Demographic Patterns and Immunophenotypic Characterization of Tumor–Immune Interactions in Canine Cutaneous Mast Cell Tumors

Demographic Patterns and Immunophenotypic Characterization of Tumor–Immune Interactions in Canine Cutaneous Mast Cell Tumors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Demographic Patterns and Immunophenotypic Characterization of Tumor–Immune Interactions in Canine Cutaneous Mast Cell Tumors Sudchaya Bhanpattanakul, Theerawat Tharasanit, Achariya Sailasuta, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6766464/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Canine cutaneous mast cell tumors (MCTs) exhibit heterogeneous biological behaviors, with increasing evidence implicating the tumor immune microenvironment (TIME) in disease progression. This study evaluated biopsy-confirmed cases of canine MCTs to investigate associations between tumor grade and host factors, and to characterize grade-related differences in immune marker expression. Tumors were histologically graded by the Kiupel system and analyzed using immunohistochemistry for CD4, CD8, CD25, CD57, MHC I, MHC II, PD-1, PD-L1, and mast cell tryptase. Quantitative assessments included positive cell counts, percentages, H-scores, total stained areas, and inter-marker correlations. Demographic analysis revealed significant associations between tumor grade and both age and sex, whereas breed and anatomical location showed no statistical correlation. Low grade (LG) tumors demonstrated greater infiltration of CD4⁺, CD8⁺, CD57⁺ T cells and higher MHC I/II expression, consistent with enhanced antigen presentation and immune activity. In contrast, high grade (HG) tumors exhibited increased CD25⁺, PD-1⁺, and PD-L1⁺ expression, suggesting a more immunosuppressive phenotype. Correlation analysis highlighted coordinated immune marker expression in LG tumors and disrupted immune architecture in HG tumors. These findings reveal distinct immunophenotypic and demographic features across MCT grades and underscore the potential of immune profiling for prognostication and therapeutic planning in canine oncology. Biological sciences/Immunology Health sciences/Biomarkers Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Canine cutaneous mast cell tumors (MCTs) are among the most common malignant skin neoplasms in dogs, exhibiting wide biological heterogeneity that challenges prognostication and therapeutic planning 1 , 2 . While histologic grading systems, such as the Kiupel two-tier classification, offer valuable prognostic insight, increasing attention has turned to the tumor immune microenvironment (TIME) as a determinant of tumor behavior and therapeutic responsiveness 3 , 4 . In parallel, host-related factors such as age, sex, breed, and tumor location have also been investigated for their potential associations with MCT grade and biological behavior. However, comprehensive demographic analyses that integrate clinical variables with histologic and immunologic profiles have not been comprehensively addressed in the veterinary literature. The interaction between tumor cells and host immunity is regulated by a dynamic sequence of events, including antigen presentation, T cell activation, immune effector infiltration, and immune suppression. Antigen presentation is primarily mediated by major histocompatibility complex (MHC) class I and II molecules, which present tumor-derived peptides to CD8⁺ cytotoxic and CD4⁺ helper T lymphocytes, respectively. However, many tumors evade immune detection through the downregulation of MHC molecule expression, a well-recognized mechanism of immune escape 5 – 7 . Tumor-infiltrating lymphocytes (TILs) including CD4⁺, CD8⁺, CD25⁺ regulatory T cells (Tregs), and CD57⁺ terminal effector cells play essential roles in immune surveillance. Increased CD8⁺ and CD57⁺ cell infiltration is generally associated with favorable outcomes, while a high density of CD25⁺ Tregs is often linked to immunosuppressive environments and tumor progression 8 – 10 . Moreover, expression of immune checkpoint molecules such as PD-1 on T cells and PD-L1 on tumor or stromal cells contributes to T cell exhaustion and immune escape 11 – 13 . Despite increasing interest, the immune landscape of canine MCTs remains underexplored. Prior studies have reported immunologic parallels between canine and human tumors, including immune marker expression in canine melanoma, lymphoma, and mammary carcinoma 3 , 14 . However, comprehensive profiling of immune marker expression in relation to MCT grade has been limited. The objective of this study was to characterize the immunophenotypic profiles of canine cutaneous mast cell tumors (MCTs) using immunohistochemistry (IHC) for MHC I and II, CD4, CD8, CD25, CD57, PD-1, PD-L1, and mast cell tryptase. In addition, a demographic analysis of biopsy-confirmed cases was conducted to explore potential associations between tumor grade and host factors, including age, sex, breed, and anatomical tumor location. By quantitatively comparing immune marker expression between low-grade and high-grade tumors and assessing inter-marker relationships, this work aimed to elucidate grade-associated differences in immune infiltration, antigen presentation, and immune modulation. These findings may provide novel insights into MCT pathogenesis and support the development of immune-based diagnostic and therapeutic strategies in veterinary oncology. RESULTS 1. Demographic Correlations in Canine Mast Cell Tumors A total of 318 biopsy confirmed cases of MCTs were analyzed, comprising 234 low grade (LG; 73.6%) and 84 high-grade (HG; 26.4%) tumors. Distributions of MCT grades were evaluated across breed, age, sex, and anatomical location (Table 1 ). Mixed breed dogs represented the largest proportion, with 130 LG (55.6%) and 54 HG (64.3%) tumors. Among purebreds, French Bulldogs, Golden Retrievers, Shih Tzus, and Labrador Retrievers showed a higher prevalence of LG tumors. Notably, Chihuahuas displayed an equal representation of LG and HG tumors (5 cases each; 2.1%), suggesting a lack of grade-related predisposition in this breed. Most MCTs occurred in dogs aged 6–10 years (132 LG [56.4%], 35 HG [41.7%]), followed by those aged 11–15 years (70 LG [29.9%], 32 HG [38.1%]). Younger dogs (0–5 years) accounted for 27 LG (11.5%) and 10 HG (11.9%) cases, while very few cases were seen in dogs over 15 years of age. Female dogs comprised 112 LG (47.9%; 20 intact, 92 spayed) and 52 HG (61.9%; 16 intact, 36 spayed) cases, while males accounted for 122 LG (52.1%; 106 intact, 16 castrated) and 32 HG (38.1%; 30 intact, 2 castrated). A higher proportion of HG tumors was observed in spayed females compared to other sex categories. For the tumor location results, trunk was the most common site of MCTs (123 LG [52.6%], 33 HG [39.3%]), followed by hind limbs (36 LG [15.4%], 19 HG [22.6%]) and mammary glands (23 LG [9.8%], 12 HG [14.3%]). High-grade tumors were relatively overrepresented in inguinal, genital, and mammary regions, suggesting an association between anatomical site and tumor grade. Table 1 Demographic summary of canine cutaneous MCT cases Low grade (%) High grade (%) Breed Mixed 130 (40.88) 54 (16.98) French Bulldog 16 (5.03) 4 (1.25) Golden Retriever 13 (4.08) 1 (0.31) Shih tzu 10 (3.14) 4 (1.25) Labrador Retriever 11 (3.45) 3 (0.94) Thai Bangkaew 8 (2.51) 4 (1.25) Chihuahua 5 (1.57) 5 (1.57) Pug 9 (2.83) 1 (0.31) Others 32 (10.06) 8 (2.51) Age (years) 0–5 27 (8.5) 10 (3.14) 6–10 131 (41.51) 36 (11.01) 11–15 70 (22.01) 32 (10.06) 16–20 5 (1.57) 6 (1.88) 20 up 1 (3.14) 0 (0) Sex Male 106 (33.33) 30 (9.43) Castrated male 16 (5.03) 2 (0.62) Female 20 (6.28) 16 (5.03) Spayed female 92 (28.93) 36 (11.32) Tumor locations Trunk 123 (38.67) 33 (10.37) Hind limb 36 (11.32) 19 (5.97) Mammary gland 23 (7.23) 12 (3.77) Fore limb 18 (5.66) 8 (2.51) Scrotal area 18 (5.66) 7 (2.2) Head 13 (4.08) 3 (0.94) Genital (female) 3 (0.94) 2 (0.63) 2. Statistical Analysis of Demographic Correlations Chi-square tests were performed to assess the associations between mast cell tumor (MCT) grade and demographic factors, including breed, age, sex, and anatomical tumor location (Fig. 1 ). Age showed a statistically significant association with MCT grade (χ² = 8.37, p = 0.039). High-grade tumors were more frequently observed in dogs older than 10 years, particularly in the 11–15 year age group. Sex was significantly associated with tumor grade (χ² = 9.71, p = 0.021). High-grade tumors were more common in spayed females and intact males, indicating a possible hormonal influence on tumor progression. Breed was not significantly associated with MCT grade (χ² = 6.55, p = 0.256), although descriptive analysis suggested numerical differences across breeds. Tumor location did not show a statistically significant relationship with tumor grade (χ² = 6.45, p = 0.375), despite a higher proportion of high-grade tumors in mammary and genital regions. These results indicate that age and sex are statistically significant factors influencing the grade of canine MCTs in this cohort, while breed and tumor location, although clinically relevant, did not demonstrate statistical significance. 3. Quantitative Evaluation of Histochemical and Immunohistochemical Staining The extent and intensity of positive histochemistry and immunoreactivity for each marker demonstrated distinct variation between low-grade and high-grade mast cell tumors, indicating grade-associated differences in the tumor immune microenvironment (Supplementary File 2, Fig. S2 -6). Quantitative immunohistochemical analysis revealed distinct expression patterns of immune and mast cell–associated markers between LG and high-grade HG canine cutaneous mast cell tumors (Fig. 2 ). LG tumors demonstrated significantly higher infiltration of CD4⁺ and CD8⁺ T cells, with increased positive cell counts (p < 0.01), percentages (p < 0.01), H-scores (p < 0.001), and stained areas (p < 0.01). CD4 and CD8 localization in LG tumors was primarily cytoplasmic or membranous, whereas HG tumors exhibited scattered, focal, or nuclear staining. CD57⁺ cells were also significantly more abundant in LG tumors (p < 0.001), suggesting preserved immune surveillance. In contrast, CD25⁺ cells were markedly increased in HG tumors (p < 0.01), reflecting enhanced regulatory T cell (Treg) presence and potential immune suppression. Total nucleated cell counts were higher in LG tumors for CD4 (p < 0.01) but did not differ significantly for other markers. Tryptase expression was significantly greater in LG tumors across all parameters (p < 0.001), indicating higher granule retention or lower degranulation. Toluidine blue staining similarly showed increased mast cell granularity in LG lesions (p < 0.01), while localization data were limited. Antigen-presenting capacity was markedly reduced in HG tumors, as evidenced by lower expression of MHC class I and II molecules across cell count, percentage, H-score, and stained area metrics (p < 0.01). LG tumors displayed primarily cytoplasmic or membranous MHC localization, whereas HG tumors exhibited more heterogeneous or compartmentalized patterns. Conversely, immune checkpoint markers PD-1 and PD-L1 were significantly upregulated in HG tumors across all measured parameters (p < 0.001). PD-1 localization was cytoplasmic in LG tumors but nuclear or mixed in HG lesions, while PD-L1 staining in HG tumors was predominantly membranous. These patterns are indicative of enhanced immune checkpoint activation and immune evasion in HG MCTs. Overall, LG tumors exhibited more strong immune infiltration, preserved antigen presentation, and mast cell granularity, whereas HG tumors showed increased immune suppression and altered immunoarchitecture. 