In vitro inhibitory effect of trichostatin A on canine grade 3 mast cell tumor

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect skin and soft tissue in dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. Trichostatin A (TSA), an antifungal antibiotic, has shown inhibitory effects on the proliferation and induction of apoptosis in various types of cancer cells. In order to evaluate the potential of trichostatin A as a therapeutic drug, cells of grade 3 MCT were cultured and treated with concentrations of 1 nM to 400 nM of TSA. MTT assay and trypan blue exclusion assays were performed to estimate cell growth and cell viability, and cell cycle analysis was evaluated. TSA treatment showed a reduction in numbers of viable cells and an increase of cell death by apoptosis. The cell cycle analysis showed an increase of hypodiploid cells and a reduction of G0/G1 and G2/M -phases. According to these results, trichostatin A may be an interesting potential chemotherapeutic agent for the treatment of canine MCT.

In vitro chemosensitivity of canine mast cell tumors grades II and III to all-trans-retinoic acid (ATRA).

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect the skin and soft tissue of dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. While retinoids are well recognized as promising antitumor agents, there have been only a few reports about retinoids` effect on canine cancers. The aim of this study was to investigate the chemosensitivity of MCT grades II and III to all-trans retinoic acid (ATRA). Immediately after surgical resection, MCT were prepared for primary culture. Samples of MCTs were also fixed in formalin for histopathology and grading according to the classification of Patnaik et al. (Veterinary Pathology 21(5):469-474, 1984). The best results were obtained when neoplastic mast cells were co-cultivated with fibroblasts. Cultured mast cells were, then, treated with concentrations of 10(-4) to 10(-7) M of ATRA, in order to evaluate their chemosensitivity to this retinoid. MTT assay was performed to estimate cell growth and death. The highest level of mast cell chemosensivity was obtained at the dose of 10(-4) M (p < 0,002). MCT of grades II or III were equally susceptible to the treatment with ATRA. Cell death was observed on the first 24 h until 48 h. According to these results, ATRA may be a potential chemotherapeutic agent for the treatment of canine MCT.

Cholinergic neurons of fetal rat telencephalon in aggregating cell culture respond to NGF as well as to protein kinase C-activating tumor promoters.

Serum-free aggregating cell cultures of fetal rat telencephalon treated with the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) showed a dose-dependent, persistent stimulation of the enzymes choline acetyltransferase (ChAT), glutamic acid decarboxylase and glutamine synthetase. After elimination of the proliferating cells by treatment of the cultures with Ara-C (0.4 microM) only the cholinergic marker enzyme, ChAT, could be stimulated by tumor promoters. The non-promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate proved to be inactive in these cultures, whereas the potent non-phorbol tumor promoter, mezerein, produced an even greater stimulatory effect than PMA. Since PMA and mezerein are potent and specific activators of protein kinase C, the present results suggest a role for this second messenger in the development of cholinergic telencephalon neurons. Stimulation of ChAT required prolonged exposure (48 h) of the cultures to PMA and the responsiveness of the cholinergic neurons to the tumor promoters decreased with progressive cellular maturation. The cholinergic telencephalon neurons showed the same pattern of responsiveness for tumor promoters as for nerve growth factor (NGF). However, the combined treatment with NGF and either PMA or mezerein produced an additive stimulatory effect, suggesting somewhat different mechanisms of action.

Peritoneal effusion in a dog secondary to visceral mast cell tumor: A case report

BACKGROUND: Mast cell tumor, one of the most common skin tumors in dogs, may also be found in visceral sites (mainly spleen and liver). When a visceral mast cell tumor is present, neoplastic mast cells may be found in any effusion secondary to the tumor. Therefore, the diagnosis may be made by cytologic analysis of the effusion. CASE: An 8-year-old, spayed, female Siberian husky presented with a peritoneal effusion secondary to a visceral mast cell tumor. Seven months earlier, the dog had presented with a cutaneous nodule diagnosed as a well-differentiated mast cell tumor. The peritoneal fluid was classified as a transudate. Numerous neoplastic mast cells were found in the effusion. Although the mast cell tumor presented with characteristics of the well-differentiated tumor, its biologic behavior was that of a malignant tumor. CONCLUSION: Care should be taken to evaluate the prognosis of mast cell tumors in dogs since their biologic behavior is extremely variable.

Visceral mast cell tumor in a captive black jaguar (Panthera onca)

Little is known about neoplasia in the jaguar (Panthera onca), the largest American feline. A captive black jaguar was diagnosed at necropsy with a mastocytic form of visceral mast cell tumor similar to that which occurs in domestic cats. This animal had no previous clinical disease and died during anesthesia for a routine dental treatment.

