Cancer Influences the Elemental Composition of the Myocardium More Strongly than Conjugated Linoleic Acids-Chemometric Approach to Cardio-Oncological Studies.

The aim of the study was to verify in a cardio-oncological model experiment if conjugated linoleic acids (CLA) fed to rats with mammary tumors affect the content of selected macro- and microelements in their myocardium. The diet of Sprague-Dawley females was supplemented either with CLA isomers or with safflower oil. In hearts of rats suffering from breast cancer, selected elements were analyzed with a quadrupole mass spectrometer with inductively coupled plasma ionization (ICP-MS). In order to better understand the data trends, cluster analysis, principal component analysis and linear discriminant analysis were applied. Mammary tumors influenced macro- and microelements content in the myocardium to a greater extent than applied diet supplementation. Significant influences of diet (p = 0.0192), mammary tumors (p = 0.0200) and interactions of both factors (p = 0.0151) were documented in terms of Fe content. CLA significantly decreased the contents of Cu and Mn (p = 0.0158 and p = 0.0265, respectively). The level of Ni was significantly higher (p = 0.0073), which was more pronounced in groups supplemented with CLA. The obtained results confirmed antioxidant properties of CLA and the relationship with Se deposition. Chemometric techniques distinctly showed that the coexisting pathological process induced differences to the greater extent than diet supplementation in the elemental content in the myocardium, which may impinge on cardiac tissue's susceptibility to injuries.

Characterization of multicellular breast tumor spheroids using image data-driven biophysical mathematical modeling.

Multicellular tumor spheroid (MCTS) systems provide an in vitro cell culture model system which mimics many of the complexities of an in vivo solid tumor and tumor microenvironment, and are often used to study cancer cell growth and drug efficacy. Here, we present a coupled experimental-computational framework to estimate phenotypic growth and biophysical tumor microenvironment properties. This novel framework utilizes standard microscopy imaging of MCTS systems to drive a biophysical mathematical model of MCTS growth and mechanical interactions. By extending our previous in vivo mechanically-coupled reaction-diffusion modeling framework we developed a microscopy image processing framework capable of mechanistic characterization of MCTS systems. Using MDA-MB-231 breast cancer MCTS, we estimated biophysical parameters of cellular diffusion, rate of cellular proliferation, and cellular tractions forces. We found significant differences in these model-based biophysical parameters throughout the treatment time course between untreated and treated MCTS systems, whereas traditional size-based morphometric parameters were inconclusive. The proposed experimental-computational framework estimates mechanistic MCTS growth and invasion parameters with significant potential to assist in better and more precise assessment of in vitro drug efficacy through the development of computational analysis methodologies for three-dimensional cell culture systems to improve the development and evaluation of antineoplastic drugs.

Blood Interactions, Pharmacokinetics, and Depth-Dependent Ablation of Rat Mammary Tumors with Photoactivatable, Liposomal Doxorubicin.

Photosensitizers can be integrated with drug delivery vehicles to develop chemophototherapy agents with antitumor synergy between chemo- and photocomponents. Long-circulating doxorubicin (Dox) in porphyrin-phospholipid (PoP) liposomes (LC-Dox-PoP) incorporates a phospholipid-like photosensitizer (2 mole %) in the bilayer of Dox-loaded stealth liposomes. Hematological effects of endotoxin-minimized LC-Dox-PoP were characterized via standardized assays. In vitro interaction with erythrocytes, platelets, and plasma coagulation cascade were generally unremarkable, whereas complement activation was found to be similar to that of commercial Doxil. Blood partitioning suggested that both the Dox and PoP components of LC-Dox-PoP were stably entrapped or incorporated in liposomes. This was further confirmed with pharmacokinetic studies in Fischer rats, which showed the PoP and Dox components of the liposomes both had nearly identical, long circulation half-lives (25-26 hours). In a large orthotopic mammary tumor model in Fischer rats, following intravenous dosing (2 mg/kg Dox), the depth of enhanced Dox delivery in response to 665 nm laser irradiation was over 1 cm. LC-Dox-PoP with laser treatment cured or potently suppressed tumor growth, with greater efficacy observed in tumors 0.8 to 1.2 cm, compared with larger ones. The skin at the treatment site healed within approximately 30 days. Taken together, these data provide insight into nanocharacterization and photo-ablation parameters for a chemophototherapy agent.