4. Correlation Patterns of Immune Marker Expression in Low- and High-Grade Mast Cell Tumors To explore the relationships among immunologic and histologic markers within the tumor microenvironment, Spearman’s rank correlation analysis was conducted on five key immunohistochemical parameters: number of positive cells, percentage of positive cells, H-score, total immunoreactive area, and total nucleated cells (Figs. 3 and 4 ). In low-grade MCTs, multiple strong and statistically significant correlations were observed among T-cell–associated markers (CD4, CD8, CD25) and antigen-presenting molecules (MHC class I and II). Specifically, CD4 and CD25 demonstrated consistently high correlation coefficients across all parameters (ρ = 0.63–0.78, p < 0.01), supporting the notion of synchronized helper and regulatory T-cell infiltration. CD8 also showed moderate-to-strong correlations with both MHC I and II in positive cell count and H-score analyses (ρ > 0.5, p < 0.05), indicative of coordinated cytotoxic T-cell activity in low-grade tumors. MHC II further correlated strongly with total nucleated cell counts and positive cell density, reinforcing its role in antigen presentation within structured immune contexts. Conversely, markers such as CD57 and mast cell tryptase demonstrated relatively weak or non-significant correlations with T-cell markers, suggesting compartmentalized expression unrelated to adaptive immune infiltration. In contrast, high-grade MCTs exhibited a markedly attenuated correlation profile. While some associations between CD4, CD8, and MHC molecules persisted, the overall magnitude and significance of correlations were reduced. CD8 expression displayed weak or negligible correlations with MHC markers and CD4 in several parameters (ρ 0.05), suggesting a breakdown of coordinated T-cell response. PD-1 and PD-L1 showed inconsistent or modest correlations with T-cell markers in both grades, although slightly higher PD-L1 inter-marker correlations were observed in high-grade cases, potentially reflecting immune escape mechanisms or stromal remodeling. A comparison of total nucleated cell correlations further underscored these distinctions. In low-grade MCTs, total nucleated counts were moderately correlated across markers especially between CD4, CD25, and MHC II, consistent with organized immune infiltration. In high-grade tumors, these associations were weak and non-significant, suggesting greater variability in immune cell distribution and density within the tumor microenvironment. Overall, this comparative correlation analysis reveals distinct immunoarchitectural dynamics between tumor grades (Fig. 5 ). Low-grade MCTs exhibit well-structured and synchronous immune marker expression, characteristic of coordinated host immune surveillance. In contrast, high-grade MCTs are defined by fragmented or decoupled marker relationships, which may reflect immune evasion, loss of immune control, or increased microenvironmental heterogeneity. This comparative analysis highlights marker relationships that may differ with tumor grade and reflect underlying immunopathological differences between indolent and aggressive MCTs. Discussion The analysis of 318 canine mast cell tumor (MCT) cases revealed significant associations between tumor grade and host factors, particularly age and sex, while breed and tumor location were not statistically significant. Chi-square analysis confirmed that age (p = 0.039) and sex (p = 0.021) were significantly associated with tumor grade. High-grade tumors were more common in dogs over 10 years of age, supporting the notion that age-related processes such as chronic inflammation and genomic instability may promote malignancy 15 . Sex-based trends showed a greater prevalence of high-grade tumors in spayed females and intact males, aligning with prior studies suggesting hormonal influences may modulate tumor progression, although mechanistic pathways remain unclear 16 . Breed distribution showed numerical differences, with mixed breeds comprising a large proportion of both grades and purebreds such as French Bulldogs and Golden Retrievers more often affected by low-grade tumors. However, the lack of statistical significance (p = 0.256) suggests breed alone may not reliably predict tumor grade, contrasting with previous reports of breed predisposition 1 , 15 . Mixed-breed dogs account for a substantial proportion of MCT cases in Thailand, reflecting their overall high tumor incidence. Studies from Bangkok, Chiang Mai, and Chonburi consistently identify MCTs as the most common cutaneous neoplasms, with mixed-breed dogs frequently affected alongside older and male dogs 17 – 19 . These findings underscore the need to include mixed-breed populations in MCT surveillance and research efforts in Southeast Asia. Tumor location, though not statistically significant (p = 0.375), showed a tendency for high-grade tumors to occur more frequently in genital and mammary regions. These findings are consistent with reports indicating site-specific biological behavior possibly influenced by local vascularization or immune responses 20 . Collectively, the data underscore the prognostic value of age and sex in canine MCTs and highlight the potential clinical relevance of integrating demographic factors into diagnostic and treatment strategies. This study provides a comprehensive immunophenotypic comparison between low-grade and high-grade canine cutaneous mast cell tumors (MCTs), highlighting significant differences in immune cell infiltration, antigen presentation, and immune regulatory marker expression. LG tumors were characterized by increased numbers and proportions of CD4+, CD8+, CD57+, and mast cell tryptase positive cells, along with elevated H-scores and staining areas, indicating a more immunologically active tumor microenvironment. These findings are consistent with prior studies demonstrating that strong T-cell infiltration, particularly of CD4 + helper and CD8 + cytotoxic subsets, is associated with better differentiation and less aggressive tumor behavior in both canine and human malignancies 21 , 22 . The high CD57 + cell counts in LG MCTs may reflect greater recruitment or retention of terminally differentiated effector lymphocytes, supporting an intact anti-tumor immune response 9 . The increased mast cell tryptase expression in LG tumors aligns with previous observations that well-differentiated mast cells with abundant granules are typical of lower-grade lesions, while HG tumors often show degranulation or loss of tryptase immunoreactivity 2 . Toluidine Blue staining further confirmed these differences in granularity, reinforcing the correlation between histologic grade and mast cell cytologic integrity. Conversely, HG tumors exhibited significantly higher expression of CD25, PD-1, and PD-L1, markers commonly associated with immunosuppression and regulatory cell function. Elevated CD25 expression in HG MCTs may reflect increased infiltration of regulatory T cells (Tregs), which are known to suppress effective anti-tumor responses and correlate with poor prognosis in various neoplasms 10 , 23 . Likewise, the overexpression of PD-1 and its ligand PD-L1 in HG tumors suggests activation of immune checkpoint pathways, likely contributing to immune evasion. Such expression patterns have been documented in canine MCTs and other solid tumors, with implications for both prognosis and therapeutic targeting 23 – 25 . Notably, both MHC class I and II molecules were significantly downregulated in HG tumors, with altered localization patterns. These findings imply a reduced capacity for tumor antigen presentation and T-cell priming, consistent with mechanisms of immune escape. Decreased MHC I/II expression has been described in aggressive canine MCTs 26 and in other immune-evasive tumor models 27 . The nuclear and heterogeneous cytoplasmic localization of MHC molecules and immune markers observed in HG lesions further supports disrupted immune signaling or trafficking, which may affect their immunogenicity and therapeutic responsiveness. The overall pattern of immune marker expression underscore distinct immune microenvironments between LG and HG MCTs. LG tumors appear to maintain a pro-inflammatory, immunologically engaged phenotype, while HG tumors shift toward immune modulation and suppression, potentially facilitating malignant progression. These differences support the utility of immunohistochemical profiling not only for diagnostic refinement but also for identifying candidates for immunotherapeutic intervention, particularly in high-grade cases with elevated PD-L1 or Treg markers. Further studies are warranted to explore the prognostic and therapeutic implications of these immune markers in canine MCTs, especially in relation to clinical outcomes and response to emerging immunotherapies. The tumor immune microenvironment plays a pivotal role in the biological behavior of mast cell tumors in dogs. In the present study, we applied Spearman’s rank correlation analysis to assess interrelationships among key immune markers based on positive cell counts, percentage positivity, H-score, total immunoreactive area, and total nucleated cells in low- and high-grade canine cutaneous MCTs. The analysis revealed distinct immunoarchitectural patterns between tumor grades, reflecting differences in immune coordination, regulatory signaling, and potential mechanisms of immune evasion. In low grade MCTs, strong and consistent correlations were observed among CD4, CD8, CD25, and MHC class II expression, particularly in positive cell counts and H-scores. These findings suggest a well-organized immune response comprising helper T cells (CD4+), cytotoxic T cells (CD8+), and regulatory T cells (CD25+), accompanied by antigen presentation via MHC II molecules. Such coordinated immune cell infiltration has been associated with effective immunosurveillance and tumor containment in various neoplastic settings 28 , 29 . The significant positive correlation between CD4 and CD25 may reflect the dual roles of T-helper and T-regulatory populations, both of which have been reported in canine MCTs and other tumors 30 . In contrast, high grade MCTs demonstrated fragmented correlation patterns with lower ρ values and fewer statistically significant associations. The loss of correlation between CD8 and MHC I/II may indicate impaired antigen presentation or dysfunctional T-cell recruitment, features commonly observed in tumors that evade immune destruction 31 . Notably, while PD-1 and PD-L1 expression showed only modest and inconsistent correlation with T-cell markers, PD-L1 levels were moderately elevated and correlated with tryptase and total area in high-grade tumors, supporting the involvement of immune checkpoint pathways in tumor progression and immune evasion 12 , 23 . These results consistent with previous reports of PD-L1 upregulation in more aggressive MCTs 32 . When total nucleated cell density was considered, low grade tumors again showed stronger inter-marker correlations, reinforcing the presence of a more homogeneous and structured immune infiltrate. In contrast, the lack of significant correlations in high-grade tumors may reflect cellular disorganization, immune suppression, or clonal expansion of neoplastic mast cells that outpace host immune regulation 33 . The cumulative evidence emphasizes that low grade MCTs are characterized by a synchronized and immunologically active microenvironment, whereas high grade tumors demonstrate a breakdown of coordinated immune responses. The loss of immune marker interdependence in high-grade tumors may reflect immune escape mechanisms, altered cytokine profiles, or tumor-driven modulation of infiltrating immune cells. Correlation-based