Cell culture
4T1 (Balb/C murine breast cancer cell) cells and 4T1-3R cells were cultured in RPMI1640 medium (GIBCO® invitrogen Inc., Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, HyClone® Thermo, Waltham, MA, USA), 1% Penicillin-Streptomycin Solution (100X) (Caisson Laboratories Inc., North Logan, UT, USA) and 1% L-Glutamin (200mM) (Sigma-Aldrich Co., St. Louis, MO, USA). Cells were maintained in 37℃ incubator containing 5% CO2 and were passaged every 2 days.
Plasmid construction
The PB-3R-puro plasmid was constructed from PB-2R-puro plasmid reported previously [4]. This plasmid contains three reporter genes, including mRFP, HSV1-tk and luciferase 2 (luc2) driven by human beta-actin, Hef1/HTLV, and CMV promoters, respectively. In brief, PB-2R-puro plasmid was digested by SpeI to remove the Act-mRFP fragment. The remained vector was ligated to a CMV-luc2 fragment digested from pGL4.10-luc2 plasmid (Promega Corporation, Madison, WI, USA) using NheI and XbaI. The resultant plasmid was named PB-tk-luc2-puro. Subsequently, the PB-Act-mRFP plasmid, a generous gift from Dr. Congjian Xu (Fudan University, Shanghai, PR China) [13] was digested by EcoRI to obtain an Act-mRFP fragment, which was then ligated to EcoRI digested PB-tk-luc2-puro vector to establish PB-3R-puro plasmid.
Establishment of stable cell line
PB-3R-puro and Act-PBase (from Dr. Congjian Xu) [13] were co-transfected into 4T1 cells using jetPEI polymer-based DNA transfection reagent (Polyplus-transfection Inc., New York, YK, USA). After 48 hours of transfection, cells were selected in RPMI1640 medium supplemented with 8 µg/ml puromycin for 7 days. The survived stable 4T1 cells were screened for cells expressing mRFP signals using the flow cytometry (BD FACS Calibur, BD biosciences, San Jose, CA, USA).
Luciferase reporter gene assay
Cells cultured in a 24-well plate 2 days and washed with 1X PBS twice, and then added 1X luciferase lysis buffer to lysed cells by freeze-thaw. After centrifuge, added 30 µl supernatant with 200 µl 1X luciferase assay buffer (100mM ATP, 1M dithiothreitol, Luciferin assay buffer, 100X Luciferin) in a 96-well plate. Detecting luminescence signals by Wallac 1420 (Thermal), and then normalized with protein concentration.
MTT assay
Cells (8,000) were cultured in a 96-well plate, and then treated with various concentrations of ganciclovir (GCV, Sigma-Aldrich Co., St. Louis, MO, USA). Cells were then incubated at 37℃ for 4 days followed by adding 1mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5diphenylterazoliumbromide, Sigma-Aldrich Co., St. Louis, MO, USA) in medium without FBS for 3 hours. After removal of MTT solution, cells were added with 100 µl DMSO to dissolve crystals. The plate was scanned by 570nm of absorbed wavelength in an ELISA reader (ELISA plate reader, BIO-TEK instruments, Winoski).
Synergistic tumor model
Parental 4T1 cells and derived stable cell lines (1x106 each) were resuspended in PRMI1640 medium without FBS and s.c. injected in thighs of 6-weeks female Balb/C mice (National Laboratory Animal Center, Taipei, Taiwan). Tumor volume was measured by a caliper periodically and calculated by the formula: length(mm) x width(mm)2/2. The animal experiment has been approved by the Institutional Animal Care and Use Committee of National Yang-Ming University (approval number: 981225).
In vivo bioluminescence imaging
Tumor-bearing mice were intraperitoneally injected with 150 mg/kg D-luciferin (Caliper Co., Hopkinton, MA, USA) and were anesthetized using 2% isoflurane. The mice were then placed in the IVIS50 system (Xenogen Co., Alameda, CA, USA), and the luminescent signals at the ROIs were acquired and quantified as photon/s.
Isolation of tumor cell from metastatic organs
Tumor-bearing mice were sacrificed to resect organs, which was rinsed with 1X PBS. The resected organs were cut into small pieces and placed in a 6-well plate. To avoid contamination, the penicillin-streptomycin concentration was raised to 3% and the medium was refreshed daily for 1 week. Non-tumor cells would enter senescence and growth arrest after two weeks of culture, and survived tumor cells expressing mRFP reporter gene were further verified using the flow cytometer (BD FACS Calibur, BD biosciences, San Jose, CA, USA). The selected cells were then cultured in normal RPMI medium as mentioned above.
Sphere formation assay
Fivehundred cells were counted and seeded in ultra-low attachment plate. DMEM/F12 was used as the condition medium supplemented with 10ng/ml of epithelial growth factor (EGF), 10ng/ml of basic fibroblast growth factor (bFGF), 10ng/ml of insulin, and 5ml of N2 (Gibco Inc., Grand Island, NY, USA). After incubation for 4 days, formed mammospheres were visualized and identified under a microscope.
Detection of CD44 expression
Cells were trypsinized and fixed in 1X PBS with 4% formaldehyde (3:1) at 37℃ for 10 minutes. After centrifugation, the pellet was resuspended in the incubation buffer (0.5g BSA / 100ml PBS, pH=7.4). The fluorescein-conjugated CD44 antibody (GeneTex Inc., Alton Pkwy Irvine, CA, USA) was then added and incubated at 4℃ overnight. The antibody treated cells were spun down, resuspended in 1X PBS, and subjected to the flow cytometer (BD Cytomic FC 500, San Jose, CA, USA) for detection of fluorescent signals.
In vitro invasion assay
The matrigel (Matrigel™, BD biosciences, San Jose, CA, USA) was mixed with FBS free medium (1:4) and added into the transwell (24 Well Millicell® 8.0ìm, Millipore Co., Billerica, MA, USA). The matrigel coated tranwells were placed in a 24-well plate, and incubated at 37℃ until solidified. About 1,000 cells were resuspended in 200µl FBS free medium and added into a transwell immersed in 24-well plate filled with normal medium. After incubated for 24 hours, the transwell was rinsed with 1X PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich Co., St. Louis, MO, USA) for 10 minutes. The transwell was stained with crystal violet (Sigma-Aldrich Co., St. Louis, MO, USA) for 10 minutes. The membrane of transwell was removed from the transwell and placed on a microslide to be visualized and counted under a bright field microscope.
Wound healing assay
Cells were seeded in a 6-well plate and cultured to confluence. A pipet tip was used to scrape five linear traces on the plate, which is reincubated and detected under a bright field microscope up to six hours. The area of migration (pixels) were calculated by the ImageJ software (Version 1.47). The formula for calculating the migration is: x hours of migration distance (%) = (Dx-D0)/D0, where Dx is the wound width at x hour; D0 is the wound width at 0 hour.
Cell adhesion assay
Cells (5x104) were seeded in a 12-well plate at 37℃ for 1 to 4 hours. Cells were removed from incubator followed by fixation using 4% paraformaldehyde for 10 minutes. The fixed cells were stained with 1.25% crystal violet in 75% ethanol. After rinse, the plates were dried and visualized under the bright field microscope.
Microarray analysis
Total RNA was extracted using the TRIzol® reagent (Ambion, invitrogen Inc., Carlsbad, CA, USA). Microarray (Affymetrix Chip Mouse 430 2.0) was commissioned in National Yang-Ming University VYM Genome Research Center, and the data was analyzed by Ingenuity Pathway Analysis (IPA, Ingenuity Systems Inc., Redwood City, CA, USA).
Western blotting
Protein was extracted with protein lysis buffer (50mM Tris-HCl, 120mM NaCl, 0.5% NP-40) with 2% PMSF and quantitated by Bio-Rad Protein Assay (Bio-Rad, Bio-Rad Laboratories Inc., Hercules, CA, USA). The protein samples were boiled in sampling buffer (250mM Tris-HCl pH6.8, 10% SDS, 30% glycerol, 5% β-mercapitalethanol, 0.02% bromophenol blue), and run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The gels were electro-transferred to a nitrocellulous membrane (BioTraceTM NT, Pall, Port Washington, NY, USA). The membrane was then blocked in TBS-T buffer (150mM NaCl、10mM Tris-HCl、0.1% Tween20, pH8.0) containing 4% milk followed by incubation with primary antibody, including anti-Twist-1 and anti-vimentin (GeneTex Inc., Alton Pkwy Irvine, CA, USA), anti-E-cadherin and anti-N-cadherin (Cell Signaling Technology Inc., Beverly, MA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Sigma-Aldrich Co., St. Louis, MO, USA). The secondary antibody is horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Millipore Co., Billerica, MA, USA). The antibody reacted membrane was rinsed in Western lightning plus-ECL buffer (PerkinElmer Inc., Waltham, MA, USA) and detected for the chemoluminescent signals using the ImageQuant™ LAS-4000 (GE Healthcare Bio-Science AB, Uppsala, Sweden).
Statistical Analysis
Each value represented after three independent experiments. The statistical analysis was determined by Student’s t-test, and when p<0.05 was showed as a statistically significant.