The melanoma-associated antigen gene (MAGE) is a key target in cancer immunotherapy. Given the potential of MAGE-B genes in veterinary immunotherapy for canine mammary tumors (CMTs), this study investigated the mRNA expression of MAGE-B1, -B4, -B5, and -B10 in CMT tissues and cells from dogs. Quantitative real-time PCR was used to analyze 28 CMT tissue samples, including 4 benign and 24 malignant tumors (13 simple carcinomas, 6 complex carcinomas, 3 carcinosarcomas, and 2 fibrosarcomas). Benign mixed tumor and complex carcinoma-type CMT cells were cultured and treated with a DNA methylase inhibitor (5-aza-2′-deoxycytidine; 5-aza-CdR) and a histone deacetylase inhibitor (Trichostatin A; TSA) under the following four conditions: (1) 5-aza-CdR for 72 h; (2) TSA for 24 h; (3) 5-aza-CdR for 48 h followed by TSA for 24 h; and (4) control. MAGE-B1 and -B4 showed the highest expression in the CMT samples (100% and 89.29%, respectively), followed by MAGE-B10 (82.14%). Carcinosarcomas and simple anaplastic carcinomas had significantly higher MAGE-B expression levels than simple tubulopapillary carcinomas (p < 0.05). 5-aza-CdR treatment increased MAGE-B expression, whereas TSA had a mild effect. Further research involving larger cohorts is needed to confirm these findings.

Melanoma-associated antigen-A (MAGE-A), a family of cancer/testis antigens, has been recognized as a potential target molecule for cancer immunotherapy. However, there has been very little information available with regard to this antigen in dogs. This study aimed to investigate the expression of MAGE-A in canine mammary gland tumors (CMTs) using immunohistochemistry and immunoblotting with human monoclonal MAGE-A antibody 6C1. The present study has provided evidence of cross-reactivity of the canine MAGE-A expression with the human MAGE-A antibody in CMTs. The MAGE-A antigens were expressed in moderate- and high-grade malignant CMTs (22.22%, 2/9), but no expression was observed in benign CMTs. The immunohistochemical staining of canine MAGE antigen in CMT cells showed nuclear and nuclear–cytoplasmic expression patterns that may be involved with the mitotic cell division of tumor cells. Molecular weights of the canine MAGE-A antigen presented in this study were approximately 42–62 kDa, which were close to those of other previous studies involving humans and dogs. The findings on this protein in CMTs could supply valuable oncological knowledge for the development of novel diagnostic, prognostic and immunotherapeutic tumor markers in veterinary medicine.

Metastasis-related disease is a major cause of death in canine mammary tumours (CMTs). Immunotherapy has been investigated due to the less successful outcomes of systemic therapy. This study aims to examine the expression of Programmed Cell Death Ligand-1 (PD-L1) in canine mammary tumours in dogs of Chiang Mai, Thailand, and determine the relationship between the level of mRNA expression and clinicopathologic characteristics. A total of 28 CMT samples were collected at the Small Animal Hospital, Chiang Mai University. Quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot assays were performed. The results revealed that all CMTs in this study expressed PD-L1 mRNA and PD-L1 protein. The mean relative mRNA expression showed no significant differences between groups categorized by age, tumour size, or histopathological findings. However, the mean relative mRNA expression in tumours with a TNM stage >3 was significantly lower compared to those with TNM stage ≤2. In conclusion, this study investigates the expression of PD-L1 mRNA and PD-L1 protein, particularly in malignant CMTs. The findings strongly support the potential for developing effective immunotherapy methods targeting the PD-1/PD-L1 pathway for advanced CMTs in the future. For further conclusive assessment, future studies should focus on refining immunotherapy strategies for CMT cases expressing PD-L1.