analyses offer complementary insight beyond individual marker expression, revealing the architectural and functional relationships among immune components in situ. Further studies incorporating spatial transcriptomics or multiplex immunofluorescence could enhance the resolution of tumor–immune interactions and elucidate the causal underpinnings of immune coordination versus disintegration in MCT progression. From a translational standpoint, the identification of strong marker correlations in low-grade tumors may support immunotherapeutic targeting, while disrupted marker networks in high-grade lesions could serve as biomarkers of immune escape or refractoriness to checkpoint inhibition. Materials and Methods 1. Demographic study of canine mast cell tumors This study included 318 biopsy-confirmed cases of canine cutaneous mast cell tumors (MCTs) diagnosed between 2020 and 2024 at the Small Animal Teaching Hospital, Chulalongkorn University, Thailand. All specimens used in this study were archived formalin-fixed, paraffin-embedded (FFPE) tissue samples that had been previously submitted for diagnostic purposes. No live animals were contacted, sampled, or subjected to any intervention specifically for the purpose of this research. Clinical data including signalment, diagnostic findings, and tumor history were retrieved retrospectively from hospital records. Both high-grade and low-grade MCTs were analyzed to assess potential associations between tumor grade and demographic variables, including sex, age, breed, and anatomical tumor location. Cases were excluded if they involved concurrent tumors confirmed histologically, or if histological sections or staging data for MCT were unavailable. Tumor grading was performed by two board-certified veterinary pathologists based on the Kiupel grading system for canine cutaneous MCTs 2 , using light microscopy. High-grade MCTs were defined by the presence of at least one of the following criteria in 10 high-power fields (HPFs): ≥7 mitotic figures, ≥ 3 multinucleated cells (each with ≥ 3 nuclei), ≥ 3 bizarre nuclei, or karyomegaly affecting ≥ 10% of neoplastic cells with ≥ 2-fold variation in nuclear diameter. Tumors not meeting these criteria were classified as low-grade (Supplementary File 2, Fig. S1 ). Clinical data including signalment, diagnostic findings, tumor history were retrieved from hospital records. Chi-square tests were used to evaluate associations between MCT grade and demographic variables. Statistical significance was determined to assess whether the distribution of these characteristics differed between high- and low-grade tumors. 2. Histochemical identification of mast cells using toluidine blue staining Toluidine blue staining was employed to visualize mast cells in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Sections were cut at a thickness of 4–6 µm, deparaffinized in xylene, and rehydrated through a graded ethanol series into distilled water. Staining was performed using 0.1% toluidine blue O solution (Sigma-Aldrich, Cat. No. T3260) for 2–5 minutes at room temperature, followed by a brief rinse in distilled water. Slides were subsequently dehydrated in graded ethanol, cleared in xylene, and cover slipped with a resinous mounting medium. Toluidine blue is a basic metachromatic dye that selectively stains acidic tissue components, particularly sulfated glycosaminoglycans found in mast cell granules. This results in characteristic metachromatic staining, with mast cell granules appearing purple to red-purple, while nuclei and other basophilic structures are stained blue (Supplementary File 2, Fig. S2 ). 3. Immunohistochemical detection of cellular markers in canine mast cell tumors Formalin-fixed, paraffin-embedded (FFPE) tissue specimens were sectioned at 4 µm thickness using a rotary microtome (Shandon, Anglia Scientific Instrument Ltd., Cambridge, UK). Slides were incubated at 60°C for 24 hours to facilitate tissue adherence and preparation for immunostaining. Deparaffinization was performed using xylene, followed by rehydration through a graded ethanol series. Antigen retrieval was conducted by immersing tissue sections in 0.01 M citrate buffer (pH 6.0) and heating them in a microwave oven at 750 W for 10 minutes (two 5-minute intervals). Endogenous peroxidase activity was quenched by treating the sections with 3% hydrogen peroxide (H₂O₂) in methanol for 10 minutes. Non-specific antibody binding was minimized by incubating the slides with normal horse serum (Vector Laboratories, Burlingame, CA, USA). Primary antibodies used in this study are listed in Supplementary File 1, Table S1 . Sections were incubated with primary antibodies overnight at 4°C, followed by incubation with biotinylated secondary antibodies (Vector Laboratories; dilution 1:200). Detection was carried out using the avidin-biotin-peroxidase complex (Vectastain® Elite ABC Kit, Vector Laboratories) for 30 minutes at room temperature. Visualization was achieved by applying 3,3′-diaminobenzidine tetrahydrochloride (ImmPACT® DAB, Vector Laboratories) for 5 minutes. Slides were then counterstained with Mayer’s hematoxylin. Negative controls were processed in parallel by substituting the primary antibody with phosphate-buffered saline (PBS). 4. Quantitative evaluation of histochemical and immunohistochemical staining For each tissue section, 10 random high-power field (HPF) images were acquired using an Olympus BX51TRF light microscope equipped with cellSens imaging software (Olympus Corporation, Tokyo, Japan). Immunohistochemical images were analyzed using ImageJ/Fiji software (version 2.14.0/1.54f; National Institutes of Health, Bethesda, MD, USA). Color deconvolution was applied to isolate DAB and hematoxylin staining, and positive immunoreactivity was quantified through manual threshold. In each field, total nucleated cells and positively stained cells were enumerated, and the mean H-score from at least 10 non-overlapping fields per sample was used for statistical evaluation. Immunostaining intensity and extent were assessed using the H-score method, calculated as: H-score = (1 × % weakly stained cells) + (2 × % moderately stained cells) + (3 × % strongly stained cells). This calculation provides a continuous score ranging from 0 to 300. Additionally, immunoreactivity patterns (nuclear, cytoplasmic, or membranous) and the spatial distribution of positive cells (e.g., peripheral vs. central tumor zones) were documented. All image analyses were conducted independently by two pathologists who were blinded to the experimental group assignments. 5. Statistical Analysis All statistical analyses were performed using GraphPad Prism software, version 10 (San Diego, CA, USA). Data are presented as mean ± standard error (SE). Comparisons between two groups were conducted using unpaired two-tailed Student’s t-test for parametric data and appropriate non-parametric tests where applicable. Associations between mast cell tumor grade and categorical variables including breed, age group, sex, and anatomical tumor location were assessed using Chi-square tests. Correlations between MHC expression levels, immune cell markers, and PD expression were evaluated using Spearman’s rank correlation analysis. A p-value < 0.05 was considered statistically significant for all tests. Declarations Acknowledgements We would like to express our gratitude to the staff of the Department of Veterinary Pathology and veterinarians at the Small Animal Teaching Hospital, Chulalongkorn University, Bangkok, Thailand, for their assistance with biopsy sample collection and valuable clinical data. Author contributions TK and TT conceptualised and supervised this study. SB performed the experiments. TK and SB analysed the data. TN and AS provided canine MCT cell lines. TK and SB drafted the manuscript. TK and TT reviewed and edited the original draft. All authors read and approved the final manuscript. Fundings This research was supported by the National Research Council of Thailand (N41D640002), the Chulalongkorn University Graduate Scholarship to the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, the 90th Anniversary of Chulalongkorn University, Rachadapisek Sompote Endowment Fund 2019 (CU_GR_62_77_31_07) and the Center of Excellence for Companion Animal Cancer (CE-CAC). Data availability The data used in this study are available from the corresponding author upon reasonable request Ethics declarations All tissue samples were obtained from archived diagnostic specimens submitted to the Small Animal Teaching Hospital, Chulalongkorn University. No animals were prospectively recruited, sampled, or handled for the specific purpose of this study. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Chulalongkorn University (Protocol No. 2131049), in compliance with ARRIVE guidelines. Competing interest The authors declare no competing interests. References Blackwood L et al . European consensus document on mast cell tumours in dogs and cats. Vet Comp Oncol. 10 , e1–e29 (2012). https://doi.org/10.1111/j.1476-5829.2012.00341.x Kiupel M et al. Proposal for a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. Vet Pathol 48 , 147–155 (2011). https://doi.org/10.1177/0300985810386469 Flesner BK et al . Immunotherapeutic approaches in canine oncology. Vet Sci 8 , 197 (2021). https://doi.org/10.3390/vetsci8090197 Weishaar KM et al. The tumor immune microenvironment in canine mast cell tumors: implications for therapeutic intervention. Vet Immunol Immunopathol 201 , 21–27 (2018). https://doi.org/10.1016/j.vetimm.2018.05.002 Carretero R et al. Regression of melanoma metastases after MHC class I upregulation by IFN-γ in vivo. Cancer Immunol Immunother . 57 , 1441–1450 (2008). https://doi.org/10.1007/s00262-008-0472-2 Goeppert B et al. Down-regulation of MHC class I is a frequent mechanism of immune escape and a negative prognostic marker in Merkel cell carcinoma. Int J Cancer 137 , 1597–1606 (2015). https://doi.org/10.1002/ijc.29520 Rimsza LM et al. Loss of MHC class II gene and protein expression in diffuse large B-cell lymphoma is related to decreased tumor immunosurveillance and poor patient survival. Blood 103 , 4251–4258 (2004). https://doi.org/10.1182/blood-2003-10-3437 Curiel TJ et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10 , 942–949 (2004). https://doi.org/10.1038/nm1093 Gerlini G et al. Human CD8⁺CD57⁺ lymphocytes accumulate in neoplastic lymph nodes and have cytotoxic activity against autologous melanoma. J Invest Dermatol 122 , 1350–1354 (2004). https://doi.org/10.1111/j.0022-202X.2004.22512.x Sato E et al. Intraepithelial CD8⁺ tumor-infiltrating lymphocytes and a high CD8⁺/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci USA 102 , 18538–18543 (2005). https://doi.org/10.1073/pnas.0509182102 Maekawa N et al. A canine chimeric anti-PD-L1 monoclonal antibody for immunotherapy of canine cancers. Sci Rep 10 , 2952 (2020). https://doi.org/10.1038/s41598-020-59838-6 Thompson MJ et al. Immune checkpoint molecule expression in high-grade canine mast cell tumors. Vet Comp Oncol 14 , 417–428 (2016). https://doi.org/10.1111/vco.12123 Topalian SL et al. Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. N Engl J Med 366 , 2443–2454 (2012). https://doi.org/10.1056/NEJMoa1200690 Porcellato I et al. Foxp3 and IDO1 expression in canine melanocytic tumors. Vet Pathol 56 , 371–379 (2019). https://doi.org/10.1177/0300985819828725 Welle MM et al. Canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment. Vet Dermatol 19 , 321–339 (2008). Dobson JM & Scase TJ Advances in the diagnosis and management of cutaneous mast cell tumours in dogs. J Small Anim Pract 48 , 424–431 (2007). Janma J et al. Prevalence of canine tumors in Nong Chok, Bangkok (2018–2023). ResearchGate . (2023). https://www.researchgate.net/publication/386254446 Klinhom S et al. Retrospective study of risk factors and prevalence of canine mammary tumors in Chiang Mai, Thailand. PeerJ 11 , e17077 (2023). https://doi.org/10.7717/peerj.17077 Worasitthichai S et al. Incidence of cutaneous tumors in dogs in eastern Thailand: A retrospective study. Vet Integr Sci 18 , 225–237 (2020). https://he02.tci-thaijo.org/index.php/vis/article/download/242788/166241 Villamil JA et al. Hormonal and sex impact on the epidemiology of canine lymphoma. J Cancer Epidemiol 2011 , 1–7 (2011). Letellier E & Haan S SOCS proteins in development and disease. J Cell Biol 183 , 575–585 (2008). https://doi.org/10.1083/jcb.200808124 Willmann M et al. T-cell infiltration in canine cutaneous mast cell tumors: a role of regulatory T cells in promoting tumor progression?. Vet Immunol Immunopathol 208 , 21–30 (2019). https://doi.org/10.1016/j.vetimm.2018.12.006 Maekawa N et al. Immunohistochemical analysis of PD-L1 expression in canine tumors. J Vet Med Sci 78 , 1473–1477 (2016). https://doi.org/10.1292/jvms.16-0081 Maekawa N et al. Prognostic significance of programmed cell death ligand-1 expression and tumor-infiltrating lymphocytes in canine cutaneous mast cell tumors. Vet Pathol 58 , 98–107 (2021). https://doi.org/10.1177/0300985820964673 Rütgen BC et al. Immunohistochemical evaluation of the tumor microenvironment in canine cutaneous mast cell tumors of different histologic grades. Vet Immunol Immunopathol 195 , 11–19 (2018). https://doi.org/10.1016/j.vetimm.2017.12.005 Takeuchi Y et al. Roles of regulatory T cells in cancer immunity. Int Immunol 25 , 69–77 (2013). https://doi.org/10.1093/intimm/dxs099 Zitvogel L et al. Immune parameters affecting the efficacy of chemotherapeutic regimens. Nat Rev Clin Oncol 3 , 661–670 (2006). https://doi.org/10.1038/ncponc0649 Fridman WH et al. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12 , 298–306 (2012). https://doi.org/10.1038/nrc3245 O’Sullivan T et al . Cancer immunoediting by the innate immune system in the absence of adaptive immunity. J Exp Med 216 , 1373–1385 (2019). https://doi.org/10.1084/jem.20181941 Marconato L et al. The dog as a possible animal model for human aggressive B-cell lymphoma: a review. Hematol Oncol 31 , 1–9 (2013). https://doi.org/10.1002/hon.2002 Hanahan D & Weinberg RA Hallmarks of cancer: the next generation. Cell 144 , 646–674 (2011). https://doi.org/10.1016/j.cell.2011.02.013 Yoshimura H et al. Programmed death-ligand 1 expression in canine tumors and its association with prognosis in canine oral melanoma. Vet Immunol Immunopathol 227 , 110089 (2020). https://doi.org/10.1016/j.vetimm.2020.110089 Scase TJ et al . Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis. J Vet Intern Med 23 , 251–258 (2009). https://doi.org/10.1111/j.1939-1676.2008.0263.x Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationfile1KaewamatawongMCT01.pdf SupplementaryInformationfile2KaewamatawongMCT01.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":114779,"visible":true,"origin":"","legend":"\u003cp\u003eChi-square test results illustrating the statistical association between demographic variables and mast cell tumor (MCT) grade in dogs. Bar graph displaying p-values from chi-square tests evaluating the relationship between MCT grade (low vs. high) and four demographic variables: breed, age, sex, and tumor location. Bars colored in green indicate statistically significant associations (p \u0026lt; 0.05), while those in blue indicate non-significant associations (p ≥ 0.05). The red dashed line represents the conventional threshold for statistical significance (p = 0.05).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/4def39193da20811527a8009.png"},{"id":94397057,"identity":"b9946c38-bbb6-42b2-a780-feffeff289c9","added_by":"auto","created_at":"2025-10-27 13:56:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120632,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative comparison of immunohistochemical parameters between low-grade (LG) and high-grade (HG) canine mast cell tumors. Bar graphs depict the mean values ± standard error (SE) for five quantitative parameters across various immunohistochemical markers: positive cell count, total nucleated cells, percentage of positive cells, H-score (staining intensity), and total stained area (µm²). Each marker (CD4, CD8, CD25, CD57, MHC I, MHC II, PD-1, PD-L1, and toluidine blue) is compared between LG and HG tumors. Asterisks (*) indicate statistically significant differences between groups (p \u0026lt; 0.05, p \u0026lt; 0.01, p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/5c495d082f7d5a8945007bed.png"},{"id":94397622,"identity":"67dad858-58f0-4233-b077-7de06e049869","added_by":"auto","created_at":"2025-10-27 13:56:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":275450,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman’s rank correlation among immunohistochemical parameters in low-grade canine cutaneous mast cell tumors (MCTs). Bar plots depict pairwise Spearman correlationปรับcoefficients (ρ) between markers based on: (A) number of positive cells, (B) total nucleated cells, (C) percentage of positive cells, (D) H-score and (E) total immunoreactive area (μm²). Correlation values are shown on the y-axis, and each bar represents the correlation between two distinct markers. Statistical significance is annotated using asterisks (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/1984255e4deaaf81ab7de486.png"},{"id":94398868,"identity":"bc23cf74-cbe7-4e10-8682-375ac6249d76","added_by":"auto","created_at":"2025-10-27 13:57:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":275161,"visible":true,"origin":"","legend":"\u003cp\u003eSpearman’s rank correlation among immunohistochemical parameters in high-grade canine cutaneous mast cell tumors (MCTs). Pairwise correlations among markers are visualized based on: (A) number of positive cells, (B) percentage of positive cells,(C) H-score, (D) total immunoreactive area (μm²), and(E) total nucleated cells.Each plot presents Spearman correlation coefficients (ρ) with significance levels indicated (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/7544d5326dd52fb6228f887d.png"},{"id":94397056,"identity":"5fd7a94c-e437-4ab7-8495-cf1c348e8cd3","added_by":"auto","created_at":"2025-10-27 13:56:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217433,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Spearman’s rank correlation coefficients (ρ) for immune and histologic markers between low-grade and high-grade canine cutaneous mast cell tumors. Bar plots illustrate the pairwise Spearman correlation values among markers within each of the following parameters: (A) number of positive cells, (B) percentage of positive cells, (C) H-score, (D) total immunoreactive area (μm²), and (E) total nucleated cells. Each pairwise comparison is shown separately for low-grade (green) and high-grade (purple) tumors. The height of each bar represents the correlation coefficient (ρ), and statistical significance is annotated with asterisks (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/e093574fdaab4fb89af70a39.png"},{"id":95801236,"identity":"fca1ae97-814e-45ae-b34b-320080228f42","added_by":"auto","created_at":"2025-11-13 08:24:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1854892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/e62a5c57-abc0-4e2b-8cb0-8cc158fe5ca9.pdf"},{"id":94396657,"identity":"6b5458d6-7cb0-48e7-99cb-35d875797183","added_by":"auto","created_at":"2025-10-27 13:56:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":138083,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationfile1KaewamatawongMCT01.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/8b01020d171653fedc851ee2.pdf"},{"id":94398901,"identity":"d0f755f2-b5ca-4767-bff8-a068e66a8786","added_by":"auto","created_at":"2025-10-27 13:57:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1302393,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationfile2KaewamatawongMCT01.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6766464/v1/5f9e974f597cf550b8703dd1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Demographic Patterns and Immunophenotypic Characterization of Tumor–Immune Interactions in Canine Cutaneous Mast Cell Tumors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCanine cutaneous mast cell tumors (MCTs) are among the most common malignant skin neoplasms in dogs, exhibiting wide biological heterogeneity that challenges prognostication and therapeutic planning\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. While histologic grading systems, such as the Kiupel two-tier classification, offer valuable prognostic insight, increasing attention has turned to the tumor immune microenvironment (TIME) as a determinant of tumor behavior and therapeutic responsiveness\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In parallel, host-related factors such as age, sex, breed, and tumor location have also been investigated for their potential associations with MCT grade and biological behavior. However, comprehensive demographic analyses that integrate clinical variables with histologic and immunologic profiles have not been comprehensively addressed in the veterinary literature.\u003c/p\u003e\u003cp\u003eThe interaction between tumor cells and host immunity is regulated by a dynamic sequence of events, including antigen presentation, T cell activation, immune effector infiltration, and immune suppression. Antigen presentation is primarily mediated by major histocompatibility complex (MHC) class I and II molecules, which present tumor-derived peptides to CD8⁺ cytotoxic and CD4⁺ helper T lymphocytes, respectively. However, many tumors evade immune detection through the downregulation of MHC molecule expression, a well-recognized mechanism of immune escape\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTumor-infiltrating lymphocytes (TILs) including CD4⁺, CD8⁺, CD25⁺ regulatory T cells (Tregs), and CD57⁺ terminal effector cells play essential roles in immune surveillance. Increased CD8⁺ and CD57⁺ cell infiltration is generally associated with favorable outcomes, while a high density of CD25⁺ Tregs is often linked to immunosuppressive environments and tumor progression\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Moreover, expression of immune checkpoint molecules such as PD-1 on T cells and PD-L1 on tumor or stromal cells contributes to T cell exhaustion and immune escape\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite increasing interest, the immune landscape of canine MCTs remains underexplored. Prior studies have reported immunologic parallels between canine and human tumors, including immune marker expression in canine melanoma, lymphoma, and mammary carcinoma\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, comprehensive profiling of immune marker expression in relation to MCT grade has been limited.\u003c/p\u003e\u003cp\u003eThe objective of this study was to characterize the immunophenotypic profiles of canine cutaneous mast cell tumors (MCTs) using immunohistochemistry (IHC) for MHC I and II, CD4, CD8, CD25, CD57, PD-1, PD-L1, and mast cell tryptase. In addition, a demographic analysis of biopsy-confirmed cases was conducted to explore potential associations between tumor grade and host factors, including age, sex, breed, and anatomical tumor location. By quantitatively comparing immune marker expression between low-grade and high-grade tumors and assessing inter-marker relationships, this work aimed to elucidate grade-associated differences in immune infiltration, antigen presentation, and immune modulation. These findings may provide novel insights into MCT pathogenesis and support the development of immune-based diagnostic and therapeutic strategies in veterinary oncology.