Therapeutic applications of local injection of hsa-miR-634 into canine spontaneous malignant melanoma tumors

Malignant melanoma (MM) is one of the most common tumors in both dogs and humans. As canine MM (CMM) and human MM (HMM) have similar clinical characteristics, CMM appears to be a good clinical model for HMM. We previously demonstrated that the introduction of a synthetic double-strand-microRNA-634 (miR-634) mimic triggered apoptotic cell death by directly targeting the genes associated with cytoprotective processes in various human cancer cell lines, including those of HMM. This study aimed to investigate the antitumor effects of the local administration of miR-634 on spontaneous CMMs to provide a basis for future applications of miR-634 formulations in HMM treatment. We found that miR-634 administration induced apoptosis in CMM cell lines in vitro via downregulation of Asct2, Nrf2, and survivin expression, similar to the mechanisms in HMM cell lines. Furthermore, intratumoral miR-634 administration induced antitumor effects in four of seven spontaneous CMM cases, with no adverse effects. Local administration of miR-634 to lung metastasis under ultrasound guidance induced tumor shrinkage. These results confirm the antitumor effect of the local administration of miR-634 in spontaneous CMM, a model for spontaneous HMM, thereby providing a novel treatment strategy for HMM.


INTRODUCTION
Malignant melanoma (MM) is one of the most common tumors in both dogs and humans.Canine MM (CMM) and human MM (HMM) are spontaneous and highly aggressive neoplasms that frequently metastasize to other organs [1,2].As CMM and HMM have similar clinical characteristics, CMM serves as a good clinical model to evaluate new treatment methods for HMM [3].
We previously demonstrated that the introduction of synthetic double-strand (ds)-miR-634 mimic triggered apoptotic cell death by directly targeting genes associated with cytoprotective processes, including glutaminolysis (ASCT2), autophagy (LAMP2), antioxidant activity (NRF2), anti-apoptotic activity (cIAP1, survivin, APIP, and XIAP), and mitochondrial function (OPA1 and TFAM) in various human cancer cell lines, including those of HMM [17][18][19][20][21]. Delivery of a synthetic ds-miR-634 mimic into tumor cells using lipid nanoparticles (LNPs) as the drug delivery system was therapeutically effective in a xenograft mouse model of pancreatic cancer [19].Topical application of an ointment containing ds-miR-634 mimic inhibited in vivo tumor growth without toxicity in a xenograft mouse model of cutaneous and oral squamous cell carcinoma [20,21].Thus, our previous reports suggested that ds-miR-634 mimic is a useful agent for cancer therapy.
This study aimed to investigate the local antitumor effects of Homo sapiens (hsa)-miR-634, hereinafter referred to as miR-634, on spontaneous CMMs occurring in pet dogs with an intact immune system.As CMMs have clinical similarities to HMMs, these findings can provide a basis for future applications of miR-634 formulations in HMM treatment.

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 overnight with primary antibodies at 4 °C 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 Biosciences, 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 16 G 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 (Fig. 1A).The administration into the lung metastatic lesion in case 1 was performed under ultrasound guidance (Fig. 1D).

Clinical assessment
Adverse events were assessed according to the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events v1.1 guidelines [26].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 [27]: (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.

Induction of apoptosis by miR-634 overexpression in CMM cells
We first examined whether introducing miR-634 would induce cell death in CMM cells.The results showed that when miR-634 was transfected into CMM cells, an effective induction of cell death occurred in five of six examined cell lines (CMeC-1, LMeC, CMeC-2, KMeC, and CMM2), except for CMM1 cells (Figs. 2A and S1).For CMeC-1 and LMeC cells, the levels of cleaved caspase-3 (cCas3) and PARP (cPARP), which are typical indicators of apoptotic cell death, were determined using western blotting analyses; the findings revealed markedly increased levels in miR-634-transfected cells compared with those in miR-NC-transfected cells (Fig. 2B).Furthermore, fluorescenceactivated cell sorting results demonstrated increased apoptotic cell populations following miR-634 overexpression (Fig. 2C).Notably, the expression levels of miR-634 target genes (canine Asct2, Nrf2, and survivin) were substantially downregulated after miR-634 overexpression in CMeC-1 and LMeC cells (Fig. 2B).We also found the putative binding sites corresponding to the seed sequence of miR-634 within the RNA sequences of each gene: two sites within the coding region in Asct2, one site each within the coding region and 3′-UTR in Nrf2, and two sites within 3′-UTR in survivin (Fig. 2D).These results suggested that miR-634 overexpression effectively induced apoptotic cell death by downregulating the expression of canine homologs of miR-634 target genes.