Expression of iron-related proteins in feline and canine mammary gland reveals unexpected accumulation of iron.

Dysregulation of cellular iron homeostasis in human breast cancer is reflected by the altered expression of regulatory proteins. The expressions of iron-related proteins in the mammary glands of cats and dogs have not been assessed. We evaluated the expressions of ferritin, ferroportin, hepcidin and transferrin receptor 1 in benign and malignant mammary gland lesions in cats and dogs. Iron deposition was detected using Perls' Prussian blue staining. We found no major differences in the expression of iron-related proteins between benign and malignant mammary gland lesions in either cats or dogs; however, these species exhibited accumulation of iron in benign lesions. Our findings provide an explanation for the absence of higher iron requirements by tumor cells in these animals. Further investigation of local iron homeostasis in cats and dogs and differences in their physiology compared to human breast cancer is required.

Prolactin and growth hormone immunoactivity in canine mammary adenomas and adenocarcinomas.

It is now widely accepted in human medicine that prolactin (PRL) and growth hormone (GH) function in the mammary gland in an autocrine and paracrine manner in tumour formation. The aim of this study was to compare PRL and GH immunoactivity in canine mammary tumours submitted for histopathologic evaluation. Formalin-fixed specimens from spontaneously occurring mammary adenomas and adenocarcinomas from 24 female client-owned dogs were used. Information pertaining to the reproductive status of the patient at the time of mammary tumour diagnosis was obtained from each of the submitting veterinarians. Tissues were paraffin-embedded and sectioned (5 µm) onto charged slides. All slides were deparaffinized and rehydrated. Endogenous peroxidase activity was inactivated with 3% H2 O2, and non-specific binding was blocked. Polyclonal rabbit antihuman PRL (DAKO A0569) and GH antibody (DAKO A0570) were applied at a 1:250 and 1:200 dilutions, respectively. A universal rabbit negative control (DAKO N1699) was used. Slides were then reacted with anti-rabbit horseradish peroxidase followed by Nova Red Peroxidase substrate. Slides were counter-stained with haematoxylin, dehydrated and mounted. Tumour type and reproductive status at time of tumour diagnosis were compared individually between tumours that were negative or positive for PRL and GH using a two-tailed analysis of variance. Significance was defined as p < .05. There was no significant relationship between tumour type and PRL and GH presence. In addition, reproductive status at the time of tumour removal was found to be not significant. These results vary from previous reports in canine mammary tumours and warrant further investigation.

Quantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implications.

The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumours (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR determinations in tumour tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concentrations were statistically significantly associated with tumour relapse and/or distant metastasis during follow-up and with reduced disease-free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non-IMC tumours (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histological aggressiveness and especially in cases of metastatic phenotype and poor prognosis.

Exploring new biomarkers in the tumour microenvironment of canine inflammatory mammary tumours.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (CIMC) are the most aggressive forms of mammary cancer. Current research aims to identify new therapeutic targets. Here, we investigated gene expression levels of biomarkers associated with the inflammatory microenvironment. A total of 32 formalin-fixed paraffin-embedded samples of canine mammary carcinoma (CIMC = 26; non-CIMC = 6) were used and their cDNA subjected to quantitative polymerase chain reaction (qPCR) to establish gene expression levels for mediators commonly implicated in linking carcinogenesis with inflammation. Gene expression differences between CIMC and non-CIMC types were obtained for cyclooxygenase 2 (COX-2) (P = 0.004), synuclein gamma (SNCG) (P = 0.006), tribbles 1 (P = 0.025), vascular endothelial growth factor (VEGF) (P = 0.017) and CSF1R (P = 0.045). Among these biomarkers correlations were found, particularly between SNCG and tribbles 1 (r = 0.512, P = 0.001). The efficient metastasis of CIMC is intimately linked to components in the tumour microenvironment. This study suggests that upregulation and correlation of SNCG and tribbles 1 deserves to be further explored.