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1. Demographic Correlations in Canine Mast Cell Tumors\u003c/h2\u003e\u003cp\u003eA total of 318 biopsy confirmed cases of MCTs were analyzed, comprising 234 low grade (LG; 73.6%) and 84 high-grade (HG; 26.4%) tumors. Distributions of MCT grades were evaluated across breed, age, sex, and anatomical location (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mixed breed dogs represented the largest proportion, with 130 LG (55.6%) and 54 HG (64.3%) tumors. Among purebreds, French Bulldogs, Golden Retrievers, Shih Tzus, and Labrador Retrievers showed a higher prevalence of LG tumors. Notably, Chihuahuas displayed an equal representation of LG and HG tumors (5 cases each; 2.1%), suggesting a lack of grade-related predisposition in this breed. Most MCTs occurred in dogs aged 6\u0026ndash;10 years (132 LG [56.4%], 35 HG [41.7%]), followed by those aged 11\u0026ndash;15 years (70 LG [29.9%], 32 HG [38.1%]). Younger dogs (0\u0026ndash;5 years) accounted for 27 LG (11.5%) and 10 HG (11.9%) cases, while very few cases were seen in dogs over 15 years of age. Female dogs comprised 112 LG (47.9%; 20 intact, 92 spayed) and 52 HG (61.9%; 16 intact, 36 spayed) cases, while males accounted for 122 LG (52.1%; 106 intact, 16 castrated) and 32 HG (38.1%; 30 intact, 2 castrated). A higher proportion of HG tumors was observed in spayed females compared to other sex categories. For the tumor location results, trunk was the most common site of MCTs (123 LG [52.6%], 33 HG [39.3%]), followed by hind limbs (36 LG [15.4%], 19 HG [22.6%]) and mammary glands (23 LG [9.8%], 12 HG [14.3%]). High-grade tumors were relatively overrepresented in inguinal, genital, and mammary regions, suggesting an association between anatomical site and tumor grade.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic summary of canine cutaneous MCT cases\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow grade (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh grade (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBreed\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMixed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e130 (40.88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54 (16.98)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFrench Bulldog\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16 (5.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (1.25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGolden Retriever\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (4.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (0.31)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShih tzu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10 (3.14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (1.25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLabrador Retriever\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e11 (3.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (0.94)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThai Bangkaew\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8 (2.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (1.25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChihuahua\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5 (1.57)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 (1.57)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePug\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9 (2.83)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (0.31)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOthers\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e32 (10.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8 (2.51)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (years)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u0026ndash;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27 (8.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 (3.14)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e131 (41.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36 (11.01)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u0026ndash;15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70 (22.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32 (10.06)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5 (1.57)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6 (1.88)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20 up\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1 (3.14)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e106 (33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30 (9.43)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCastrated male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16 (5.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (0.62)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20 (6.28)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16 (5.03)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSpayed female\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e92 (28.93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e36 (11.32)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTumor locations\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTrunk\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e123 (38.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33 (10.37)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHind limb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e36 (11.32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19 (5.97)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMammary gland\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23 (7.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12 (3.77)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFore limb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18 (5.66)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8 (2.51)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eScrotal area\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18 (5.66)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7 (2.2)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHead\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13 (4.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (0.94)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenital (female)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3 (0.94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (0.63)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e2. Statistical Analysis of Demographic Correlations\u003c/h3\u003e\n\u003cp\u003eChi-square tests were performed to assess the associations between mast cell tumor (MCT) grade and demographic factors, including breed, age, sex, and anatomical tumor location (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Age showed a statistically significant association with MCT grade (χ\u0026sup2; = 8.37, p\u0026thinsp;=\u0026thinsp;0.039). High-grade tumors were more frequently observed in dogs older than 10 years, particularly in the 11\u0026ndash;15 year age group. Sex was significantly associated with tumor grade (χ\u0026sup2; = 9.71, p\u0026thinsp;=\u0026thinsp;0.021). High-grade tumors were more common in spayed females and intact males, indicating a possible hormonal influence on tumor progression. Breed was not significantly associated with MCT grade (χ\u0026sup2; = 6.55, p\u0026thinsp;=\u0026thinsp;0.256), although descriptive analysis suggested numerical differences across breeds. Tumor location did not show a statistically significant relationship with tumor grade (χ\u0026sup2; = 6.45, p\u0026thinsp;=\u0026thinsp;0.375), despite a higher proportion of high-grade tumors in mammary and genital regions. These results indicate that age and sex are statistically significant factors influencing the grade of canine MCTs in this cohort, while breed and tumor location, although clinically relevant, did not demonstrate statistical significance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e3. Quantitative Evaluation of Histochemical and Immunohistochemical Staining\u003c/h3\u003e\n\u003cp\u003eThe extent and intensity of positive histochemistry and immunoreactivity for each marker demonstrated distinct variation between low-grade and high-grade mast cell tumors, indicating grade-associated differences in the tumor immune microenvironment (Supplementary File 2, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e-6). Quantitative immunohistochemical analysis revealed distinct expression patterns of immune and mast cell\u0026ndash;associated markers between LG and high-grade HG canine cutaneous mast cell tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). LG tumors demonstrated significantly higher infiltration of CD4⁺ and CD8⁺ T cells, with increased positive cell counts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), percentages (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), H-scores (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and stained areas (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). CD4 and CD8 localization in LG tumors was primarily cytoplasmic or membranous, whereas HG tumors exhibited scattered, focal, or nuclear staining. CD57⁺ cells were also significantly more abundant in LG tumors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting preserved immune surveillance. In contrast, CD25⁺ cells were markedly increased in HG tumors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), reflecting enhanced regulatory T cell (Treg) presence and potential immune suppression. Total nucleated cell counts were higher in LG tumors for CD4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but did not differ significantly for other markers. Tryptase expression was significantly greater in LG tumors across all parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating higher granule retention or lower degranulation. Toluidine blue staining similarly showed increased mast cell granularity in LG lesions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while localization data were limited.\u003c/p\u003e\u003cp\u003eAntigen-presenting capacity was markedly reduced in HG tumors, as evidenced by lower expression of MHC class I and II molecules across cell count, percentage, H-score, and stained area metrics (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). LG tumors displayed primarily cytoplasmic or membranous MHC localization, whereas HG tumors exhibited more heterogeneous or compartmentalized patterns. Conversely, immune checkpoint markers PD-1 and PD-L1 were significantly upregulated in HG tumors across all measured parameters (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PD-1 localization was cytoplasmic in LG tumors but nuclear or mixed in HG lesions, while PD-L1 staining in HG tumors was predominantly membranous. These patterns are indicative of enhanced immune checkpoint activation and immune evasion in HG MCTs. Overall, LG tumors exhibited more strong immune infiltration, preserved antigen presentation, and mast cell granularity, whereas HG tumors showed increased immune suppression and altered immunoarchitecture.