Cell lines and cell cultures
Six canine melanoma cell lines (CMeC-1, CMeC-2, KMeC, LMeC, CMM1, and CMM2) were provided by the Laboratory of Veterinary Surgery, University of Tokyo (Tokyo, Japan) [22, 23]. Cells were cultured in the Roswell Park Memorial Institute medium (RPMI 1640; WAKO, Tokyo, Japan) containing 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The cultures were maintained at 37 °C with 5% CO2.
Antibodies and reagents
Antibodies against the following proteins were used: cCas3 (#9661), cPARP (#9541), survivin (#2808; All Cell Signaling Technology, Danvers, MA, USA), β-actin (A5441; Sigma, St. Louis, MO, USA), NRF2 (sc-13032; Santa Cruz Biotechnology, Dallas, TX, USA), and ASCT2 (Proteintech, Rosemont, IL, USA).
miRNA transfection
Transfection with miRNA (10 nM) was performed using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions and as described in a previous publication [20]. The miRVana miR-634 mimic (4464066) and negative control 1 (miR-NC; 4464058) were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
Cell survival assay
Cell survival was assessed using crystal violet staining as previously described [20]. The optical density was measured at 560 nm using a microplate reader (SYNERGT H1; BioTek, VT, USA). Subsequently, the percentage absorbance in each well was measured. The optical density values of cells in control wells were set at 100% to calculate the percentage of viable cells.
Western blot analysis
Western blotting was performed as previously described [20]. Whole-cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to polyvinylidene difluoride membranes (GE HealthCare UK Ltd., Little Chalfont, UK). The membranes were then blocked with Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat dry milk for 1 h. Subsequently, they were incubated with primary antibodies and then exposed to horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) antibodies (both at 1:5000) for 3 h. Bound antibodies were visualized using a horseradish peroxidase staining solution or an ECL Western Detection Kit, according to the manufacturer’s instructions (Thermo Fisher Scientific).
Assessment of the apoptotic cell population
As described previously [20], apoptotic cells were stained using the MEBCYTO Apoptosis Kit (MBL International Corporation, Woburn, MA, USA), and cell populations were analyzed using an Accuri Flow Cytometer (BD, San Jose, CA, USA).
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Total RNA was isolated from case 1 biopsy samples using TRIsure reagent (Nippon Genetics, Tokyo, Japan) according to standard procedures described in a previous publication [20]. Tissue was collected from three locations by inserting a 16G core biopsy needle into the tumor. qRT-PCR was performed using an ABI PRISM 7500 Fast Real-time PCR System, TaqMan Universal PCR Master Mix, TaqMan Reverse Transcription Kit, and TaqMan MicroRNA Assays (miR-634; assay ID: 001576; ABI, Waltham, MA, USA), according to the manufacturer’s instructions. Gene expression values are presented as the ratio (difference in threshold cycle values) between miR-634 and an internal reference, miR-16 (assay ID: 000391).
Canine clinical trial design and interventions
The clinical trial on the effect of miR-634 was conducted between September 2018 and March 2020 at the Veterinary Teaching Hospital of Gifu University, Japan. Client-owned pet dogs with histologically confirmed CMMs and dogs displaying tumor recurrence after surgery or radiation therapy were enrolled. All dogs underwent a physical examination, laboratory evaluations (complete blood counts and serum biochemical profiles), and whole-body computed tomography (CT) scans.
Before treatment, the tumor stage was defined according to the WHO criteria for CMM [24, 25]. Treatment was discontinued in dogs when no lesion was found, when the tumor was insensitive to miR-634 and continued to grow, or when the owner expressed disinterest in continuing the study.
Treatment with miR-634
miR-634 treatment was administered alone to lesions with tumor recurrence after surgery or radiation therapy. In all cases, miR-634 (2–10 nmol) in 180 μL of Opti-MEM was mixed with 10 μL of Lipofectamine RNAiMAX, and the mixture was injected intratumorally using a tuberculin syringe after allowing the solution to stand at 15–25 °C for 10 min. miRNA was injected under sedation (medetomidine 0.03 mg/kg, midazolam 0.15 mg/kg, and butorphanol 0.1 mg/kg) once a week. For each intratumoral injection, the needle was inserted into all areas of the tumor, and miR-634 was divided into multiple doses to ensure that it was distributed as evenly as possible within the tumor (Figure 1A). The administration to the lung metastatic lesion in case 1 was performed under ultrasound guidance (Figure 1D).
Clinical assessment
Adverse events were assessed according to the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events v1.1 guidelines [28]. Adverse events were monitored throughout the study based on the reports by owners, physical examinations, and hematology and blood biochemistry results.
The tumor size was determined before commencing the treatment. CT images were acquired to determine the sizes of lung metastases, and physical examination for external signs was performed using 2- or 3-dimensional calipers. Standard Response Evaluation Criteria in Solid Tumors criteria were used for defining responses as follows [29]: 1) Complete response (CR) was defined as the disappearance of all target lesions; 2) partial response (PR) was defined as a reduction of at least 30% in the sum of diameters of target lesions from the baseline; 3) stable disease (SD) was defined as a <30% decrease or >20% increase in the sum of diameters of target lesions from the smallest sum while on treatment; and 4) progressive disease (PD) was defined as an increase of at least 20% in the sum of diameters of target lesions over the size present at the beginning of the study. Blood analyses, including complete blood count and blood biochemistry tests, were performed in all cases before and after miRNA administration.
Statistical analyses
Significance was assessed using the two-tailed Student’s t-test of Prism version 5.04 (GraphPad, La Jolla, CA, USA). Results with P ≤ 0.05 were considered statistically significant. Error bars indicate the standard deviation of triplicate experiments, and data are presented as the mean ± standard deviation.