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  • br Western Blot br Proteins were separated by

    2022-05-04


    Western Blot
    Proteins were separated by electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes using an iBlot apparatus (Invitrogen, MA). After blocking in skim milk for 1 h, the membrane was washed with TBS with Tween 20 (TBS-T) buffer at room temper-ature (RT). Primary N,N-Dimethylsphingosine against EGFR (Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam) were added, and the membranes were shaken gently for 1 h at RT. After three washes with TBS-T, the membranes were incu-bated with horseradish peroxidase (HRP)-labeled secondary antibody (anti-rabbit immunoglobulin G [IgG]) for 1 h at RT, and then they
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    were washed three times in TBS-T. Immunoreactive bands of proteins were visualized using the ECL Plus Solution (Amersham, Arlington Heights, IL, USA), and signals were recorded by autoradiography.
    Cell Viability
    Cell proliferation was evaluated using the MTT dye reduction method as described.46,47 Tumor cells were seeded in 96-well plates (5,000 cells/
    well) and incubated with DMEM (supplemented with 10% FCS and 1% penicillin/streptomycin) at 37 C and 5% CO2. After 24 h, cells were exposed to serial dilutions of cetuximab (Merck Serono, Germany) and/or C-REV at the indicated MOIs. The day of treatment was desig-nated as day 0. Cells were grown for another 1, 2, or 3 days. Viable cells were quantified using colorimetric MTT assays.
    Viral Proliferation Assay
    Cells were placed on six-well plates and incubated overnight. The following day, the cells were treated with C-REV at an MOI of 1 and incubated with various concentrations of cetuximab (5, 10, and 20 mg/mL). Then, 3 days later, cells were scraped, and the superna-tants were collected and subjected to three freeze-thaw cycles. The released virus particles were collected and serially diluted in DMEM
    without fetal bovine serum (FBS). Following a standard viral plaque assay,48,49 Vero cells were infected with serial dilutions of viruses in 6-well plates for 1 h. The viral supernatant was removed and 2% low-melting agarose was added. Cells were incubated at 37 C for 5–7 days until the plaques could be counted.
    Determination of EGFR Expression Level by Flow Cytometry
    All three CRC cell lines were treated with or without C-REV; infec-tions ran for 3 days. Cells were collected on days 1, 2, and 3 and stained with isotype control antibody (allophycocyanin [APC]) (eBioscience) and anti-human EGFR antibody (APC) (BioLegend) at 4 C for 10 min. Stained cells were washed twice with fluores-cence-activated cell sorting (FACS) buffer and fixed in 4% parafor-maldehyde. Data were acquired on a FACSCanto II (BD Biosciences) and analyzed using the FlowJo software.
    Animal Studies
    The 5- to 6-week-old male BALB/c Slc-nu/nu mice were obtained from Japan SLC (Shizuoka, Japan). All animal experiments were conducted in accordance with the guidelines issued by the Nagoya University Animal Center. Suspensions of HT-29 tumor cells (5 106 cells/100 mL) were subcutaneously injected into the backs of the mice. Tumors were allowed to reach a volume of about 100 mm3. The mice were then randomly divided into six groups of 10: control, cetuximab (G1 and G2), C-REV, and combination of cetuximab and C-REV (G1 and G2).
    Mice in the C-REV group and combination group (G1 and G2) were treated with C-REV (5 106 PFU/50 mL intratumorally [i.t.]) every 3 days for 1 week. Mice in the cetuximab group (G1 and G2) and combination group (G1 and G2) were treated with cetuximab (0.25 mg/injection intraperitoneally [i.p.]) every 3 days for 2 weeks. Cetuximab was injected 24 h before or after C-REV injection. The first 
    day of C-REV treatment was designated as day 1. Cetuximab was administered 24 h before or after the first dose of C-REV. Tumor size and body weight were measured twice a week.
    Tumor volume (V) was evaluated using the equation V = LW2/2, where L and W were tumor length and width, respectively. To deter-
    mine the presence or absence of synergy between cetuximab and C-REV, we used the FTV method.50,51 Here, the expected FTV of
    the combination treatment was divided by the observed FTV of the combination treatment, yielding a ratio that indicated the interaction (>1 indicates synergy and <1 a less than additive effect). FTV for each treatment group was obtained by dividing the mean tumor volume of the treatment group by that of the control group.
    In a separate experiment, mice bearing subcutaneous HT-29-derived tumors were randomly divided into six groups of three to receive treatment. On days 3 and 14 (2 days and 13 days after the last C-REV injection), tumor samples were collected for immunohisto-chemical staining.
    Immunohistochemistry
    Tumors samples were fixed and embedded in paraffin, and then they were sectioned at a 5-mm thickness to prepare slides for immunohistochemical staining. HSV-1 and CD31 were detected using anti-HSV-1 antibody (1/100 dilution; Imgenex, CA, USA) and anti-CD31 antibody (1/50 dilution; Abcam), respectively. Antigen retrieval was performed by autoclaving for 15 min in Tris-EDTA (pH 9.0). After blocking with 3% normal goat serum (Histofine; Nichirei Biosciences, Tokyo, Japan), sections were incu-bated overnight with the primary antibodies described above. Bio-tinylated anti-rabbit IgG (Histofine; Nichirei Biosciences) was used as the secondary antibody. HRP-3,3-diaminobenzidine (Histofine; Nichirei Biosciences) was used as the chromogenic agent. Each slide was examined by microscopy at 100 magnification. Five random fields of view in each section were chosen for histological quantifica-tion. CD31 density was evaluated by counting the number of CD31-stained microvessels within these areas.