Evaluation of vascular endothelial growth factor gene and protein expression in canine metastatic mammary carcinomas.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that could be associated with the induction of endothelial cell proliferation and metastasis. In this study, we evaluated VEGF gene and protein expression in canine mammary tumors (CMT), including metastatic carcinomas, to determine if there is an influence of this marker in the malignant processes and aggressiveness of CMT. We also compared VEGF protein levels with clinicopathological features. The VEGF gene and protein expression levels were evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples, benign mammary tumors, nonmetastatic mammary carcinomas, and metastatic mammary carcinomas. High VEGF gene and protein levels were associated with malignant tumors compared with normal mammary glands (p = 0.0089 and p < 0.0001, respectively). Benign tumors showed an increased VEGF protein expression compared with normal samples (p = 0.0467). No significant differences in VEGF gene or protein levels were detected between benign and malignant tumors or between nonmetastatic and metastatic carcinomas, suggesting an absence in the correlation of VEGF with malignant processes and aggressiveness of CMT. No correlation of VEGF expression with clinical and histopathological parameters was observed, suggesting that VEGF could be important in the beginning of the mammary gland carcinogenic process and could be related to survival time.

Molecular based subtyping of feline mammary carcinomas and clinicopathological characterization.

Molecular classification of feline mammary carcinomas (FMC) from which specific behavioral patterns may be estimated has potential applications in veterinary clinical practice and in comparative oncology. In this perspective, the main goal of this study was to characterize both the clinical and the pathological features of the different molecular phenotypes found in a population of FMC (n = 102), using the broadly accepted IHC-based classification established by St. Gallen International Expert Consensus panel. The luminal B/HER2-negative subtype was the most common (29.4%, 30/102) followed by luminal B/HER2-positive subtype (19.6%, 20/102), triple negative basal-like (16.7%, 17/102), luminal A (14.7%, 15/102), triple negative normal-like (12.7%, 13/102) and finally, HER2-positive subtype (6.9%, 7/102). Luminal A subtype was significantly associated with smaller tumors (p = 0.024) and with well differentiated ones (p < 0.001), contrasting with the triple negative basal-like subtype, that was associated with larger and poorly differentiated tumors (p < 0.001), and with the presence of necrotic areas in the tumoral lesion (p = 0.003). In the survival analysis, cats with Luminal A subtype presented the highest survival time (mean OS = 943.6 days) and animals with triple negative basal-like subtype exhibited the lowest survival time (OS mean = 368.9 days). Moreover, two thirds (64%, 32/50) of the queens with multiple primary tumors showed different molecular subtypes in each carcinoma, revealing that all independent lesions should be analyzed in order to improve the clinical management of animals. Finally, the similarities between the subtypes of feline mammary tumors and human breast cancer, reveal that feline can be a valuable model for comparative studies.

Identification and quantitation of lipid C=C location isomers: A shotgun lipidomics approach enabled by photochemical reaction.

The field of lipidomics has been significantly advanced by mass spectrometric analysis. The distinction and quantitation of the unsaturated lipid isomers, however, remain a long-standing challenge. In this study, we have developed an analytical tool for both identification and quantitation of lipid C=C location isomers from complex mixtures using online Paternò-Büchi reaction coupled with tandem mass spectrometry (MS/MS). The potential of this method has been demonstrated with an implementation into shotgun lipid analysis of animal tissues. Among 96 of the unsaturated fatty acids and glycerophospholipids identified from rat brain tissue, 50% of them were found as mixtures of C=C location isomers; for the first time, to our knowledge, the quantitative information of lipid C=C isomers from a broad range of classes was obtained. This method also enabled facile cross-tissue examinations, which revealed significant changes in C=C location isomer compositions of a series of fatty acids and glycerophospholipid (GP) species between the normal and cancerous tissues.

Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution.

The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (∼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency.

Four cases of cell cannibalism in highly malignant feline and canine tumors.

Four cases of tumors in which cell internalization was frequently visualized are reported: one feline mammary carcinoma, one feline cutaneous squamous cell carcinoma, one canine pulmonary squamous cell carcinoma and one canine pleural mesothelioma. Cell internalization was observed by cytology in two of these cases (the feline mammary tumour and the pleural effusion in the canine mesothelioma) and by histopathology in all but the canine mesothelioma. Immunohistochemical staining for pancytokeratin was positive for both internalized and host cells, while E-cadherin expression was frequently absent, although internalized cells occasionally stained positive. This cell-to-cell interaction seems to be associated with tumors displaying a strong epithelial-mesenchymal transitional phenotype, in which cancer cells become engulfed by other cancer cells. Such event could be regarded as an important hallmark of very high malignancy.

Serum and Tissue Steroid Hormone Levels in Canine Mammary Tumours: Clinical and Prognostic Implications.

Hormonal dependency of canine mammary tumours (CMT) has been studied over the last few decades. However, studies assessing the prognostic and predictive potential of serum and/or tissue steroid hormone levels are still scarce in CMT. To the best of our knowledge, this is the first report relating serum and tissue levels of steroid hormones and prognosis in dogs. Serum and tumour tissue from 45 female dogs with spontaneous CMT were included in the study. Moreover, serum and normal mammary tissue from 13 healthy female dogs were also included as controls. Steroid hormones were determined by competitive enzyme immunoassay. Overall, levels of steroid hormones in serum and tissue homogenates were significantly different between malignant and benign mammary tumours (p < 0.01), except for progesterone (P4) serum levels that revealed no statistical differences between groups. In malignant tumours, oestrone sulphate (SO4E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T) and P4 elevated tissue concentrations were significantly associated with tumour relapse and/or distant metastasis during follow-up. A significant association was found between elevated tissue SO4E1 (p = 0.003), 17ß-oestradiol (E2) (p = 0.036), DHEA (p = 0.022), A4 (p = 0.001) and P4 (p = 0.013) concentrations and shorter disease-free survival and overall survival in female dogs with malignant mammary tumours. The high levels of tissue steroids found in cases of poor prognosis open the possibility of additional new therapeutic approaches. Future clinical trials will be needed to clarify the usefulness of targeting steroid hormones in the treatment of this neoplastic disease.

Invasive ductular carcinoma in 2 rhesus macaques (Macaca mulatta).

In the United States, breast cancer is the most common malignancy among women, with an estimated lifetime incidence of approximately 12% in American women. Invasive ductal carcinoma is the most common form of breast cancer in women, accounting for approximately 60% of all breast carcinomas. Prognostic markers are used to assess aggressiveness, invasiveness, and extent of spread of a neoplasm and thus may be correlated with patient survival. Immunohistochemistry is currently widely used for this purpose, with a variety of prognostication markers available. Classic markers for breast cancer in women include estrogen and progesterone receptor steroid hormone proteins and human epidermal growth factor receptor 2. Many additional markers have been used in diagnosis and prognostication, including p53, p63, and E-cadherin and cell proliferation markers such as Ki67. Despite an estimated lifetime incidence of approximately 6.1%, naturally occurring mammary neoplasms in nonhuman primates are uncommonly reported, with only sporadic references over the past 75 y. The majority of reported tumors occur in rhesus macaques, although this prevalence has been suggested to be a consequence of their high frequency of usage in biomedical research. Here we present 2 cases of mammary carcinoma in adult female intact rhesus macaques, with cytology, histopathology, and extensive immunohistochemical analysis. According to current classifications for human breast tumors, both tumors were classified as invasive ductal carcinoma. The prognostic value of immunohistochemical markers in human breast cancer and in reported cases in nonhuman primates is discussed.

