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

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

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

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

Metabolic Symbiosis and Immunomodulation: How Tumor Cell-Derived Lactate May Disturb Innate and Adaptive Immune Responses.

The tumor microenvironment (TME) is composed by cellular and non-cellular components. Examples include the following: (i) bone marrow-derived inflammatory cells, (ii) fibroblasts, (iii) blood vessels, (iv) immune cells, and (v) extracellular matrix components. In most cases, this combination of components may result in an inhospitable environment, in which a significant retrenchment in nutrients and oxygen considerably disturbs cell metabolism. Cancer cells are characterized by an enhanced uptake and utilization of glucose, a phenomenon described by Otto Warburg over 90 years ago. One of the main products of this reprogrammed cell metabolism is lactate. "Lactagenic" or lactate-producing cancer cells are characterized by their immunomodulatory properties, since lactate, the end product of the aerobic glycolysis, besides acting as an inducer of cellular signaling phenomena to influence cellular fate, might also play a role as an immunosuppressive metabolite. Over the last 10 years, it has been well accepted that in the TME, the lactate secreted by transformed cells is able to compromise the function and/or assembly of an effective immune response against tumors. Herein, we will discuss recent advances regarding the deleterious effect of high concentrations of lactate on the tumor-infiltrating immune cells, which might characterize an innovative way of understanding the tumor-immune privilege.

Modulation of Cytokines Production by Indomethacin Acute Dose during the Evolution of Ehrlich Ascites Tumor in Mice.

The aim of the present study was to investigate the influence of a nonselective COX1/COX2 inhibitor (indomethacin) on tumor growth of Ehrlich Ascites Tumor (EAT) in mice, using as parameters the tumor growth and cytokine profile. Mice were inoculated with EAT cells and treated with indomethacin. After 1, 3, 6, 10, and 13 days the animals were evaluated for the secretion of TNFα, IL-1α, IL-2, IL-4, IL-6, IL-10, and IL-13 and PGE2 level in peritoneal cavity. The results have shown that EAT induces PGE2 production and increases tumor cells number from the 10th day. The cytokine profile showed EAT induces production of IL-6 from 10th day and of IL-2 on 13th day; the other studied cytokines were not affected in a significant way. The indomethacin treatment of EAT-bearing mice inhibited the tumor growth and PGE2 synthesis from the 10th day. In addition, the treatment of EAT-bearing mice with indomethacin has stimulated the IL-13 production and has significantly inhibited IL-6 in the 13th day of tumor growth. Taken together, the results have demonstrated that EAT growth is modulated by PGE2 and the inhibition of the tumor growth could be partly related to suppression of IL-6 and induction of IL-13.

c-Kit immunoexpression patterns differ in melanotic and amelanotic canine oral melanomas / Análise qualitativa quanto à diferença da imunoexpressão do gene c-Kit em melanomas melânicos e amelânicos da cavidade oral em cães

Melanomas are the most common oral malignancy in dogs. Cell proliferation and connexin expression has been shown to differ in canine melanotic and amelanotic oral melanomas. This study aimed to analyze the c-Kit protein expression in melanotic and amelanotic melanomas from canine buccal cavity. A total of 34 canine buccal melanomas (19 melanotic and 15 amelanotic).were collected. The amelanotic melanomas presented faster evolution and higher incidence of metastasis than melanotic tumors. A significantly higher number of c-Kit positive cells were observed in amelanotic neoplasms. In addition, the intensity of c-Kit immunolabeling was predominantly stronger in amelanotic melanomas. These results confirm a potential role for c-Kit in canine oral melanomas with clear differences in expression patterns between the two histological types of tumor, melanotic and amelanotic. This study highlights the importance of a detailed study of c-Kit mutations in canine oral melanomas to better understand the molecular mechanisms implicated in the development of this disease...

Melanomas são as mais frequentes neoplasias malignas da cavidade bucal de cães. Sabe-se que a proliferação de células e expressão de conexina diferem em melanomas melanóticos e amelanóticos da cavidade bucal de cães. Este estudo analisou a expressão da proteína c-Kit em melanomas melanóticos e amelanóticos da cavidade bucal canina. Um total de 34 melanomas bucais caninos (19 melanóticos e 15 amelanóticos) foram coletados. Os melanomas amelanóticos apresentaram evolução mais rápida e maior incidência de metástase. Foi constatado um número significativamente maior de células positivas para c-Kit em neoplasias amelanóticas. Além disso, a intensidade de imunomarcação de c-Kit foi predominantemente mais forte em melanomas amelanóticos. Estes resultados confirmam um papel potencial para c-Kit em melanomas orais caninos, com diferenças claras em padrões de expressão entre os dois tipos histológicos de tumor, melanóticos e amelanóticos. Este trabalho destaca a importância de um estudo detalhado das mutações c-Kit em melanomas orais caninos para ser possível a melhor compreensão dos mecanismos moleculares envolvidos no desenvolvimento da doença...