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e4. Correlation Patterns of Immune Marker Expression in Low- and High-Grade Mast Cell Tumors\u003c/h3\u003e\n\u003cp\u003eTo explore the relationships among immunologic and histologic markers within the tumor microenvironment, Spearman\u0026rsquo;s rank correlation analysis was conducted on five key immunohistochemical parameters: number of positive cells, percentage of positive cells, H-score, total immunoreactive area, and total nucleated cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn low-grade MCTs, multiple strong and statistically significant correlations were observed among T-cell\u0026ndash;associated markers (CD4, CD8, CD25) and antigen-presenting molecules (MHC class I and II). Specifically, CD4 and CD25 demonstrated consistently high correlation coefficients across all parameters (ρ\u0026thinsp;=\u0026thinsp;0.63\u0026ndash;0.78, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), supporting the notion of synchronized helper and regulatory T-cell infiltration. CD8 also showed moderate-to-strong correlations with both MHC I and II in positive cell count and H-score analyses (ρ\u0026thinsp;\u0026gt;\u0026thinsp;0.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicative of coordinated cytotoxic T-cell activity in low-grade tumors. MHC II further correlated strongly with total nucleated cell counts and positive cell density, reinforcing its role in antigen presentation within structured immune contexts. Conversely, markers such as CD57 and mast cell tryptase demonstrated relatively weak or non-significant correlations with T-cell markers, suggesting compartmentalized expression unrelated to adaptive immune infiltration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, high-grade MCTs exhibited a markedly attenuated correlation profile. While some associations between CD4, CD8, and MHC molecules persisted, the overall magnitude and significance of correlations were reduced. CD8 expression displayed weak or negligible correlations with MHC markers and CD4 in several parameters (ρ\u0026thinsp;\u0026lt;\u0026thinsp;0.3, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting a breakdown of coordinated T-cell response. PD-1 and PD-L1 showed inconsistent or modest correlations with T-cell markers in both grades, although slightly higher PD-L1 inter-marker correlations were observed in high-grade cases, potentially reflecting immune escape mechanisms or stromal remodeling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA comparison of total nucleated cell correlations further underscored these distinctions. In low-grade MCTs, total nucleated counts were moderately correlated across markers especially between CD4, CD25, and MHC II, consistent with organized immune infiltration. In high-grade tumors, these associations were weak and non-significant, suggesting greater variability in immune cell distribution and density within the tumor microenvironment.\u003c/p\u003e\u003cp\u003eOverall, this comparative correlation analysis reveals distinct immunoarchitectural dynamics between tumor grades (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Low-grade MCTs exhibit well-structured and synchronous immune marker expression, characteristic of coordinated host immune surveillance. In contrast, high-grade MCTs are defined by fragmented or decoupled marker relationships, which may reflect immune evasion, loss of immune control, or increased microenvironmental heterogeneity. This comparative analysis highlights marker relationships that may differ with tumor grade and reflect underlying immunopathological differences between indolent and aggressive MCTs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe analysis of 318 canine mast cell tumor (MCT) cases revealed significant associations between tumor grade and host factors, particularly age and sex, while breed and tumor location were not statistically significant. Chi-square analysis confirmed that age (p\u0026thinsp;=\u0026thinsp;0.039) and sex (p\u0026thinsp;=\u0026thinsp;0.021) were significantly associated with tumor grade. High-grade tumors were more common in dogs over 10 years of age, supporting the notion that age-related processes such as chronic inflammation and genomic instability may promote malignancy\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Sex-based trends showed a greater prevalence of high-grade tumors in spayed females and intact males, aligning with prior studies suggesting hormonal influences may modulate tumor progression, although mechanistic pathways remain unclear\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Breed distribution showed numerical differences, with mixed breeds comprising a large proportion of both grades and purebreds such as French Bulldogs and Golden Retrievers more often affected by low-grade tumors. However, the lack of statistical significance (p\u0026thinsp;=\u0026thinsp;0.256) suggests breed alone may not reliably predict tumor grade, contrasting with previous reports of breed predisposition\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Mixed-breed dogs account for a substantial proportion of MCT cases in Thailand, reflecting their overall high tumor incidence. Studies from Bangkok, Chiang Mai, and Chonburi consistently identify MCTs as the most common cutaneous neoplasms, with mixed-breed dogs frequently affected alongside older and male dogs\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These findings underscore the need to include mixed-breed populations in MCT surveillance and research efforts in Southeast Asia.\u003c/p\u003e\u003cp\u003eTumor location, though not statistically significant (p\u0026thinsp;=\u0026thinsp;0.375), showed a tendency for high-grade tumors to occur more frequently in genital and mammary regions. These findings are consistent with reports indicating site-specific biological behavior possibly influenced by local vascularization or immune responses\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Collectively, the data underscore the prognostic value of age and sex in canine MCTs and highlight the potential clinical relevance of integrating demographic factors into diagnostic and treatment strategies.\u003c/p\u003e\u003cp\u003eThis study provides a comprehensive immunophenotypic comparison between low-grade and high-grade canine cutaneous mast cell tumors (MCTs), highlighting significant differences in immune cell infiltration, antigen presentation, and immune regulatory marker expression. LG tumors were characterized by increased numbers and proportions of CD4+, CD8+, CD57+, and mast cell tryptase positive cells, along with elevated H-scores and staining areas, indicating a more immunologically active tumor microenvironment. These findings are consistent with prior studies demonstrating that strong T-cell infiltration, particularly of CD4\u0026thinsp;+\u0026thinsp;helper and CD8\u0026thinsp;+\u0026thinsp;cytotoxic subsets, is associated with better differentiation and less aggressive tumor behavior in both canine and human malignancies\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The high CD57\u0026thinsp;+\u0026thinsp;cell counts in LG MCTs may reflect greater recruitment or retention of terminally differentiated effector lymphocytes, supporting an intact anti-tumor immune response\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The increased mast cell tryptase expression in LG tumors aligns with previous observations that well-differentiated mast cells with abundant granules are typical of lower-grade lesions, while HG tumors often show degranulation or loss of tryptase immunoreactivity\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Toluidine Blue staining further confirmed these differences in granularity, reinforcing the correlation between histologic grade and mast cell cytologic integrity.\u003c/p\u003e\u003cp\u003eConversely, HG tumors exhibited significantly higher expression of CD25, PD-1, and PD-L1, markers commonly associated with immunosuppression and regulatory cell function. Elevated CD25 expression in HG MCTs may reflect increased infiltration of regulatory T cells (Tregs), which are known to suppress effective anti-tumor responses and correlate with poor prognosis in various neoplasms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Likewise, the overexpression of PD-1 and its ligand PD-L1 in HG tumors suggests activation of immune checkpoint pathways, likely contributing to immune evasion. Such expression patterns have been documented in canine MCTs and other solid tumors, with implications for both prognosis and therapeutic targeting\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Notably, both MHC class I and II molecules were significantly downregulated in HG tumors, with altered localization patterns. These findings imply a reduced capacity for tumor antigen presentation and T-cell priming, consistent with mechanisms of immune escape. Decreased MHC I/II expression has been described in aggressive canine MCTs\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and in other immune-evasive tumor models\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The nuclear and heterogeneous cytoplasmic localization of MHC molecules and immune markers observed in HG lesions further supports disrupted immune signaling or trafficking, which may affect their immunogenicity and therapeutic responsiveness.\u003c/p\u003e\u003cp\u003eThe overall pattern of immune marker expression underscore distinct immune microenvironments between LG and HG MCTs. LG tumors appear to maintain a pro-inflammatory, immunologically engaged phenotype, while HG tumors shift toward immune modulation and suppression, potentially facilitating malignant progression. These differences support the utility of immunohistochemical profiling not only for diagnostic refinement but also for identifying candidates for immunotherapeutic intervention, particularly in high-grade cases with elevated PD-L1 or Treg markers. Further studies are warranted to explore the prognostic and therapeutic implications of these immune markers in canine MCTs, especially in relation to clinical outcomes and response to emerging immunotherapies.\u003c/p\u003e\u003cp\u003eThe tumor immune microenvironment plays a pivotal role in the biological behavior of mast cell tumors in dogs. In the present study, we applied Spearman\u0026rsquo;s rank correlation analysis to assess interrelationships among key immune markers based on positive cell counts, percentage positivity, H-score, total immunoreactive area, and total nucleated cells in low- and high-grade canine cutaneous MCTs. The analysis revealed distinct immunoarchitectural patterns between tumor grades, reflecting differences in immune coordination, regulatory signaling, and potential mechanisms of immune evasion. In low grade MCTs, strong and consistent correlations were observed among CD4, CD8, CD25, and MHC class II expression, particularly in positive cell counts and H-scores. These findings suggest a well-organized immune response comprising helper T cells (CD4+), cytotoxic T cells (CD8+), and regulatory T cells (CD25+), accompanied by antigen presentation via MHC II molecules. Such coordinated immune cell infiltration has been associated with effective immunosurveillance and tumor containment in various neoplastic settings\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The significant positive correlation between CD4 and CD25 may reflect the dual roles of T-helper and T-regulatory populations, both of which have been reported in canine MCTs and other tumors\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In contrast, high grade MCTs demonstrated fragmented correlation patterns with lower ρ values and fewer statistically significant associations. The loss of correlation between CD8 and MHC I/II may indicate impaired antigen presentation or dysfunctional T-cell recruitment, features commonly observed in tumors that evade immune destruction\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Notably, while PD-1 and PD-L1 expression showed only modest and inconsistent correlation with T-cell markers, PD-L1 levels were moderately elevated and correlated with tryptase and total area in high-grade tumors, supporting the involvement of immune checkpoint pathways in tumor progression and immune evasion\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These results consistent with previous reports of PD-L1 upregulation in more aggressive MCTs\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. When total nucleated cell density was considered, low grade tumors again showed stronger inter-marker correlations, reinforcing the presence of a more homogeneous and structured immune infiltrate. In contrast, the lack of significant correlations in high-grade tumors may reflect cellular disorganization, immune suppression, or clonal expansion of neoplastic mast cells that outpace host immune regulation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe cumulative evidence emphasizes that low grade MCTs are characterized by a synchronized and immunologically active microenvironment, whereas high grade tumors demonstrate a breakdown of coordinated immune responses. The loss of immune marker interdependence in high-grade tumors may reflect immune escape mechanisms, altered cytokine profiles, or tumor-driven modulation of infiltrating immune cells. Correlation-based analyses offer complementary insight beyond individual marker expression, revealing the architectural and functional relationships among immune components in situ.