weight. The proteins were transferred to a PVDF membrane followed by soaking the membrane in methanol. The membrane was placed in 5% skimmed milk powder prepared by TBST for non-specific antigen blocking, and then Rabbit anti rat E-cadherin antibody (Merck, Germany, diluted in 1:2000), Rabbit anti rat Vimentin (Merck, Germany, diluted in1:500), Rabbit anti rat N-cadherin antibody (Merck, Germany, diluted in 1:500), or Rabbit anti rat β-actin antibodies were added (Merck, Germany, diluted in 1:2000). The membrane was washed three times with TBST, then Sheep anti rabbit IgG (Merck, Germany, diluted in 1:5000) labeled by HRP (Merck, Germany) was added for incubation followed by 3x TBST wash. After the film dried slightly, it was incubated with supersignal chemiluminescent reagent (Merck, Germany). The membrane was placed in a dark box and exposed together with the X-ray film (Merck, Germany). The exposure time was about 1 min. X-light was photographed after developing and fixing, and the gray value of the strip was analyzed by gel image analysis software IMAGE-J.All measurements were performed in triplicate, and the experiments were repeated three times independently
7. Animal experiment
Six-week-old female BALB/c nude mice were obtained from Silaike Laboratory Animal Co., Ltd,Shanghai, China. The protocol was authorized by the Animal Care and Use Committee of Xi’an Jiaotong University. In order to evaluate whether CD133+ HPCs affected the growth of breast cancer in vivo, a 1×107 suspension cells of MCF-7 and MDA-MB-231 breast cancer cells were taken respectively, mixed with CD133+/- cells, and inoculated to the nude mice (n=8 in each group). In the experimental and negative control group, breast cancer cells and CD133+/- cells were added in a 20:1 ratio. For the negative control group, breast cancer cells and CD133- HPCs cell suspensions were added in a 20:1 ratio. For the blank control group, only breast cancer cells were inoculated. Tumor growth was monitored every 2 days for 30 days after inoculation, and the tumor volume (volume = length×width2×0.5) was measured. Each group of eight mice were housed in separated individual standard cleaned cages under automatically controlled air conditioning 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 endpoint, mice were euthanized by inhalation of CO2 followed by cervical dislocation. On the 30th day, mice were sacrificed and the tumor tissues were collected, stained with hematoxylin and eosin (H&E) and observed under an optical microscope.
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 statistical significance.