Visceral mast cell tumor and mastocythemia in a dog

Mast cell tumor manifests as a localized proliferation of mast cells in the skin, or less frequently as a systemic disorder, which may be accompanied by the presence of neoplastic mast cells in the peripheral blood (mastocythemia). In some cases, the neoplastic circulating mast cells originate in the bone marrow, designated as mast cell leukemia, rarely observed in dogs, or the cells may arise from visceral mast cell tumors, characterizing systemic mastocytosis. The aim of this report was to describe a case of a six-year-old female German shepherd dog presenting with history of anorexia, hematemesis and diarrhea. The blood work revealed intense mastocythemia (43%), with degranulated mast cells, and anisocytosis. At necropsy, white nodular lesions in the thymic region and an infiltrative mass in mesenteric and abdominal lymph nodes were observed. Those lymph nodes were enlarged and off-white. Histopathological examination revealed neoplastic mast cells in the liver, spleen, lymph nodes, kidneys, lungs, gastric and enteric mucosae, and adrenal glands. The clinical, hematological and histopathological findings were compatible with mastocythemia, associated with a moderately differentiated visceral mast cell tumor.

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

Immunological microenvironment in prostate cancer: high mast cell densities are associated with favorable tumor characteristics and good prognosis

BACKGROUND: Number of intratumoral mast cells predicts survival in various cancers. The prognostic significance of such mast cells in surgically treated prostate cancer is unknown. METHODS: Mast cell densities were determined in prostate cancer samples of more than 2,300 hormone-naïve patients using a tissue microarray format in correlation with clinical follow-up data. Mast cells were visualized immunohistochemically (c-kit). All patients were homogeneously treated by radical prostatectomy at a single institution. RESULTS: Mast cells were present in 95.9% of the tumor samples. Median mast cell number on the tissue spot was 9 (range: 0-90; median density: 31 mast cells/mm(2)). High mast cell densities were significantly associated with more favorable tumors having lower preoperative prostate-specific antigen (P = 0.0021), Gleason score (P < 0.0001) and tumor stage (P < 0.0001) than tumors with low mast cell densities. Prostate-specific antigen recurrence-free survival significantly (P = 0.0001) decreased with decline of mast cell density showing poorest outcome for patients without intratumoral mast cells. In multivariate analysis mast cell density narrowly missed to add independent prognostic information (P = 0.0815) for prostate-specific antigen recurrence. CONCLUSION: High intratumoral mast cell density is associated with favorable tumor characteristics and good prognosis in prostate cancer. This finding is consistent with a role of mast cells in the immunological host-defense reaction on prostate cancer. Triggering mast cell activity might expand immunotherapeutic strategies in prostate cancer.

Nuclear morphometry in cytopathology: a prognostic indicator for canine cutaneous mast cell tumors

Twenty-nine canine cutaneous mast cell tumors (MCTs) were morphometrically analyzed with regard to mean nuclear area (MNA) using cytopathology smears. The results showed a correlation between MNA and survival. When graded into 2 morphometrically different groups, there were statistically significant differences among high- and low-grade MCTs, regarding both Romanowsky-type stain and hematoxylin and eosin. Cytomorphometry could also separate histologic grade II tumors with better prognosis from the more aggressive MCTs. The results indicated that nuclear morphometry on cytopathology preparations can predict the biological behavior of cutaneous MCTs in dogs in an independent manner, yielding a rapid and reproducible diagnosis, which renders the method useful for veterinary oncology.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Canine Subcutaneous Mast Cell Tumors: Cellular Proliferation and KIT Expression as Prognostic Indices

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers-including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)-as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytoplasmic and nuclear morphometric parameters in cytologic preparations of canine cutaneous mast cell tumors

Thirty fine-needle biopsy (FNB) samples from 28 dogs subjected to surgical resection of cutaneous mast cell tumors (MCTs) were stained with Giemsa. At least 100 neoplastic cells from each cytology slide were evaluated by morphometric analysis. The parameters were: area, perimeter of the cell, cytoplasm, nucleus and circumference factor. MCTs of grade III had a mean cellular area of 231.70 μm2 ± 57.1, and grade II had a mean of 252.30 μm2 ± 55.0. Cellular perimeter was 61.20 ± 7.1 in grade II and 59.1 ± 8.6 in grade III. Cellular parameters were not statistically different between grades (p>.05). Mean nuclear area was 88.90 μm2 ± 19 in grade III and 72.30 μm2 ± 13.9 in grade II, with statistical difference between grades (P =.011). Mean nuclear perimeter was 32.40 ìm ± 3.0 in grade II and 35.70 ìm ± 4.0 in grade III, with statistical difference between grades (P =.018). Mean nuclear circumference factor was 1.0 ± 0.33 in grade II and 1.1 ± 0.28 in grade III, with no statistical difference between grades (P = 0.78). Nuclear-tocytoplasmic ratio in grade II was 0.29 ±.07 and 0.39 ±.08 in grade III, with statistical difference (P =.02). The number of binucleated and multinucleated cells and mitotic figures was significantly increased in grade III MCTs (P <.001). In conclusion, the number of mitotic figures, presence of binucleation and multinucleation, and nuclear-to-cytoplasmic ratio can help to guide a profile of MCT aggressiveness in cytologic preparations.

Ki67/KIT double immunohistochemical staining in cutaneous mast cell tumors from Boxer dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.