\u003c/p\u003e\u003cp\u003eFurther studies incorporating spatial transcriptomics or multiplex immunofluorescence could enhance the resolution of tumor\u0026ndash;immune interactions and elucidate the causal underpinnings of immune coordination versus disintegration in MCT progression. From a translational standpoint, the identification of strong marker correlations in low-grade tumors may support immunotherapeutic targeting, while disrupted marker networks in high-grade lesions could serve as biomarkers of immune escape or refractoriness to checkpoint inhibition.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e1. Demographic study of canine mast cell tumors\u003c/h2\u003e\u003cp\u003eThis study included 318 biopsy-confirmed cases of canine cutaneous mast cell tumors (MCTs) diagnosed between 2020 and 2024 at the Small Animal Teaching Hospital, Chulalongkorn University, Thailand. All specimens used in this study were archived formalin-fixed, paraffin-embedded (FFPE) tissue samples that had been previously submitted for diagnostic purposes. No live animals were contacted, sampled, or subjected to any intervention specifically for the purpose of this research. Clinical data including signalment, diagnostic findings, and tumor history were retrieved retrospectively from hospital records. Both high-grade and low-grade MCTs were analyzed to assess potential associations between tumor grade and demographic variables, including sex, age, breed, and anatomical tumor location. Cases were excluded if they involved concurrent tumors confirmed histologically, or if histological sections or staging data for MCT were unavailable. Tumor grading was performed by two board-certified veterinary pathologists based on the Kiupel grading system for canine cutaneous MCTs\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, using light microscopy. High-grade MCTs were defined by the presence of at least one of the following criteria in 10 high-power fields (HPFs): \u0026ge;7 mitotic figures, \u0026ge;\u0026thinsp;3 multinucleated cells (each with \u0026ge;\u0026thinsp;3 nuclei), \u0026ge;\u0026thinsp;3 bizarre nuclei, or karyomegaly affecting\u0026thinsp;\u0026ge;\u0026thinsp;10% of neoplastic cells with \u0026ge;\u0026thinsp;2-fold variation in nuclear diameter. Tumors not meeting these criteria were classified as low-grade (Supplementary File 2, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Clinical data including signalment, diagnostic findings, tumor history were retrieved from hospital records. Chi-square tests were used to evaluate associations between MCT grade and demographic variables. Statistical significance was determined to assess whether the distribution of these characteristics differed between high- and low-grade tumors.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003e2. Histochemical identification of mast cells using toluidine blue staining\u003c/h3\u003e\n\u003cp\u003eToluidine blue staining was employed to visualize mast cells in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Sections were cut at a thickness of 4\u0026ndash;6 \u0026micro;m, deparaffinized in xylene, and rehydrated through a graded ethanol series into distilled water. Staining was performed using 0.1% toluidine blue O solution (Sigma-Aldrich, Cat. No. T3260) for 2\u0026ndash;5 minutes at room temperature, followed by a brief rinse in distilled water. Slides were subsequently dehydrated in graded ethanol, cleared in xylene, and cover slipped with a resinous mounting medium. Toluidine blue is a basic metachromatic dye that selectively stains acidic tissue components, particularly sulfated glycosaminoglycans found in mast cell granules. This results in characteristic metachromatic staining, with mast cell granules appearing purple to red-purple, while nuclei and other basophilic structures are stained blue (Supplementary File 2, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3. Immunohistochemical detection of cellular markers in canine mast cell tumors\u003c/h2\u003e\u003cp\u003eFormalin-fixed, paraffin-embedded (FFPE) tissue specimens were sectioned at 4 \u0026micro;m thickness using a rotary microtome (Shandon, Anglia Scientific Instrument Ltd., Cambridge, UK). Slides were incubated at 60\u0026deg;C for 24 hours to facilitate tissue adherence and preparation for immunostaining. Deparaffinization was performed using xylene, followed by rehydration through a graded ethanol series. Antigen retrieval was conducted by immersing tissue sections in 0.01 M citrate buffer (pH 6.0) and heating them in a microwave oven at 750 W for 10 minutes (two 5-minute intervals). Endogenous peroxidase activity was quenched by treating the sections with 3% hydrogen peroxide (H₂O₂) in methanol for 10 minutes. Non-specific antibody binding was minimized by incubating the slides with normal horse serum (Vector Laboratories, Burlingame, CA, USA). Primary antibodies used in this study are listed in Supplementary File 1, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Sections were incubated with primary antibodies overnight at 4\u0026deg;C, followed by incubation with biotinylated secondary antibodies (Vector Laboratories; dilution 1:200). Detection was carried out using the avidin-biotin-peroxidase complex (Vectastain\u0026reg; Elite ABC Kit, Vector Laboratories) for 30 minutes at room temperature. Visualization was achieved by applying 3,3\u0026prime;-diaminobenzidine tetrahydrochloride (ImmPACT\u0026reg; DAB, Vector Laboratories) for 5 minutes. Slides were then counterstained with Mayer\u0026rsquo;s hematoxylin. Negative controls were processed in parallel by substituting the primary antibody with phosphate-buffered saline (PBS).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4. Quantitative evaluation of histochemical and immunohistochemical staining\u003c/h2\u003e\u003cp\u003eFor each tissue section, 10 random high-power field (HPF) images were acquired using an Olympus BX51TRF light microscope equipped with cellSens imaging software (Olympus Corporation, Tokyo, Japan). Immunohistochemical images were analyzed using ImageJ/Fiji software (version 2.14.0/1.54f; National Institutes of Health, Bethesda, MD, USA). Color deconvolution was applied to isolate DAB and hematoxylin staining, and positive immunoreactivity was quantified through manual threshold. In each field, total nucleated cells and positively stained cells were enumerated, and the mean H-score from at least 10 non-overlapping fields per sample was used for statistical evaluation.\u003c/p\u003e\u003cp\u003eImmunostaining intensity and extent were assessed using the H-score method, calculated as:\u003c/p\u003e\u003cp\u003eH-score = (1 \u0026times; % weakly stained cells) + (2 \u0026times; % moderately stained cells) + (3 \u0026times; % strongly stained cells). This calculation provides a continuous score ranging from 0 to 300. Additionally, immunoreactivity patterns (nuclear, cytoplasmic, or membranous) and the spatial distribution of positive cells (e.g., peripheral vs. central tumor zones) were documented. All image analyses were conducted independently by two pathologists who were blinded to the experimental group assignments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e5. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using GraphPad Prism software, version 10 (San Diego, CA, USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Comparisons between two groups were conducted using unpaired two-tailed Student\u0026rsquo;s t-test for parametric data and appropriate non-parametric tests where applicable. Associations between mast cell tumor grade and categorical variables including breed, age group, sex, and anatomical tumor location were assessed using Chi-square tests. Correlations between MHC expression levels, immune cell markers, and PD expression were evaluated using Spearman\u0026rsquo;s rank correlation analysis. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant for all tests.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to the staff of the Department of Veterinary Pathology and veterinarians at the Small Animal Teaching Hospital, Chulalongkorn University, Bangkok, Thailand, for their assistance with biopsy sample collection and valuable clinical data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTK and TT conceptualised and supervised this study. SB performed the experiments. TK and SB analysed the data. TN and AS provided canine MCT cell lines. TK and SB drafted the manuscript. TK and TT reviewed and edited the original draft. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Council of Thailand (N41D640002), the Chulalongkorn University Graduate Scholarship to the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, the 90th Anniversary of Chulalongkorn University, Rachadapisek Sompote Endowment Fund 2019 (CU_GR_62_77_31_07) and the Center of Excellence for Companion Animal Cancer (CE-CAC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used in this study are available from the corresponding author upon reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tissue samples were obtained from archived diagnostic specimens submitted to the Small Animal Teaching Hospital, Chulalongkorn University. No animals were prospectively recruited, sampled, or handled for the specific purpose of this study. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Chulalongkorn University (Protocol No. 2131049), in compliance with ARRIVE guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBlackwood L \u003cem\u003eet al\u003c/em\u003e. European consensus document on mast cell tumours in dogs and cats. \u003cem\u003eVet Comp Oncol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e1\u0026ndash;e29 (2012). https://doi.org/10.1111/j.1476-5829.2012.00341.x\u003c/li\u003e\n\u003cli\u003eKiupel M \u003cem\u003eet al.\u003c/em\u003e Proposal for a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. \u003cem\u003eVet Pathol\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 147\u0026ndash;155 (2011). https://doi.org/10.1177/0300985810386469\u003c/li\u003e\n\u003cli\u003eFlesner BK \u003cem\u003eet al\u003c/em\u003e. Immunotherapeutic approaches in canine oncology. \u003cem\u003eVet Sci\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 197 (2021). https://doi.org/10.3390/vetsci8090197\u003c/li\u003e\n\u003cli\u003eWeishaar KM \u003cem\u003eet al.