Cell lines
A canine anaplastic astrocytoma cell line (J3T-1), which was gifted from Prof. Kurozumi (Hamamatsu University School of Medicine), was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque, Inc., Kyoto, Japan) with 10% fetal bovine serum (Thermo Fisher Scientific) and maintained at 37℃ in a humidified incubator with 5% CO2 (Inoue et al 2012). The cell line was tested for mycoplasma by using EZ-PCR Mycoplasma detection kit (Biological industries, Kibbutz Beit Haemek, Israel). The number of viable cells was determined by performing the trypan blue dye exclusion test.
Reagents and antibodies
Afatinib or dacomitinib (Tokyo Chemical Industry, Tokyo, Japan), which was resolved in dimethyl sulfoxide (DMSO), was added to the cell culture media to determine its effect on cell growth and protein expression. Z-VAD-FMK (Medical and biological laboratories, Tokyo, Japan) was used to inhibit pan-caspase activities at a dose of 25 µM. DMSO was used as a negative control.
The following antibodies were used: anti-p-ERBB4 mouse monoclonal antibody (1:200, Cat# sc-81491, Santa Cruz Biotechnology, Dallas, TX, USA), anti-ERBB4 mouse monoclonal antibody (1:200, Cat# sc-8050, Santa Cruz Biotechnology), anti-p-Akt (Ser473) rabbit monoclonal antibody (1:1000, Cat.# 4060, Cell Signaling Technology, Denvers, MA, USA), anti-Akt rabbit monoclonal antibody (1:1000, Cat.# 4691, Cell Signaling Technology), anti-p-ERK1/2 (Thr202/Tyr204) rabbit monoclonal antibody (1:1000, Cat.# 4370, Cell Signaling Technology), anti-ERK1/2 rabbit monoclonal antibody (1:1000, Cat.# 4695, Cell Signaling Technology), and anti-cleaved caspase-3 rabbit monoclonal antibody (1:1000, Cat.# 9664, Cell Signaling Technology). Horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling. The loading control was prepared by re-incubating the same membrane with an anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA).
Western blotting
Total protein was extracted from whole cells as previously described (Noguchi et al 2011). Protein content was measured with a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Lysate protein measuring 10 µg was separated for western blotting via Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Then, the protein was electroblotted onto a polyvinylidene fluoride membrane (PerkinElmer Life Sciences, Boston, MA, USA). Details of the method used after blotting have been previously described (Noguchi et al 2011). The antibodies were properly diluted with Tris buffered saline with Tween 20 (TBS-T) containing 2% bovine serum albumin and 0.01% sodium azide.
Flowcytometric analysis
The cells were treated with afatinib or dacomitinib with or without Z-VAD-FMK for 24 h. Then, the cells were stained using an Annexin V-FITC Apoptosis detection kit (NACALAI TESQUE, INC., Kyoto, Japan), according to the manufacturer’s protocol. Quantification of the number of apoptotic cells was performed by CytoFLEX (Beckman Coulter, Brea, CA, USA).
Orthotopic mice xenograft model
2.5x105 J3T-1 cells were prepared in 2 µl DMEM for stereotactic injection into 5 weeks old nude mice (Japan SLC, Hamamatsu, Japan). According to the previous study (Torrini et al 2022), a Hamilton syringe was introduced into a burr hole 2.5 mm lateral, 1mm anterior of bregma and 3.0 mm down into the striatum using a stereotactic device (Narishige, Tokyo, Japan), as shown in Fig. 3a. Tumor cells were then stereotactically injected at a rate of 1 µL/10 s using a microinjector (Narishige). From the 7th day after the transplantation, each drug was orally administered 5 consecutive days a week for 5 weeks using a cannula. Each drug was dissolved in 0.5% methyl cellulose solution (FUJIFILM Wako Pure Chemical, Osaka, Japan), which was used as a control. Afatinib and dacomitinib were administered at 20 mg/kg and 15 mg/kg, and doses were determined based on FDA interview form and the previous study (Yoshioka et al 2019), respectively. When any neurologic abnormalities or > 20% reduction of the body weight of mice were observed, the mice were euthanized. The brains of all of the mice were histopathologically evaluated. The animal experiment protocol was approved by the Committee for Animal Research and Welfare of Osaka Prefecture University.
Statictics
Each examination was performed in triplicate. All calculated data were compared by using the unpaired 2-tailed Student’s t-test or one-way ANOVA following Tukey’s method using Microsoft Excel add-in software, Statcel3 (OMS publication, Tokyo, Japan). The survival time of mice was assessed by log-rank test using GraphPad Prism8. A p-value of < 0.05 was considered statistically significant.