Cell proliferation and expression of connexins differ in melanotic and amelanotic canine oral melanomas.

Melanoma is a malignant neoplasm occurring in several animal species, and is the most frequently found tumor in the oral cavity in dogs. Melanomas are classified into two types: melanotic and amelanotic. Prior research suggests that human amelanotic melanomas are more aggressive than their melanotic counterparts. This study evaluates the behavior of canine melanotic and amelanotic oral cavity melanomas and quantifies cell proliferation and the expression of connexins. Twenty-five melanomas (16 melanotic and 9 amelanotic) were collected from dogs during clinical procedures at the Veterinary Hospital of the School of Veterinary Medicine and Animal Science of the University of São Paulo, Brazil. After diagnosis, dogs were followed until death or euthanasia. Histopathology confirmed the gross melanotic or amelanotic characteristics and tumors were classified according to the WHO. HMB45 or Melan A immunostainings were performed to confirm the diagnosis of amelanotic melanomas. Cell proliferation was quantified both by counting mitotic figures and PCNA positive nuclei. Expressions of connexins 26 and 43 were evaluated by immunohistochemistry, qRT-PCR and Western blot. Dogs bearing amelanotic melanomas presented a shorter lifespan in comparison to those with melanotic melanomas. Cell proliferation was significantly higher in amelanotic melanomas. Expressions of Connexins 26 and 43 were significantly reduced in amelanotic melanomas. The results presented here suggest that oral cavity melanotic and amelanotic melanomas differ regarding their behavior, cell proliferation and connexin expression in dogs, indicating a higher aggressiveness of amelanotic variants.

Canine mammary tumors in Santos, Brazil: clinicopathological and survival profile / Neoplasias mamárias caninas em Santos, Brasil: perfil clinicopatológico e de sobrevida

Tumors of the mammary glands are the most common neoplasms in dogs in our country; however, there are few Brazilian reports dedicated to clinicopathological and survival studies about this disease. This report aims the clinical and pathological study of canine mammary tumors in the Santos Metropolitan Region, an area in Sao Paulo state with an estimated canine population of 120,000 animals. Data of 14,298 dogs were collected retrospectively from the medical records of the Veterinary Medical Teaching Hospital of the Metropolitan University of Santos – São Paulo – Brazil. During the study period, from records of 317 females with histopathological diagnosis of neoplasia, 170 were mammary epithelial lesions distributed in 13 benign tumors, 152 malignant (89.4% of diagnosis) and 5 non-neoplasic epithelial lesions (ductal hyperplasia). The highest prevalent malignant tumor was tubular carcinoma (38.2% of diagnosis) and Grade I tumors, corresponding to 73.0% of all diagnosis. The results have shown clinical staging of canine mammary neoplasms as an important prognostic survival factor and, in a multivariate analysis, tumor diameter, tumor grade, adjuvant chemotherapy and recurrence as covariates with predictive value for survival. Moreover, the high prevalence of tubular carcinoma qualifies the canine population of Santos as a promising model for the translational study of this disease.

Os tumores das glândulas mamárias são as neoplasias mais comuns em cadelas em nosso país; no entanto, são poucos os trabalhos brasileiros dedicados ao estudo clinicopatológico e de sobrevida nesta doença. O presente trabalho teve por objetivo o estudo clínico e patológico dos tumores mamários caninos na Região Metropolitana de Santos, uma área no estado de São Paulo com uma população canina estimada em 120 mil animais. Dados de 14.298 cães foram coletados retrospectivamente dos prontuários médicos do Hospital Veterinário da Universidade Metropolitana de Santos – São Paulo – Brasil. Durante o período do estudo, foram atendidas 317 fêmeas com diagnóstico histopatológico de neoplasia, dos quais, 170 se referiam a lesões mamárias epiteliais distribuídas em 13 tumores benignos, 152 malignos (89,4% dos diagnósticos) e 5 lesões epiteliais não-neoplásicas (hiperplasia ductal). O tumor mais frequente foi o carcinoma tubular (38,2% dos tumores malignos) e tumores de grau I, respondendo por 73,0% do total diagnosticado. Estudos de sobrevida apontaram para o estadiamento clínico das neoplasias mamárias caninas como importante fator prognóstico, e na análise multivariada, diâmetro do tumor, grau histológico, quimioterapia adjuvante e recorrência apresentaram-se como covariáveis com valor preditivo de sobrevida. Levando-se em conta a elevada prevalência de carcinoma tubular simples na população canina de Santos, pode-se considerá-la como promissor modelo translacional para o estudo da doença.