Background: Melatonin has oncostatic actions and IL-25 is active in inflammatory processes that induce apoptosis in tumor cells Aim: The aim of this study was to evaluate melatonin and IL-25 in metastatic (CF-41) and non-metastatic (CMT-U229) canine mammary tumor cells cultured as monolayers and tridimensional structures. Materials and Methods: The cells were treated with melatonin, IL-25 and IL-17B silencing gene and performed cell viability, gene and protein expression of caspase-3 and VEGFA (Vascular endothelial growth factor A) and an apoptosis membrane protein array. Results: Treatment with 1 mM of melatonin reduced cell viability of both tumor cell lines, all treatments alone and combined significantly increased caspase-3 cleaved and proteins involved in the apoptotic pathway and reduced pro-angiogenic VEGFA, confirming the effectiveness of these potential promising treatments. Conclusion: This is the first study evaluating the potential use of these strategies in CF-41 and CMT-U229 cell lines and together encourages subsequent in vitro and in vivo studies for further exploration of clinical applications.

Cancer stem-like cells as cells with ability to self-renewal and potential to differentiate into various types of cells are known to be responsible for tumour initiation, recurrence and drug resistance. Hence a comprehensive research is concentrated on discovering cancer stem-like cells biology and interdependence between them and other cells. The aim of our study was to evaluate the impact of macrophages on cancer stem-like cells in canine mammary carcinomas. As recent studies indicated presence of macrophages in cancer environment stimulates cancer cells into more motile and invasive cells by acquisition of macrophage phenotypes. From two canine mammary tumour cell lines, CMT-U27 and P114 cancer stem-like cells were stained with Sca1, CD44 and EpCAM monoclonal antibodies and isolated. Those cells were next co-cultured with macrophages for 5 days and used for further experiments. Canine Gene Expression Microarray revealed 29 different expressed transcripts in cancer stem-like cells co-cultured with macrophages compared to those in mono-culture. Up-regulation of C-C motif chemokine 2 was considered as the most interesting for further investigation. Additionally, those cells showed overexpression of genes involved in non-canonical Wnt pathway. The results of 3D tubule formation in endothelial cells induced by cancer stem-like cells co-cultured with macrophages compared to cancer stem-like cells from mono-cultures and with addition of Recombinant Canine CCL2/MCP-1 revealed the same stimulating effect. Based on those results we can conclude that macrophages have an impact on cancer stem-like cells increasing secretion of pro-angiogenic factors.

Cancer stem cells (CSCs) display both unique self-renewal ability as well as the ability to differentiate into many kinds of cancer cells. They are supposed to be responsible for cancer initiation, recurrence and drug resistance. Despite the fact that a variety of methods are currently employed in order to target CSCs, little is known about the regulation of their phenotype and biology by miRNAs. The aim of our study was to assess miRNA expression in canine mammary cancer stem-like cells (expressing stem cell antigen 1, Sca-1; CD44 and EpCAM) sorted from canine mammary tumour cell lines (CMT-U27, CMT-309 and P114). In order to prove their stem-like phenotype, we conducted a colony formation assay that confirmed their ability to form colonies from a single cell. Profiles of miRNA expression were investigated using Agilent custom-designed microarrays. The results were further validated by real-time rt-PCR analysis of expression of randomly selected miRNAs. Target genes were indicated and analysed using Kioto Encyclopedia of Genes and Genomes (KEGG) and BioCarta databases. The results revealed 24 down-regulated and nine up-regulated miRNAs in cancer stem-like cells compared to differentiated tumour cells. According to KEGG and BioCarta databases, target genes (n=240) of significantly down-regulated miRNAs were involved in transforming growth factor-beta signaling, mitogen-activated protein kinases (MAPK) signaling pathway, anaplastic lymphoma receptor tyrosine kinase (ALK) and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1A) pathways. The analysis of single-gene overlapping with different pathways showed that the most important genes were: TGFBR1, TGFBR2, SOS1, CHUK, PDGFRA, SMAD2, MEF2A, MEF2C and MEF2D. All of them are involved in tumor necrosis factor-beta signaling and may indicate its important role in cancer stem cell biology. Increased expression of TGFBR2, SMAD2, MEF2A and MEF2D in canine mammary cancer stem-like cells was further confirmed by real-time-qPCR. The results of our study point at epigenetic differences between cancer stem-like cells and differentiated tumour cells, which may be important not only for veterinary medicine but also for comparative oncology.

Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms.

The diverse effects of progesterone on female reproductive tissues are mediated by the progesterone receptor (PR), a member of the nuclear receptor family of ligand-dependent transcription factors. Thus, PR is an important therapeutic target in female reproduction and in certain endocrine dependent cancers. This paper reviews our understanding of the mechanism of action of the most widely used PR antagonist RU486. Although RU486 is a competitive steroidal antagonist that can displace the natural hormone for PR, it’s potency derives from additional “active antagonism” that involves inhibiting the activity of PR hormone agonist complexes in trans through heterodimerization and competition for binding to progesterone response elements on target DNA, and by recruitment of corepressors that have the potential to actively repress gene transcription. An additional functional role for PR has recently been defined whereby a subpopulation of PR in the cytoplasm or cell membrane is capable of mediating rapid progesterone induced activation of certain signal transduction pathways in the absence of gene transcription. This paper also reviews recent results on the mechanism of the extra-nuclear action of PR and the potential biological roles and implications of this novel PR signaling pathway.

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

There is an increasing need for more accurate prognostic and predictive markers in veterinary oncology because of an increasing number of treatment options, the increased financial costs associated with treatment, and the emotional stress experienced by owners in association with the disease and its treatment. Numerous studies have evaluated potential prognostic and predictive markers for veterinary neoplastic diseases, but there are no established guidelines or standards for the conduct and reporting of prognostic studies in veterinary medicine. This lack of standardization has made the evaluation and comparison of studies difficult. Most important, translating these results to clinical applications is problematic. To address this issue, the American College of Veterinary Pathologists' Oncology Committee organized an initiative to establish guidelines for the conduct and reporting of prognostic studies in veterinary oncology. The goal of this initiative is to increase the quality and standardization of veterinary prognostic studies to facilitate independent evaluation, validation, comparison, and implementation of study results. This article represents a consensus statement on the conduct and reporting of prognostic studies in veterinary oncology from veterinary pathologists and oncologists from around the world. These guidelines should be considered a recommendation based on the current state of knowledge in the field, and they will need to be continually reevaluated and revised as the field of veterinary oncology continues to progress. As mentioned, these guidelines were developed through an initiative of the American College of Veterinary Pathologists' Oncology Committee, and they have been reviewed and endorsed by the World Small Animal Veterinary Association.

The aim of the study was to identify the genes responsible for the high growth rate and antiapoptotic potential in selected canine mammary cancer cells. cDNA canine microarrays were used to compare the transcriptome in simple carcinoma CMT-U27 and spindle-cell tumor CMT-U309 cell lines. In CMT-U27 cell line the growth rate (shorter cell cycle), anti-apoptotic potential (higher expression of Bcl-2) was higher and spontaneous and induced apoptosis was lower. Comparison of transcriptomes revealed 333 genes which expression differed similarly. We focused on genes involved in cell proliferation, adhesion and apoptosis, and selected 29 of them. The high growth rate and anti-apoptotic potential in CMT-U27 cells was associated with enhanced expression of genes (at the level of transcripts) involved in Ca(2+) signaling pathway (Calmodulin 1, 2, 3 and SPSB2) and growth hormone cellular pathway. The low-proliferative and pro-apoptotic phenotype of CMTU309 cells was more dependent on TGFbeta, neuregulin 1 pathways and adhesion-related molecules.

Female dogs are frequently affected by mammary tumors, both carcinomas and sarcomas. The mechanisms behind mammary-tumor formation and the high degree of heterogeneity are not understood. To provide insight into this issue, it is important to determine the properties of the cells forming the different types of tumors. One question is if individual neoplastic cells can give rise to phenotypically distinct tumor types, i.e., show plasticity. We studied 3 different tumors (a spindle-cell tumor, an osteosarcoma, and a carcinoma) and followed the change of lineage marker expression between the primary canine mammary tumors, the clones derived from the corresponding tumors and in tumors generated after inoculation of tumor clones into nude mice ( n = 75). Inoculation of clones derived from the spindle-cell tumor gave rise to spindle-cell tumors in nude mice. Several of these contained bone tissue, a sign of plasticity. Clones derived from the osteosarcoma were negative for a panel of lineage markers but, when inoculated into nude mice, they were able to form bone, again a sign of plasticity. In contrast to the primary carcinoma, most of the clones derived thereof lacked keratin expression, but keratin expression was recovered in most of the tumors formed after inoculation of clones into nude mice. Moreover, tumors generated from the carcinoma clones, in contrast to the primary tumor, were positive for smooth-muscle-cell markers. Our results point to plasticity in canine mammary tumors, as shown both by morphologic criteria and by expression patterns for lineage specific markers.