Fucoidan inhibited 4T1 mouse breast cancer cell growth in vivo and in vitro via downregulation of Wnt/ß-catenin signaling.

Fucoidan is a sulfated polysaccharide derived from brown algae and is known to possess anticancer properties. However, the relationship between fucoidan and ß-catenin, one of the key components of the Wnt signaling pathway, in mouse breast cancer remains poorly characterized. In this study, mouse breast cancer cells (4T1) were exposed to fucoidan to investigate the relationship between fucoidan and the Wnt/ß-catenin signaling pathway in vivo and in vitro. We found that fucoidan significantly inhibited cell growth, increased cell death, and induced G1 cell cycle arrest in 4T1 cells. Fucoidan also reduced ß-catenin expression and T cell factor/lymphoid-enhancing factor reporter activity. Furthermore, fucoidan downregulated the expression of downstream target genes such as c-myc, cyclin D1, and survivin. Intraperitoneal injection of fucoidan in tumor-bearing mice reduced the tumor volume and weight. Fucoidan induced aberrant downregulation of ß-catenin in tumor tissues with a significant increase in apoptosis. Thus, our data suggested that fucoidan exerts its anticancer activity through downregulation of Wnt/ß-catenin signaling. Fucoidan may be an effective therapy for the chemoprevention and treatment of mouse breast cancer.

Heterologous expression and functional characterization of matrix metalloproteinase-11 from canine mammary tumor.

Matrix metalloproteinases (MMPs) are reported to be involved in tumor growth, apoptosis, angiogenesis, invasion, and development of metastases. These are zinc containing metalloproteases, known for their role in extracellular matrix degradation. MMP-11 (stromelysin3) is reported to be highly expressed in breast cancer, therefore it may act as marker enzyme for breast cancer progression. The present work was carried out to produce recombinant canine (Canis lupus familiaris) MMP-11 lacking the signal and propeptide in E. coli by optimizing its expression and purification in biologically active form and to functionally characterize it. A bacterial protein expression vector pPROEX HTc was used. The MMP-11 mature peptide encoding gene was successfully cloned and expressed in E. coli and the purified recombinant enzyme was found to be functionally active. The recombinant enzyme exhibited caseinolytic activity and could be activated by Trypsin and 4-Amino phenyl mercuric acetate (APMA). However Ethylene diamine tertra acetate (EDTA) inhibited the enzyme's caseinolytic activity. The recombinant enzyme degraded extracellular matrix constituents and facilitated migration of MDCK (Madin-Darby canine kidney) cells through BD Biocoat Matrigel invasion chambers. These results suggest that in vivo MMP-11 could play a significant role in the turnover of extracellular matrix constituents.

Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential.

Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs.

Fractionated photothermal antitumor therapy with multidye nanoparticles.

PURPOSE: Photothermal therapy is an emerging cancer treatment paradigm which involves highly localized heating and killing of tumor cells, due to the presence of nanomaterials that can strongly absorb near-infrared (NIR) light. In addition to having deep penetration depths in tissue, NIR light is innocuous to normal cells. Little is known currently about the fate of nanomaterials post photothermal ablation and the implications thereof. The purpose of this investigation was to define the intratumoral fate of nanoparticles (NPs) after photothermal therapy in vivo and characterize the use of novel multidye theranostic NPs (MDT-NPs) for fractionated photothermal antitumor therapy. METHODS: The photothermal and fluorescent properties of MDT-NPs were first characterized. To investigate the fate of nanomaterials following photothermal ablation in vivo, novel MDT-NPs and a murine mammary tumor model were used. