Breast cancer cell line MCF-7 was kindly donated by Prof. Shuren Zhang (Department of Immunology, Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences). MCF-7 cells were maintained in RPMI1640 culture (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin G, and 100 μg/ml streptomycin. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.
SP cell isolation
Cells were detached from cell culture flasks with 0.25% trypsin, and viable cells were counted with trypan blue and collected for inoculation into NOD/SCID mice. The remaining cells were stained with the fluorescent dye Hoechst 33342 (Sigma) at a concentration of 5 μg/mL (37°C for 90 min) as described by Goodell et al.[29
] After washing with HBSS/2% FBS, the cells were incubated with 1 μg/ml propidium iodide to exclude dead cells, cell analysis and sorting were performed on a FACS Vantage SE (Becton Dickinson) by using a dual-wavelength analysis (blue, 420-470 nm; red, 660- 680 nm). We collected both MCF-7 SP and non-SP cells for the experiment.
Tumor formation in an animal model and drug intervention
For the tumor formation assay, the NOD/SCID female mice (5-6 weeks old) were purchased from the Animal Institute of Peking Union Medical College and maintained under standard conditions according to the guidelines of the Institutional Animal Care and Use Committee of Peking University. The mice were allowed to adapt to the new environment for one week. We first identified the tumorigenicity of SP cells. Unsorted, SP and non-SP cells were collected, and cells were resuspended in PBS/Matrigel (BD Biosciences) (1:1) ranging from 103 to 5 × 106 cells per 100 μl. Cells were then injected s.c. into the bilateral mammary pads of the mice. The mice were received an estradiol supplement (0.4 mg/kg s.c., Sigma) every 10 days until the end of the experiment after cell injection. The mice without tumors were examined visually everyday. Throughout the study, mice were weighed and tumors were measured with a caliper twice a week. Tumor volumes were calculated using the formula (length×width2/2). When the xenograft tumors grew to proper size, the mice were euthanized and a portion of the s.c. tumor tissue was collected, fixed in 4% formalin, and embedded in paraffin for H&E staining to assess tumor pathology.
For the drug administration assay, an identical protocol was followed. The mice were randomized into three groups (6 in each group). SP cells were resuspended in PBS/Matrigel (BD Biosciences) (1:1) with 1 × 104
cells per 100 μl. 1 × 104
cells were then injected s.c. into the right mammary fat pad of each mouse at day 0. The CKI group was injected i.p. with CKI (courtesy of the Shanxi Zhengdong Pharmaceutical Co. LTD., Z14021230, China), (2 ml/kg, diluted with saline in a final volume of 200 ul) every two days, and the control group was administered with the same volume of 200 ul saline every two days beginning from 24 hours after xenotransplantation, while the DDP group was applied with DDP (courtesy of the Yunnan Supertrack Bio- pharmaceutical Corporation, H53021740, China), (5 mg/kg, diluted with saline in a final volume of 200 ul, dose according to Hardman et al.[30
]) for three times at Day1, Day 8, Day 15 post inoculation.
Quantitative RT-PCR (QRT-PCR) analysis
To assess the expression levels of β-catenin, LEF1, TCF4, CyclinD1, c-Myc, total RNA from cells/tumors was extracted by Trizol (Invitrogen) according to the manufacturer's instructions. RNA (2 μg) was quantified by spectrophotometry (DU640, Backman, USA), and reverse transcribed into cDNA using a RevertiAid™ First Strand cDNA Synthesis Kit (Fermentas, CA) according to the manufacturer's instructions. Reactions were performed using SYBR Green I Master Mix(Applied Biosystems, CA) on a GeneAmp 7500 TaqMAN PCR (Applied Biosystems, CA). PCR conditions were: initial denaturation at 95°C for 10 min followed by 40 cycles: 95°C,25 s; 55°C, 25 s and 72°C,50 s with a final extension at 72°C for 5 min. The sequences of the primers used were as follows: β-actin forward, 5'-GAGACCTTCAACACCCCAGCC-3' and reverse,
5'-AATGTCACGCACGATTTCCC-3'; β-catenin forward, 5'-AAGGTCTGAGGAGCAGCTTC-3' and reverse, 5'-TGGACCATAACTGCAGCCTT-3'; LEF1 forward, 5'-CTACCACGACAAGGCCAGAG-3' and reverse, 5'-CAGTGAGGATGGGTAGGGTTG-3' and TCF4 forward 5'-TCCCACCACATCATACGCTACAC-3', and reverse,
5'- TCGCTTGCTCTTCTCTGGACAG-3'. CyclinD1 forward, 5'-CGATGCCAACCTCCTCAACGAC-3' and reverse, 5'-CCAGCATCCAGGTGGCGACG-3' and c-Myc forward 5'-CAGCAAACCTCCTCAGCC-3', and reverse, 5'-ATTGTTTTCCAACTCCGGGAT-3'.
The amount of each target gene in a given sample was normalized to the level of β-actin in that sample. The 2-ΔΔCT
method was applied to analyze the relative changes in gene expression [31
Western blot assay
Tumors were ground and lysed with the Keygen Total Protein Extraction Kit (KGP250, Keygen Serving Science, China) on ice. Tissue debris was removed by centrifugation at 4°C for 5 min. Tissue extracts were collected, and the protein concentration was determined by using the BCA Protein Assay Kit (KGPBCA, Keygen serving science, China). 60 μg of protein was run on SDS/PAGE gel and, after electrophoresis, the proteins were transferred to a PVDF membrane. Primary antibodies including anti-β-catenin (BD Bioscience, USA), anti-wnt1 (ab15251, Abcam, UK), anti-CyclinD1 (ab6125, Abcam, UK), anti-c-Myc (ab32, Abcam, UK) were applied, followed by incubation with secondary antibodies (Goat Anti-rabbit IgG, ZB2301; Goat Anti-mouse IgG, ZB2305, Zhongshan Golden Bridge Biotechnology CO., LTD., China). Blots were developed by ChemiDoc XRS System (Bio-Rad, USA).
Student's independent-samples t-test, one-way ANOVA, and χ2-test were used for statistical analysis by SPSS 10.0 software (SPSS, China, 657180). P < 0.05 was considered significant.