The expressions of mhc class i and class ii in biopsy tissues by immunohistochemistry and the effect of interferon-gamma (ifn-γ) on in vitro established canine cutaneous mast cell tumors

Canine mast cell tumors (MCT), common skin tumors in dogs, present difficulties in treatment, emphasizing the need for effective therapeutic protocols. The cancer-immunity cycle's complex interactions between cancer and the immune system play a crucial role in tumor growth. Tumors often evade immune response by downregulating MHC expression, hindering T-cell recognition. Cancer immunotherapy is a promising approach that utilizes the patient's immune system for combating cancer. Interferon-gamma (IFN-γ), a potent immune substance, plays a crucial role in enhancing MHC expression and immune functionality, crucial for T-cell recognition and cancer cell elimination. Limited research exists on MHC expression, tumor immune response, and IFN-γ impact in canine cutaneous MCT. To fulfill this research gap, we conducted three in vitro studies from canine MCT samples. Study 1 assessed MHC expression and its correlation with CD4+ and CD8+ T cells in canine cutaneous MCT tissues using immunohistochemistry (IHC). Results showed significantly higher MHC I and CD8 T lymphocyte levels in low grade MCT compared to high grade MCT. Although not statistically significant, low-grade MCT exhibited higher MHC II and CD4+ T lymphocytes. Positive correlations between MHC I and CD8 T lymphocytes, and between MHC II and CD4+ T lymphocytes were observed, suggesting a potential link in low-grade MCT. These findings support the notion that increased MHC expression in cancers correlates with elevated tumor-infiltrating lymphocytes, contributing to tumor control and indicating a more favorable prognosis. This study highlights the potential importance of MHC expression in canine cutaneous MCT for prognosis and therapy. Study 2 aimed to establish and characterize a novel in-house canine cutaneous MCT cell line, named C18, and compare it with three reference cell lines (CMMC, VIMC, CoMS). C18, derived from high grade cutaneous MCT, exhibited consistent gene expression, including c-kit, tryptase, FcεRIα, and FcεRIβ, maintained over 140 passages. In contrast to reference lines, The C18 showed the longest doubling time and the smallest spheroid size, indicating lower metabolic activity and growth. The C18 exhibited c-kit internal tandem duplication (ITD) in exon 11 and nine single nucleotide polymorphisms (SNPs) in five genes (c-kit, HYAL4, SEL1L, SPAM1, and TRAF3). In terms of tumor sensitivity, C18 showed more susceptibility to immune cell-mediated cytotoxicity compared to the other reference cell lines, suggesting its potential in cancer immunotherapy research. Study 3 optimized in vitro conditions to enhance MHC expression and assess cellular responses in canine MCT cell lines following IFN-γ treatment within an MCT-PBMCs co-culture system. All four lines, including the C18 cell line and three references, showed increased MHC expression with 10 IU/mL IFN-γ for 48 hours. IFN-γ induced apoptosis in MCT cell lines during co-culture with PBMCs, particularly at a high Target: Effector (T: E) ratio of 1:100, suggesting its anti-tumor potential for canine MCT. Variations in MHC expression and apoptosis rates were observed among cell lines, with cutaneous-origin lines exhibiting higher MHC expression and greater apoptosis propensity. These findings imply potential biological variations and treatment responses. The data highlight IFN-γ’s potential as an anti-cancer agent for canine MCTs, with co-culturing indicating increased apoptosis compared to untreated cells. In conclusion, our study highlights tumor heterogeneity in canine cutaneous MCT, noting higher MHC expression and increased lymphocyte infiltration in low grade cases. The established C18 cell line holds promise as an in vitro model for evaluating IFN-γ’s impact on MHC expression and studying various aspects of canine MCT. Additionally, our in vitro findings highlight variations among canine MCT cell lines in tumor characteristics and cellular responses to IFN-γ in an MCT-PBMCs co-culture system.