\u003c/em\u003e The tumor immune microenvironment in canine mast cell tumors: implications for therapeutic intervention. \u003cem\u003eVet Immunol Immunopathol \u003c/em\u003e\u003cstrong\u003e201\u003c/strong\u003e, 21\u0026ndash;27 (2018). https://doi.org/10.1016/j.vetimm.2018.05.002\u003c/li\u003e\n\u003cli\u003eCarretero R \u003cem\u003eet al.\u003c/em\u003e Regression of melanoma metastases after MHC class I upregulation by IFN-\u0026gamma; in vivo. \u003cem\u003eCancer Immunol Immunother\u003c/em\u003e.\u003cstrong\u003e57\u003c/strong\u003e, 1441\u0026ndash;1450 (2008). https://doi.org/10.1007/s00262-008-0472-2\u003c/li\u003e\n\u003cli\u003eGoeppert B \u003cem\u003eet al.\u003c/em\u003e Down-regulation of MHC class I is a frequent mechanism of immune escape and a negative prognostic marker in Merkel cell carcinoma. \u003cem\u003eInt J Cancer\u003c/em\u003e \u003cstrong\u003e137\u003c/strong\u003e, 1597\u0026ndash;1606 (2015). https://doi.org/10.1002/ijc.29520\u003c/li\u003e\n\u003cli\u003eRimsza LM \u003cem\u003eet al.\u003c/em\u003e Loss of MHC class II gene and protein expression in diffuse large B-cell lymphoma is related to decreased tumor immunosurveillance and poor patient survival. \u003cem\u003eBlood \u003c/em\u003e\u003cstrong\u003e103\u003c/strong\u003e, 4251\u0026ndash;4258 (2004). https://doi.org/10.1182/blood-2003-10-3437\u003c/li\u003e\n\u003cli\u003eCuriel TJ \u003cem\u003eet al.\u003c/em\u003e Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 942\u0026ndash;949 (2004). https://doi.org/10.1038/nm1093\u003c/li\u003e\n\u003cli\u003eGerlini G \u003cem\u003eet al.\u003c/em\u003e Human CD8⁺CD57⁺ lymphocytes accumulate in neoplastic lymph nodes and have cytotoxic activity against autologous melanoma. \u003cem\u003eJ Invest Dermatol\u003c/em\u003e \u003cstrong\u003e122\u003c/strong\u003e, 1350\u0026ndash;1354 (2004). https://doi.org/10.1111/j.0022-202X.2004.22512.x\u003c/li\u003e\n\u003cli\u003eSato E \u003cem\u003eet al.\u003c/em\u003e Intraepithelial CD8⁺ tumor-infiltrating lymphocytes and a high CD8⁺/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. \u003cem\u003eProc Natl Acad Sci USA\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 18538\u0026ndash;18543 (2005). https://doi.org/10.1073/pnas.0509182102\u003c/li\u003e\n\u003cli\u003eMaekawa N \u003cem\u003eet al.\u003c/em\u003e A canine chimeric anti-PD-L1 monoclonal antibody for immunotherapy of canine cancers. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2952 (2020). https://doi.org/10.1038/s41598-020-59838-6\u003c/li\u003e\n\u003cli\u003eThompson MJ \u003cem\u003eet al.\u003c/em\u003e Immune checkpoint molecule expression in high-grade canine mast cell tumors. \u003cem\u003eVet Comp Oncol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 417\u0026ndash;428 (2016). https://doi.org/10.1111/vco.12123\u003c/li\u003e\n\u003cli\u003eTopalian SL \u003cem\u003eet al.\u003c/em\u003e Safety, activity, and immune correlates of anti\u0026ndash;PD-1 antibody in cancer. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e366\u003c/strong\u003e, 2443\u0026ndash;2454 (2012). https://doi.org/10.1056/NEJMoa1200690\u003c/li\u003e\n\u003cli\u003ePorcellato I \u003cem\u003eet al.\u003c/em\u003e Foxp3 and IDO1 expression in canine melanocytic tumors. \u003cem\u003eVet Pathol\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 371\u0026ndash;379 (2019). https://doi.org/10.1177/0300985819828725\u003c/li\u003e\n\u003cli\u003eWelle MM \u003cem\u003eet al.\u003c/em\u003e Canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment. \u003cem\u003eVet Dermatol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 321\u0026ndash;339 (2008).\u003c/li\u003e\n\u003cli\u003eDobson JM \u0026amp; Scase TJ Advances in the diagnosis and management of cutaneous mast cell tumours in dogs. \u003cem\u003eJ Small Anim Pract\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 424\u0026ndash;431 (2007).\u003c/li\u003e\n\u003cli\u003eJanma J \u003cem\u003eet al.\u003c/em\u003e Prevalence of canine tumors in Nong Chok, Bangkok (2018\u0026ndash;2023). \u003cem\u003eResearchGate\u003c/em\u003e. (2023). https://www.researchgate.net/publication/386254446\u003c/li\u003e\n\u003cli\u003eKlinhom S \u003cem\u003eet al.\u003c/em\u003e Retrospective study of risk factors and prevalence of canine mammary tumors in Chiang Mai, Thailand. \u003cem\u003ePeerJ \u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e, e17077 (2023). https://doi.org/10.7717/peerj.17077\u003c/li\u003e\n\u003cli\u003eWorasitthichai S \u003cem\u003eet al.\u003c/em\u003e Incidence of cutaneous tumors in dogs in eastern Thailand: A retrospective study. \u003cem\u003eVet Integr Sci\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 225\u0026ndash;237 (2020). https://he02.tci-thaijo.org/index.php/vis/article/download/242788/166241\u003c/li\u003e\n\u003cli\u003eVillamil JA \u003cem\u003eet al.\u003c/em\u003e Hormonal and sex impact on the epidemiology of canine lymphoma. \u003cem\u003eJ Cancer Epidemiol\u003c/em\u003e \u003cstrong\u003e2011\u003c/strong\u003e, 1\u0026ndash;7 (2011).\u003c/li\u003e\n\u003cli\u003eLetellier E \u0026amp; Haan S SOCS proteins in development and disease. \u003cem\u003eJ Cell Biol\u003c/em\u003e \u003cstrong\u003e183\u003c/strong\u003e, 575\u0026ndash;585 (2008). https://doi.org/10.1083/jcb.200808124\u003c/li\u003e\n\u003cli\u003eWillmann M \u003cem\u003eet al.\u003c/em\u003e T-cell infiltration in canine cutaneous mast cell tumors: a role of regulatory T cells in promoting tumor progression?. \u003cem\u003eVet Immunol Immunopathol\u003c/em\u003e \u003cstrong\u003e208\u003c/strong\u003e, 21\u0026ndash;30 (2019). https://doi.org/10.1016/j.vetimm.2018.12.006\u003c/li\u003e\n\u003cli\u003eMaekawa N \u003cem\u003eet al.\u003c/em\u003e Immunohistochemical analysis of PD-L1 expression in canine tumors. \u003cem\u003eJ Vet Med Sci\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 1473\u0026ndash;1477 (2016). https://doi.org/10.1292/jvms.16-0081\u003c/li\u003e\n\u003cli\u003eMaekawa N \u003cem\u003eet al.\u003c/em\u003e Prognostic significance of programmed cell death ligand-1 expression and tumor-infiltrating lymphocytes in canine cutaneous mast cell tumors. \u003cem\u003eVet Pathol\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 98\u0026ndash;107 (2021). https://doi.org/10.1177/0300985820964673\u003c/li\u003e\n\u003cli\u003eR\u0026uuml;tgen BC \u003cem\u003eet al.\u003c/em\u003e Immunohistochemical evaluation of the tumor microenvironment in canine cutaneous mast cell tumors of different histologic grades. \u003cem\u003eVet Immunol Immunopathol\u003c/em\u003e \u003cstrong\u003e195\u003c/strong\u003e, 11\u0026ndash;19 (2018). https://doi.org/10.1016/j.vetimm.2017.12.005\u003c/li\u003e\n\u003cli\u003eTakeuchi Y \u003cem\u003eet al.\u003c/em\u003e Roles of regulatory T cells in cancer immunity. \u003cem\u003eInt Immunol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 69\u0026ndash;77 (2013). https://doi.org/10.1093/intimm/dxs099\u003c/li\u003e\n\u003cli\u003eZitvogel L \u003cem\u003eet al.\u003c/em\u003e Immune parameters affecting the efficacy of chemotherapeutic regimens. \u003cem\u003eNat Rev Clin Oncol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 661\u0026ndash;670 (2006). https://doi.org/10.1038/ncponc0649\u003c/li\u003e\n\u003cli\u003eFridman WH \u003cem\u003eet al.\u003c/em\u003e The immune contexture in human tumours: impact on clinical outcome. \u003cem\u003eNat Rev Cancer\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 298\u0026ndash;306 (2012). https://doi.org/10.1038/nrc3245\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Sullivan T \u003cem\u003eet al\u003c/em\u003e. Cancer immunoediting by the innate immune system in the absence of adaptive immunity. \u003cem\u003eJ Exp Med\u003c/em\u003e \u003cstrong\u003e216\u003c/strong\u003e, 1373\u0026ndash;1385 (2019). https://doi.org/10.1084/jem.20181941\u003c/li\u003e\n\u003cli\u003eMarconato L \u003cem\u003eet al.\u003c/em\u003e The dog as a possible animal model for human aggressive B-cell lymphoma: a review. \u003cem\u003eHematol Oncol\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 1\u0026ndash;9 (2013). https://doi.org/10.1002/hon.2002\u003c/li\u003e\n\u003cli\u003eHanahan D \u0026amp; Weinberg RA Hallmarks of cancer: the next generation. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 646\u0026ndash;674 (2011). https://doi.org/10.1016/j.cell.2011.02.013\u003c/li\u003e\n\u003cli\u003eYoshimura H \u003cem\u003eet al.\u003c/em\u003e Programmed death-ligand 1 expression in canine tumors and its association with prognosis in canine oral melanoma. \u003cem\u003eVet Immunol Immunopathol\u003c/em\u003e \u003cstrong\u003e227\u003c/strong\u003e, 110089 (2020). https://doi.org/10.1016/j.vetimm.2020.110089\u003c/li\u003e\n\u003cli\u003eScase TJ \u003cem\u003eet al\u003c/em\u003e. Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis. \u003cem\u003eJ Vet Intern Med \u003c/em\u003e\u003cstrong\u003e23\u003c/strong\u003e, 251\u0026ndash;258 (2009). https://doi.org/10.1111/j.1939-1676.2008.0263.x\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6766464/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6766464/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCanine cutaneous mast cell tumors (MCTs) exhibit heterogeneous biological behaviors, with increasing evidence implicating the tumor immune microenvironment (TIME) in disease progression. This study evaluated biopsy-confirmed cases of canine MCTs to investigate associations between tumor grade and host factors, and to characterize grade-related differences in immune marker expression. Tumors were histologically graded by the Kiupel system and analyzed using immunohistochemistry for CD4, CD8, CD25, CD57, MHC I, MHC II, PD-1, PD-L1, and mast cell tryptase. Quantitative assessments included positive cell counts, percentages, H-scores, total stained areas, and inter-marker correlations. Demographic analysis revealed significant associations between tumor grade and both age and sex, whereas breed and anatomical location showed no statistical correlation. Low grade (LG) tumors demonstrated greater infiltration of CD4⁺, CD8⁺, CD57⁺ T cells and higher MHC I/II expression, consistent with enhanced antigen presentation and immune activity. In contrast, high grade (HG) tumors exhibited increased CD25⁺, PD-1⁺, and PD-L1⁺ expression, suggesting a more immunosuppressive phenotype. Correlation analysis highlighted coordinated immune marker expression in LG tumors and disrupted immune architecture in HG tumors. These findings reveal distinct immunophenotypic and demographic features across MCT grades and underscore the potential of immune profiling for prognostication and therapeutic planning in canine oncology.\u003c/p\u003e","manuscriptTitle":"Demographic Patterns and Immunophenotypic Characterization of Tumor–Immune Interactions in Canine Cutaneous Mast Cell Tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-26 00:36:05","doi":"10.21203/rs.3.rs-6766464/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66ae034f-6933-43a5-9607-e26b623fb289","owner":[],"postedDate":"October 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56582879,"name":"Biological sciences/Immunology"},{"id":56582880,"name":"Health sciences/Biomarkers"},{"id":56582881,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-11-12T12:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-26 00:36:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6766464","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6766464","identity":"rs-6766464","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}