Targeting canine mammary neoplastic epithelial cells with a reengineered anthrax toxin: first study.

Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000 ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.

Inhibitory Effects of Alpha-Connexin Carboxyl-Terminal Peptide on Canine Mammary Epithelial Cells: A Study on Benign and Malignant Phenotypes.

Mammary cancer is highly prevalent in non-castrated female dogs. Cell-to-cell communication is an important mechanism to maintain homeostasis, and connexins are proteins that assemble to form the communicating gap junctions. In many cancers, communication capacity is reduced; several approaches are being tested in order to increase the communication capacity in cancer cells and, therefore, alter their viability. This study analyzed the effects of the alpha-connexin carboxyl-terminal peptide (αCT1) on canine mammary non-neoplastic and neoplastic epithelial cells. Seven canine epithelial mammary cell lines were used. Among these, one was a normal canine epithelial mammary cell line (LOEC-NMG), two canine mammary adenomas (LOEC-MAd1 and LOEC-MAd2), and four canine mammary adenocarcinomas (LOEC-MCA1, LOEC-MCA2, LOEC-MCA3 and CF41). The αCT1 corresponds to a short Cx43 C-terminal sequence linked to an internalization sequence called the antennapedia. After 24 h of incubation, the medium containing different αCT1 peptide concentrations was added to the cells, and only the culture medium was used for control. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to quantify cell viability before treatment and 48, 72, and 96 h after the treatment. Results showed that the normal mammary epithelial cell line (LOEC-NMG) was resistant to treatment with αCT1, which is consistent with a previous study on human mammary cell lines. One of the adenoma cell lines (LOEC-MAd2) was also resistant to treatment with αCT1, although the other (LOEC-MAd1) was susceptible to treatment, mostly at 72 h after treatment. Regarding the four canine adenocarcinoma cell lines, they differ regarding the susceptibility to the treatment with αCT1. Three cell lines, canine mixed adenocarcinoma (LOEC-MCA1), canine complex adenocarcinoma (LOEC-MCA2), and commercial canine mammary adenocarcinoma cell line CF41, were susceptible to treatment with αCT1, while one canine mammary adenocarcinoma cell line (LOEC-MCA3) was resistant to treatment. In most αCT1 treated cell lines, Cx43 was strongly detected in cell membranes by immunofluorescence. We propose that αCT1 restored the cell-to-cell communication capacity of neoplastic cells and induced inhibitory effects on cell viability.

Is placental glutathione S-Transferase (GST-P) a suitable biomarker for oral carcinogenesis: A scoping review.

Placental Glutathione S-Transferase (GST-P) can be considered a useful marker of not only of preneoplastic lesion in rat hepatocarcinogenesis and hamster pancreatic carcinogen but also as a potential marker for premalignant and malignant lesions in cases of buccal pouch mucosa. In this context, the aim of this review is to elucidate the following question whether the GST-P is a suitable biomarker for oral carcinogenesis. A total of 16 studies were carefully selected. Our results demonstrate that GST-P expression is a useful and coherent marker for oral carcinogenesis. Regarding the samples, most studies evaluated hamsters, two evaluated GST-P expression in rats and three evaluated GST-P expression in human cells. All studies demonstrated positive findings allowing us to consider such studies reliable. In summary, our conclusion is that GST-P can be a suitable biomarker for oral carcinogenesis.

Resveratrol can induce differentiating phenotypes in canine oral mucosal melanoma cells.

This report described the differentiation induction of canine oral mucosal melanoma (OMM) cells by resveratrol. Exposure of canine OMM cells to resveratrol (maximum dose: 50 µM and treatment period: 72 hr) induced differentiating features like melanocytes, and enhanced chemosensitivity against cisplatin, but alone had no influence on cell viability. Additionally, resveratrol significantly enhanced mRNA expression of key melanoma differentiation markers such as microphthalmia-associated transcription factor (MITF). Of several inhibitors against mitogen-activated protein kinase subtypes, only the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, induced melanocyte-like morphological change and enhanced MITF mRNA expression. Furthermore, resveratrol also suppressed JNK activation in OMM cells by approximately 33%. Overall, these findings suggest that resveratrol induces differentiation in canine OMM cells, due to the inhibition of JNK signaling.

