The Chinese hamster ovary cell line CHO-s (Sanyou, Shanghai, China) and human mammary epithelial cell line MCF-10A (American Type Culture Collection, New York, USA) were maintained in DMEM/F12 medium (Gibco, Grand Island, USA), supplemented with 10% (v/v) FBS (Gibco, Auckland, NZ). The human embryonic kidney cell line HEK293T kept in our laboratory was cultured in DMEM high glucose medium (Gibco, New York, USA), supplemented with 10% (v/v) FBS. The human breast cancer cell lines MDA-MB-231/MDA-MB-468 (American Type Culture Collection) were maintained in L-15 medium (Gibco, New York, USA) supplemented with 10% (v/v) FBS. HUVECs (ScienCell, San Diego, CA) were maintained in endothelial culture medium (ECM, ScienCell) supplemented with 5% (v/v) FBS and 1% (v/v) endothelial cell growth supplement (ECGS, ScienCell). All cells were maintained at 37 °C in a humidifed atmosphere with 5% CO2.
Binding affinity and kinetic analysis
SHH002-hu1 was expressed in CHO-s cells and purified by Protein G affinity chromatograph (GE Healthcare, Buckinghamshire, UK), followed by the analysis of SDS-PAGE and SEC-HPLC. The binding kinetics of SHH002-hu1 to rhFzd1/2/5/7/8 was measured with Fortebio Octet Red96 (PALL, USA). Firstly, SHH002-hu1 was captured by the Anti-Human Fab-CH1 2nd Generation Sensor (PALL, USA). Then, rhFzd1/2/5/7/8 was injected at different concentrations into running buffer (KB buffer: 0.1% BSA + 0.05% Tween 20 dissolved in PBS, pH 7.2), and capture was done at a constant flow rate. As for SHH002 (the murine antibody targeting Fzd7 generated by us), the antibody was captured by the Anti-Biotin Sensor (PALL, USA) after conjugated with biotin. Sensorgrams were obtained at each concentration, and the association rate constant (ka) and dissociation rate constant (kd) were calculated by the instrument algorithm. Finally, the equilibrium dissociation constant (KD) was calculated from the ratio of rate constants kd/ka.
IF assay for the binding of SHH002-hu1
The gene of human Fzd7 was linked with the lentiviral vector (HBLV-GFP-PURO, Hanbio Biotechnology, China) to obtain the recombinant plasmid HBLV-h-Fzd7-3*flag-GFP-PURO. HEK293T cells stably overexpressing Fzd7 (Fzd7 OE) were obtained through virus infection and screening. HEK293T cells (blank/vector control/Fzd7 OE), MDA-MB-231/MDA-MB-468 cells were inoculated into 6-well plate. When cell confluence reached 70-80%, the cells were incubated with SHH002-hu1 and then Goat Anti-Human IgG H&L (Alexa Fluor 647, Abcam, USA). Subsequently, images were taken by an OLYMPUS fluorescence microscope (Olympus, Tokyo, Japan).
In vivo dynamics and targeting capability by NIR imaging
5-week-old female BALB/c-nude mice were purchased from Shanghai Lab. Animal Research Center, China. 1 × 107 MDA-MB-231 cells were injected into the mammary fat pads of mice. When the average tumor volume reached 100 mm3, mice were randomized into 2 groups (n = 5 for each group). IRB-NHS fluorescence probing (Keyuandi Biotechnology, Shanghai, China) was incubated with SHH002-hu1 for 2 h to form NIRB-SHH002-hu1 fluorescence probe. NIRB-SHH002-hu1 (50 nmol/kg) was then intravenously injected into TNBC tumor-bearing mice. Additionally, free SHH002-hu1 (2.5 μmol/kg) was mixed with NIRB-SHH002-hu1 (50 nmol/kg) to evaluate the competitive blocking. After intravenous injection, fluorescence images were taken by IVIS Spectrum CT imaging system (PerkinElmer, USA) at different time intervals. The tumor/normal tissue ratios were analyzed from the region of interests (ROI).