BACKGROUND Mammary tumors are the most common type of tumor in female dogs. The histopathological diagnosis is usually made by a hematoxylin-eosin (HE) staining of the tumor, which then requires a pathologist's judgment for assessment of malignancy. The purpose of this study was to investigate an alternative silver staining of some argyrophilic nucleolar organizer regions (AgNOR) for improving the diagnostic accuracy with mammary tumors. HYPOTHESIS There is a correlation between the histopathological diagnosis by AgNOR count and AgNOR area in canine mammary tumors. ANIMALS Seventy-three canine mammary tumors from 33 female dogs. MATERIALS AND METHODS The AgNOR staining was evaluated retrospectively in 73 canine mammary tumors with a parallel HE staining as a "Gold Standard." Both a quantitative manual counting method and a qualitative computerized morphometric method were tested. RESULT The result from both methods indicated a clinically relevant difference in the mean values of the AgNOR in the following 4 categories: malignant, benign, hyperplastic, and normal mammary tissue. The counting method was superior, with 89% of the cases given a correct diagnosis of a malignant or a nonmalignant canine mammary tumor. The 2 methods were then compared to test their ability to classify the tumors correctly. Again, the counting method was the most reliable method, with a sensitivity of 80% and a specificity of 76% when the upper 50% of the AgNOR counts were presumed malignant. CONCLUSION AND CLINICAL IMPORTANCE The results indicated that an AgNOR test could be an aid to pathologists as a prognostic indicator or to assist them in deciding between a benign or a malignant diagnosis in questionable cases.

Lipid-rich carcinomas occurred in seven female dogs. Affected dogs were purebred (all but one), intact (all but one), and between 4 and 13 years of age. Five of them had a history of parity, one had pseudopregnancy, and none had received contraceptive steroids. The tumors were single (five cases) or multiple (two cases) well-circumscribed masses of different sizes (varying from I to 6 cm in diameter), composed of solid nests and cords of tumor cells separated by a moderate amount of stroma. The tumor cells contained either multiple and small or large and solitary vacuoles that pushed the nucleus to the periphery of the cell (signet-ring cell). A glandular epithelial immunophenotype (cytokeratins 5 and 8 and 8 and 18) was observed in the majority of tumor cells. All tumors lacked both estrogen and progesterone receptors, and five out of seven tumors gave rise to local recurrence and proximal or distant metastases or both.

It has now been well documented that the normal and tumorous canine mammary glands can be extra-pituitary sites of substantial growth hormone (GH) synthesis. Until now, attempts to reproduce the GH synthesis in-vitro using canine mammary explants or mammary tumor cells have not been successful. Therefore, the response of CMT-U335 canine mammary tumor cells to administered porcine GH (pGH) was investigated as an in-vitro model to study the possible effects of GH synthesis on this site. CMT-U335 cells spontaneously express the growth hormone receptor (GHR) as well as the prolactin receptor (PRLR). Twenty five minutes after administration, GH induced, in a dose-dependent manner, phosphorylation of the transcription factors Stat5a and Stat5b. Clear phosphorylation was induced by 10(-7) M and 10(-8) M pGH, with virtually no phosphorylation at 10(-9) M pGH. A similar dose-dependent phosphorylation of Stat5a by ovine prolactin was found in these cells. Although at high concentrations binding of pGH to the canine PRLR can occur (albeit with a low pKa), the similar dose-dependent effect of oPRL on Stat5a phosphorylation indicated that pGH signaled through the GHR. Remarkably, pGH induced a moderately decreased proliferation of CMT-U335 tumor cells, which may indicate that GH induces differentiation in these tumor cells. The GH-induced activation of Stat5a and Stat5b in these cells, as part of the JAK/Stat signal transduction pathway, is consistent with mammary GH playing a role in autocrine and/or paracrine stimulation of (tumorous) mammary cells.