Diagnosis, Prognosis, and Treatment of Canine Hemangiosarcoma: A Review Based on a Consensus Organized by the Brazilian Association of Veterinary Oncology, ABROVET.

Hemangiosarcoma is a mesenchymal neoplasm originating in the endothelial cells of blood vessels; they can be classified as non-visceral and visceral types. Non-visceral hemangiosarcomas can affect the skin, subcutaneous tissues, and muscle tissues; visceral hemangiosarcomas can affect the spleen, liver, heart, lungs, kidneys, oral cavity, bones, bladder, uterus, tongue, and retroperitoneum. Among domestic species, dogs are most affected by cutaneous HSA. Cutaneous HSA represents approximately 14% of all HSA diagnosed in this species and less than 5% of dermal tumors, according to North American studies. However, Brazilian epidemiological data demonstrate a higher prevalence, which may represent 27 to 80% of all canine HSAs and 13.9% of all skin neoplasms diagnosed in this species. Cutaneous HSA most commonly affects middle-aged to elderly dogs (between 8 and 15 years old), with no gender predisposition for either the actinic or non-actinic forms. The higher prevalence of cutaneous HSA in some canine breeds is related to lower protection from solar radiation, as low skin pigmentation and hair coverage lead to greater sun exposure. Actinic changes, such as solar dermatosis, are frequent in these patients, confirming the influence of solar radiation on the development of this neoplasm. There are multiple clinical manifestations of hemangiosarcoma in canines. The diagnostic approach and staging classification of cutaneous HSAs are similar between the different subtypes. The definitive diagnosis is obtained through histopathological analysis of incisional or excisional biopsies. Cytology can be used as a presurgical screening test; however, it has little diagnostic utility in cases of HSA because there is a high risk of blood contamination and sample hemodilution. Surgery is generally the treatment of choice for dogs with localized non-visceral HSA without evidence of metastatic disease. Recently, electrochemotherapy (ECT) has emerged as an alternative therapy for the local ablative treatment of different neoplastic types; the use of radiotherapy for the treatment of dogs with cutaneous HSA is uncommon. There is greater consensus in the literature regarding the indications for adjuvant chemotherapy in subcutaneous and muscular HSA; doxorubicin is the most frequently used antineoplastic agent for subcutaneous and muscular subtypes and can be administered alone or in combination with other drugs. Other therapies include antiangiogenic therapy, photodynamic therapy, the association of chemotherapy with the metronomic dose, targeted therapies, and natural products. The benefits of these therapies are presented and discussed. In general, the prognosis of splenic and cardiac HSA is unfavorable. As a challenging neoplasm, studies of new protocols and treatment modalities are necessary to control this aggressive disease.

Unraveling the Risk Factors and Etiology of the Canine Oral Mucosal Melanoma: Results of an Epidemiological Questionnaire, Oral Microbiome Analysis and Investigation of Papillomavirus Infection.

Oral mucosal melanoma (OMM) is the most common oral cancer in dogs and is very aggressive in this species; its risk factors and etiology are yet to be determined. This study aimed to unravel the risk factors for the development of OMM in dogs and to investigate the possible presence of papillomaviruses as an etiological factor. A case-control study was conducted in 15 dogs with OMM and 15 paired controls whose owners answered an epidemiological questionnaire. Oral swabs from the same dogs were subjected to 16S rRNA sequencing for microbiome analyses. In addition, DNA fragments of OMM had their DNA extracted and amplified by polymerase chain reaction in an attempt to detect canine papillomaviruses. The gingiva was the most frequent anatomical site (47%) of OMM, and most tumors were stage III when diagnosed. Most dogs bearing OMM and the controls had grade 3 periodontal disease, and this factor, along with tartar treatment and tooth brushing, did not differ between cases and controls. Most dogs with OMM and most controls had contact with smokers; there was no statistically significant difference. Canine papillomaviruses were not detected among OMM cases. Tannerella forsythia and Porphyromonas gingivalis were significantly increased in case dogs compared to the controls. As these bacteria are reportedly involved in the pathogenesis of periodontal disease and esophageal cancer in humans, we suggest that they might be risk factors for the development of canine OMM. The limitations of this study include the low number of dogs, and therefore, further studies on canine OMM with larger numbers of animals are encouraged.