Cell proliferation assay
4 × 103 HEK293T/MCF-10A/MDA-MB-231/MDA-MB-468 cells were seeded into a 96-well plate to attach. Then, different concentrations of SHH002-hu1 were added and pre-incubated for 2 h, after which recombinant Wnt3a (R&D, Minneapolis, MN, USA) was added at a final concentration of 200 ng/mL. The SHH002-hu1 untreated group with Wnt3a induced was as vehicle control. After incubation for 48 h, cell viability was quantified by MTT assay and the proliferation rate was expressed as percentage of the vehicle control (100 %).
Luciferase reporter assay
The β-catenin/TCF-driven transcriptional activity was assessed by transient transfection of MDA-MB-231/MDA-MB-468 cells with the TOP-FLASH/FOP-FLASH luciferase reporter assay and a Renilla luciferase transfection control reporter. MDA-MB-231/MDA-MB-468 cells were seeded into 48-well plates and transfected with TOP-FLASH/FOP-FLASH plasmid using Lipofectamine™ 2000 (Invitrogen, Cartsbad, CA, USA). Then, the cells were treated with SHH002-hu1 (100 nmol/L)/FH535 (10 μmol/L, absin, China), 2 h later, Wnt3a (200 ng/mL) was added. The luciferase activities were measured at 24 h with the Dual-Glo luciferase assay reporter system (Promega, Madison, WI, USA).
IF assay for the location of β-catenin
5×105 MDA-MB-231/MDA-MB-468 cells were seeded on cover slips in 6-well plates and allowed to adhere. When reaching 70% confluence, cells were treated with SHH002-hu1 (100 nmol/L)/FH535 (10 μmol/L), 2 h later, Wnt3a (200 ng/mL) was added. After another 22 h, cells were fixed and then incubated with α-β-catenin (Cell Signaling Technology, USA) followed by FITC-conjugated secondary antibody. Images were taken by an OLYMPUS fluorescence microscope at 400-times magnification after the incubation with DAPI stain solution (Sangon Biotech, Shanghai, China).
Western blot assay
The whole cell proteins of MDA-MB-231/MDA-MB-468 cells were extracted from cells using RIPA buffer (Beyotime, Shanghai, China) and the nuclear extracts were prepared with a NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, USA). Western blots were probed with α-phospho LRP6 (Ser1490), α-LRP6, α-β-catenin, α-E-cadherin, α-N-cadherin, α-Vimentin, α-Snail, α-Histone H3, α-c-Myc, α-cyclin D1, α-Axin, α-CD44, α-HIF-1a, α-Glut1 (Cell Signaling Technology, USA), α-VEGFA (Abcam, Cambridge, UK), α-β-actin.
Tube formation assay
2 × 104 HUVECs were seeded into the 96-well plate coated with matrigel (Corning, Bedford, USA), and added with the supernatants obtained from SHH002-hu1 treated MDA-MB-231/MDA-MB-468 cells. After 8-h incubation, endothelial tube formation was photographed with an inverted OLYMPUS microscope, 10 ng/mL VEGF165 (Sino Biological, Beijing, China) treated HUVECs were as control. The endothelial tubes were counted with Image-Pro-Plus program and the tube formation rate quantified on the basis of the control.
Transwell invasion and wound healing assays
1 × 104 of MDA-MB-231/MDA-MB-468 cells suspended in serum-free medium were plated into the upper wells of 24-well transwell chamber (Millipore, Billerica, USA) coated matrigel, and then treated with Bevacizumab (200 nmol/L, Roche, France)/Bevacizumab (200 nmol/L) + SHH002-hu1 (100 nmol/L)/Bevacizumab (200 nmol/L) + FH535 (10 μmol/L). The lower chambers were filled with complete medium. 12 h later, the invaded cells were then fixed and stained. Images were taken using an OLYMPUS inverted microscope. Invaded cells were counted using Image-Pro-Plus program and invasion percentages quantified on the basis of untreated control.