Cases
In total, seven dogs aged 9-17 years were included in this study.Their clinical characteristics are summarized in Table 1.These cases were classified in accordance with the TNM classification [ 24,25]: two dogs had stage I, one dog had stage II, one dog had stage III, and three dogs had stage IV CMM.All cases had undergone surgery or radiation therapy prior to miRNA clinical trials, and miRNA was administered locally to recurrent lesions (Table 1).

Delivery of miR-634 into tumor cells
The miR-634 expression levels in the tumor tissues were quantified via qRT-PCR before (pretreatment) and after (treatment) the local administration of miR-634 for 22 times (Fig. 3).miR-634 expression in the tumor tissues after treatment increased compared with that in the pretreated samples (Fig. 3).Weekly local miR-634 administration maintained high miR-634 expression levels in tumor tissues.

Clinical responses and adverse events
The effects of miR-634 administration are summarized in Table 2. Three, two, and three lesions attained a PR, SD, and PD state, respectively.The median treatment period was 53 d (range 23-652 d).miRNA was administered a median of seven times (range 4-76 times) at 2-10 nmol per dose.
Information regarding treatment effects in case 1 is shown in Fig. 1 as a representative example.For primary oral lesions, miR-634 was administered locally into the tumor under sedation (Fig. 1A), and CT scans were performed before and after treatment to evaluate tumor size (Fig. 1B, C).The same method of administration to the primary lesion and method of determining efficacy were used for all cases.In case 1, a lung metastatic lesion was detected 84 days after starting miRNA administration to the primary lesion in the oral cavity.The lung lesion was subjected to radiation therapy; however, it continued to grow after treatment.Therefore, the local administration of miR-634 to the lung lesion was started 412 days after administering miR-634 to the oral lesion; the results revealed tumor shrinkage (Fig. 1E, F).All administrations of miR-634 to lung metastases were performed under ultrasound guidance (Fig. 1D).Furthermore, routine physical examinations and blood tests were performed on all participants to determine any side effects.Blood tests showed no evidence of serious hepatic or renal injury associated with miR-634 administration (Table S1).In case 3, the locally administered target lesion was in partial remission, but tumor progression was observed at other sites, with worsening disseminated intravascular coagulation and elevated C-reactive protein levels.

DISCUSSION
In this study, we found that miR-634 administration induced apoptosis in CMM cell lines in vitro via regulation of Asct2, Nrf2, and survivin expression, similar to the mechanisms in HMM cell lines [19].Furthermore, intratumoral miR-634 administration to canine cases with spontaneous CMMs resulted in antitumor effects in four of seven cases.
In this study, miR-634 was transfected into six different cell lines derived from CMMs, and cell proliferation was inhibited in five of them.Further examination of the target gene and apoptotic marker expression showed downregulated levels of Asct2, Nrf2, and survivin, the corresponding human target genes of miR-634, as well as an increase in apoptotic cell numbers.These results suggested that miR-634 induced apoptosis in CMM cell lines through the same mechanisms that we reported in other human and murine malignant tumor cell lines, including HMM [17][18][19][20][21].This conclusion is crucial because it suggests the compatibility of the miR-634 treatment technique for CMM cases with HMM cases.On the other hand, CMM1 cells were less susceptible to the cell death effect of miR-634.This could be attributed to the excessive expression of the target gene, which may have led to inadequate suppression of its expression.Alternatively, miRNA634 may have a high efflux effect outside the cell.However, these assumptions are yet to be confirmed.Based on these in vitro results, we tested the local antitumor effects of intratumoral miR-634 administration to dogs with spontaneous CMMs and subsequently found clinical benefits in four of seven cases.In case 1, miR-634-induced tumor shrinkage was observed not only in the primary oral lesion but also in the lung metastatic lesion, demonstrating the effectiveness of miR-634 against distant metastatic lesions.This preliminary study was performed to establish a method for the treatment of distant metastatic lesions through intravenous systemic administration, and the results obtained present the beneficial effects of this technology.
In case 1, tumor tissue samples were collected before and after the local administration of miR-634 for 22 times, and the miR-634 expression level in each tissue was measured.The results suggested that weekly local administration of miR-634 to tumors can maintain high expression levels of miR-634.Likewise, our previous study on local intratumoral administration of miRNAs to dogs with spontaneous CMMs demonstrated that the administration of synthetic miR-205 has antitumor effects [28].The results of this and previous studies suggested that local sensitization of miRNAs is beneficial because it induces local antitumor effects in cases with spontaneous CMM and that no subsequent adverse reactions are observed.In this study, we did not observe any findings suggestive of inflammation in any of the cases.This suggests that the local administration of miR-634 is unlikely to cause off-target effects or cytokine bursts, which can be problematic when nucleic acid drugs are administered.Therefore, miR-634 local administration may be a safe option for CMM treatment.
Future studies should focus on the development of a drug delivery system that is less invasive and more capable of sustained sensitization as an alternative to local injection.In fact, an miR-634 ointment formulation has shown promising results in a mouse model of human cutaneous and oral squamous cell carcinoma [20,21].miR-634 ointment formulations can be used as the drug delivery system to permanently sensitize tumors to miRNAs as they are less invasive and easier to administer than intratumoral injections.In HMM, such formulations may hold promise as a new, minimally invasive treatment option.For further therapeutic applications against CMM, it is necessary to confirm the antitumor effects of intravenous systemic administration, as CMM is not only localized at one site but also metastasizes to the lungs, a major clinical problem.Systemic intravenous administration of miR-634, using LNPs as the drug delivery system, showed anticancer effects in a mouse model of pancreatic cancer [19].Although local administration was performed as a preliminary study for systemic administration, further pursuit of LNP-mediated delivery of miR-634 in CMM treatment is needed to verify its anticancer effects.
In the development of novel therapeutics, spontaneous canine tumors are useful in bridging the gap between murine and human tumors because of their diversity, which cannot be generated in mouse models.Moreover, many canine neoplastic diseases have biological dynamics similar to those of spontaneous human tumors.Clinical trials in canine tumor cases can confirm drug safety and efficacy, such as studies on the effects of ivermectin, an anti-parasitic drug, and masitinib, a molecular drug targeting c-kit and plateletderived growth factor receptor [29].This enables the development of comparable drugs for humans with reduced risks, which would otherwise require a large amount of development funds.
In conclusion, this study demonstrated that miR-634 administration induced apoptosis in CMM cell lines, similar to those in HMM, by regulating the expression of Asct2, Nrf2, and survivin.Furthermore, intratumoral administration of miR-634 to cases with spontaneous CMM resulted in antitumor effects in four of seven cases.These results suggested a local antitumor effect of miR-634 in spontaneous CMM, a model for spontaneous HMM, to provide a basis for the application of miR-634 administration to HMM.

Fig. 1
Fig. 1 Antitumor activity of miR-634 in spontaneous canine malignant melanomas.A Intratumoral local administration of miR-634.Primary lesions in the oral cavity were administered locally under visual inspection.B Representative contrast computed tomography (CT) image of an oral tumor (yellow arrowhead) before miR-634 administration.C Representative contrast CT image of the same oral tumor (yellow arrowhead) 582 days after starting miR-634 administration.D Ultrasound findings of pulmonary metastatic lesions.Local administration of miR-634 to lung metastases was performed under ultrasound guidance.E Representative contrast CT image of the lung metastatic lesion (red arrowhead) before miR-634 administration.F Representative contrast CT image of the same lung metastatic lesion (red arrowhead) 148 days after starting miR-634 administration.

Fig. 2
Fig. 2 Induction of apoptosis by miR-634 overexpression in canine melanoma cells.A Phase-contrast images and growth rates of miR-NCor miR-634-transfected cells.Error bars indicate standard deviations of triplicate experiments.Scale bars denote 20 µm.Data are presented as the mean ± standard deviation (two-tailed Student's t-test; *P < 0.0001).B Western blotting analysis of miR-NC-or miR-634-transfected cells.C Fluorescence-activated cell sorting (FACS) analysis of the apoptotic cell population.Error bars indicate standard deviations of triplicate experiments.D Putative binding sites within miR-634 target genes in canines.The seed sequence within miR-634 and putative binding sites of miR-634 within the mRNA sequences of each gene are underlined.CDS, coding sequence.

Fig. 3
Fig. 3 Delivery of miR-634 into tumor cells.Pretreatment: Before miR-634 administration.Treatment: 148 days after starting miR-634 administration.Enforced expression of miR-634 in case 1 biopsy samples following treatment was confirmed via quantitative reverse transcription polymerase chain reaction.miR-634 expression was normalized to that of miR-16.Error bars indicate standard deviations.Data are presented as the mean ± standard deviation.

Table 1 .
Clinical characteristics of canine malignant melanoma cases treated with miR-634.CM castrated male, F female, M male, MD miniature dachshund, MS miniature schnauzer, RT radiation therapy, ST Shih Tzu, Sx surgery, TP toy poodle.Stage: Clinical staging of dogs with melanoma in accordance with the WHO guidelines.Stage I: <2 cm in diameter, with no evidence of lymph node metastases.Stage II: 2-4 cm in diameter, with no evidence of lymph node metastases.Stage III: >4 cm in diameter or lymph node metastases.Stage IV: Any size with evidence of distant metastases.

Table 2 .
Effects of intratumoral administration of miR-634 on canine melanomas.LML lung metastatic lesion, PD progressive disease, PL primary lesion, PR partial response, SD stable disease.