Vet-ICD-O-Canine-1, a System for Coding Canine Neoplasms Based on the Human ICD-O-3.2.

Cancer registries are fundamental tools for collecting epidemiological cancer data and developing cancer prevention and control strategies. While cancer registration is common in the human medical field, many attempts to develop animal cancer registries have been launched over time, but most have been discontinued. A pivotal aspect of cancer registration is the availability of cancer coding systems, as provided by the International Classification of Diseases for Oncology (ICD-O). Within the Global Initiative for Veterinary Cancer Surveillance (GIVCS), established to foster and coordinate animal cancer registration worldwide, a group of veterinary pathologists and epidemiologists developed a comparative coding system for canine neoplasms. Vet-ICD-O-canine-1 is compatible with the human ICD-O-3.2 and is consistent with the currently recognized classification schemes for canine tumors. It comprises 335 topography codes and 534 morphology codes. The same code as in ICD-O-3.2 was used for the majority of canine tumors showing a high level of similarity to their human counterparts (n = 408). De novo codes (n = 152) were created for specific canine tumor entities (n = 126) and topographic sites (n = 26). The Vet-ICD-O-canine-1 coding system represents a user-friendly, easily accessible, and comprehensive resource for developing a canine cancer registration system that will enable studies within the One Health space.

Imatinib Mesylate for the Treatment of Canine Mast Cell Tumors: Assessment of the Response and Adverse Events in Comparison with the Conventional Therapy with Vinblastine and Prednisone.

Mast cell tumors (MCTs) are common neoplasms in dogs, and treatments for these diseases include surgery, polychemotherapy and targeted therapy with tyrosine kinase inhibitors. This study aimed to evaluate the response and the adverse events of treatment with imatinib mesylate (IM) compared to conventional therapy using vinblastine and prednisolone (VP) in canine cutaneous MCTs. Twenty-four dogs were included in the study; 13 animals were treated with IM and 11 with VP. Tumor tissue samples were submitted for histological diagnosis, grading and KIT immunostaining. The response to treatment was assessed by tomographic measurements according to VCOG criteria. Adverse events were classified according to VCOG-CTCAE criteria. The IM and VP groups had dogs with similar breeds, gender, ages, MCT localization, WHO stages and lymph node metastasis profiles. Most MCTs were grade 2/low and had KIT- patterns 2 and 3. The objective response rate (ORR) was significantly higher (30.79%) in the IM group then in VP group (9.09%). Adverse events (AE) in IM group were all grade 1, significantly different from VP. In conclusion, IM presented better ORR and less severe adverse events when compared to VP, representing a suitable option for the treatment of low-grade canine MCTs.

Diagnosis, Prognosis and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors.

Mast cell tumors (MCTs) are hematopoietic neoplasms composed of mast cells. It is highly common in dogs and is extremely important in the veterinary oncology field. It represents the third most common tumor subtype, and is the most common malignant skin tumor in dogs, corresponding to 11% of skin cancer cases. The objective of this critical review was to present the report of the 2nd Consensus meeting on the Diagnosis, Prognosis, and Treatment of Canine Cutaneous and Subcutaneous Mast Cell Tumors, which was organized by the Brazilian Association of Veterinary Oncology (ABROVET) in August 2021. The most recent information on cutaneous and subcutaneous mast cell tumors in dogs is presented and discussed.

Towards progressive regulatory approaches for agricultural applications of animal biotechnology.