3 × 104 MDA-MB-231/MDA-MB-468 cells were placed into each well of Culture-Insert (Ibidi, Martinsried, Germany) in 24-well plate. After adherence, the Culture-Insert was removed and Bevacizumab (200 nmol/L)/Bevacizumab (200 nmol/L) + SHH002-hu1 (100 nmol/L)/Bevacizumab (200 nmol/L) + FH535 (10 μmol/L) dissolved in serum-free medium was added into the wells. Images were taken with OLYMPUS inversion fluorescence microscope at 0, 8, 16 h after the addition of the treatments. The wound migrated distances were measured with Image-Pro-Plus program and calculated as follows: Ln = (L0-Ltime)/2.
Cell xenografts in nude mice assay
The xenograft tumors of MDA-MB-231/MDA-MB-468 cells in nude mice were established as described above. When the average tumor volume reached 50 mm3, mice were randomized into 5 groups (n = 5 for each group), and the administration began: (1) PBS control; (2) 5 mg/kg Bevacizumab (intravenous injection, twice a week); (3) 5 mg/kg SHH002-hu1 (intravenous injection, twice a week); (4) 5 mg/kg Bevacizumab + 5 mg/kg SHH002-hu1; (5) 5 mg/kg Bevacizumab + 10 mg/kg Docetaxel (intravenous injection, every 3 days). Tumors were measured by digital calipers periodically and the tumor volume was determined as V = (length × width2)/2. At the end of drug treatment, the mice were humanely euthanized and tumors were harvested for further studies.
IF and immunohistochemistry (IHC) analysis
For ALDH1/Hypoxyprobe and β-catenin/Hypoxyprobe double labeling, mice were injected intravenously with 60 mg/kg of the pimonidazole solution, 90 min later, the mice were euthanatized and tumor tissues were removed and snap-frozen. Frozen tissue sections were then interrogated with FITC-conjugated α-pimonidazole (Hypoxyprobe Inc., USA) and α-ALDH1 (Abcam, USA)/β-catenin (Cell Signaling Technology, USA) followed by respective Cy3-conjugated secondary IgG. Coverslips were then mounted with DAPI stain solution. For Periodic Acid Schiff (PAS)-CD31 double IHC staining, paraffin sections were cut into 5 μm sections and fixed in 4% paraformaldehyde. The sections were firstly incubated with α-CD31 (Cell Signaling Technology, USA), then exposed to 1% sodium periodate and incubated with PAS (BestBio, Shanghai, China).
Secondary nude mouse xenograft model
MDA-MB-231/MDA-MB-468 tumors tissues were minced into small pieces and processed with collagenase for 2 h at 37°C. After centrifugation, the cell pellets were trypsinized and passed through an 80-μm filter to produce single-cell suspension, then living cells were sorted out by fluorescence-activated cell sorting. Each nude mouse was inoculated with 1 × 104 cells from control/Bevacizumab-treated/Bevacizumab + SHH002-hu1-treated tumors in one of the inguinal mammary fat pads. The growth of tumors was monitored and tumor sizes were measured weekly.
Sphere formation assay
The single-cell suspension was produced as above, then 5 × 103 dissociated tumor cells were seeded on ultralow attachment 6-well plates in serum-free medium DM-EM/F12 (Gibco, Grand Island, USA) supplemented with B27 (Gibco, Grand Island, USA), 20 ng/mL human recombinant fibroblast growth factor (FGF), and 20 ng/mL epidermal growth factor (EGF, Sino Biological Inc., Beijing, China). The mammospheres (diameter > 60 μm) were counted under an OLYMPUS inversion fluorescence microscope.
All data were presented as the mean ± standard deviation (SD). Differences between multiple groups were analyzed by the student’s t test and p values of 0.05 or less were considered statistically significant.