Traditional breeding techniques, applied incrementally over thousands of years, have yielded huge benefits in the characteristics of agricultural animals. This is a result of significant, measurable changes to the genomes of those animal species and breeds. Genome editing techniques may now be applied to achieve targeted DNA sequence alterations, with the potential to affect traits of interest to production of agricultural animals in just one generation. New opportunities arise to improve characteristics difficult to achieve or not amenable to traditional breeding, including disease resistance, and traits that can improve animal welfare, reduce environmental impact, or mitigate impacts of climate change. Countries and supranational institutions are in the process of defining regulatory approaches for genome edited animals and can benefit from sharing approaches and experiences to institute progressive policies in which regulatory oversight is scaled to the particular level of risk involved. To facilitate information sharing and discussion on animal biotechnology, an international community of researchers, developers, breeders, regulators, and communicators recently held a series of seven virtual workshop sessions on applications of biotechnology for animal agriculture, food and environmental safety assessment, regulatory approaches, and market and consumer acceptance. In this report, we summarize the topics presented in the workshop sessions, as well as discussions coming out of the breakout sessions. This is framed within the context of past and recent scientific and regulatory developments. This is a pivotal moment for determination of regulatory approaches and establishment of trust across the innovation through-chain, from researchers, developers, regulators, breeders, farmers through to consumers.

Methylene blue and photodynamic therapy for melanomas: Inducing different rates of cell death (necrosis and apoptosis) in B16-F10 melanoma cells according to methylene blue concentration and energy dose.

BACKGROUND: Photodynamic therapy (PDT) is a cancer treatment based on the interaction between the photosensitizing agent methylene blue (MB), light, and molecular oxygen. MB has antibacterial properties and can to bind to melanin. Here, we investigated whether MB based PDT (MB-PDT) could decrease viability and induce death of murine melanoma B16-F10 cells. METHODS: B16-F10 cells were incubated with different concentrations of MB (0, 1, or 2 µg/mL) and exposed to a diode red laser with a wavelength of 660 nm and power output of 100 mW/cm2. The energy dose and density varied from 0 J and 0 J/cm2 to 100.8 J and 720 J/cm². Cell viability was measured using the trypan blue exclusion assay of cell viability and confirmed by performing an MTT assay. The morphological type and cell death rates were determined using fluorescence microscopy with acridine orange and ethidium bromide. The presence and rate of apoptosis were evaluated via Annexin V-Alexa Fluor/propidium iodide staining and flow cytometry analysis. RESULTS: MB-PDT decreased cell viability and increased cell death (necrosis and apoptosis) in a drug- and light-dose dependent manner. Morphological characteristics of necrosis were observed immediately after treatment, and apoptotic characteristics were observed after 3 h. The apoptosis and necrosis rates varied with the drug and light doses, with 2 µg/mL MB and a 100.8 J energy dose inducing the highest rates. CONCLUSIONS: We demonstrated that MB-PDT reduced murine melanoma B16-F10 cell viability and induced cell death in a drug- and light-dose dependent manner.

Quantification of Global DNA Methylation in Canine Melanotic and Amelanotic Oral Mucosal Melanomas and Peripheral Blood Leukocytes From the Same Patients With OMM: First Study.

Oral mucosal melanomas (OMMs) are aggressive and resistant cancers of high importance in veterinary oncology. Amelanotic OMM produces comparatively less melanin and is considered to be more aggressive than melanotic OMM. Global DNA methylation profiles with hypomethylated or hypermethylated patterns have both been associated with aggressive neoplasms; however, global DNA hypomethylation seems to correlate to higher aggressiveness. Accordingly, global DNA methylation in peripheral blood leukocytes has been investigated to understand the role of systemic or environmental factors in cancer development. This study aimed to quantify global DNA methylation in canine melanotic and amelanotic OMM samples and in the peripheral blood leukocytes of the same dogs. Tumor tissue samples were collected from 38 dogs, of which 19 were melanotic and 19 were amelanotic OMM. These were submitted to immunohistochemistry (IHC) with anti-5-methylcytosine (5mC) and anti-Ki67 primary antibodies. Ki67- and 5mC-positive nuclei were manually scored with the help of an image analysis system. Peripheral blood samples were collected from 18 among the 38 OMM-bearing dogs and from 7 additional healthy control dogs. Peripheral blood leukocytes were isolated from the 25 dogs, and DNA was extracted and analyzed by high-performance liquid chromatography (HPLC) for global DNA methylation. The pattern of global DNA methylation in both canine melanotic and amelanotic OMM indicated higher percentages of weakly or negatively stained nuclei in most of the OMM cells, presuming predominant global DNA hypomethylation. In addition, Ki67 counts in amelanotic OMM were significantly higher than those in melanotic OMM (p < 0.001). Global DNA methylation different immunostaining patterns (strong, weak or negative) correlated with Ki67 scores. Global DNA methylation in circulating leukocytes did not differ between the 9 melanotic and 9 amelanotic OMM or between the 18 OMM-bearing dogs and the 7 healthy dogs. This study provides new information on canine melanotic and amelanotic OMM based on global DNA methylation and cell proliferation.

Intratumoral (Poly-ICLC) Therapy for Dogs with Advanced Cancers: First Report on Clinical Effectiveness, Quality of Life, and Adverse Events.

Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC) is a synthetic double-stranded viral RNA analog widely tested as a component of human therapeutic cancer vaccines and as a standalone agent for treating human cancers. However, there are no reports on the use of poly-ICLC for treating canine cancers. This study aimed to investigate the clinical efficacy, quality of life (QL), and adverse events of poly-ICLC treatment in dogs with advanced cancers. The treatment protocol consisted of weekly intratumoral doses of poly-ICLC. The canine patients underwent clinical, laboratory, and imaging tests, and their owners answered weekly QL questionnaires. Fourteen canine patients with different types of spontaneous advanced tumors were enrolled. Most dogs had received prior conventional therapies. Five dogs received at least 12 doses of poly-ICLC: the injected tumor was stable in three dogs, there was a partial response in one, and the injected tumor significantly enlarged in the other. The QL scoring remained stable or increased in most cases. Mild adverse events related to poly-ICLC were observed in 10 of the 14 patients. The data showed that intratumoral poly-ICLC therapy was well tolerated in dogs with advanced cancers, with clinical benefit and improved QL scores observed in some dogs.

Effects of Alpha-Connexin Carboxyl-Terminal Peptide (aCT1) and Bowman-Birk Protease Inhibitor (BBI) on Canine Oral Mucosal Melanoma (OMM) Cells.

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin-chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 µM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 µg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 µM) and BBI (400 µg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

Quantification of Global DNA Methylation in Canine Mammary Gland Tumors via Immunostaining of 5-Methylcytosine: Histopathological and Clinical Correlations.

Mammary tumors are the most prevalent neoplasms in non-neutered female dogs, with genetic and epigenetic alterations contributing to canine mammary carcinogenesis. This study quantified global DNA methylation in 5-methylcytosine (5mC)-immunostained canine mammary tumor samples and established histopathological and clinical correlations. A total of 91 formalin-fixed paraffin-embedded mammary tumor samples from female dogs were retrospectively selected and subjected to immunohistochemistry using an anti-5mC mouse monoclonal antibody. We evaluated 5mC+ stained nuclei of neoplastic epithelial cells in canine mammary glands to obtain semiquantitative histoscores based on staining intensity. Survival rates were estimated based on owners' or veterinary records. Histological samples comprised 28 and 63 benign and malignant canine mammary gland tumors, respectively. Results revealed significant differences between global DNA methylation patterns when mammary samples were categorized as benign or malignant (p = 0.024), with hypomethylated patterns more prevalent in malignant tumors and those with higher relapse behavior (p = 0.011). Of note, large diameter (>5 cm) tumors revealed a lower methylation pattern (p = 0.028). Additionally, we found non-statistically significant differences when tumors were grouped by histopathological characteristics, clinical parameters, or survival. These findings propose global DNA methylation assessment as a promising tool for detecting canine mammary tumors with relapse propensity.

Inhibitory Effects of a Reengineered Anthrax Toxin on Canine and Human Osteosarcoma Cells.

Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.

Inhibitory Effects of a Reengineered Anthrax Toxin on Canine Oral Mucosal Melanomas.

Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.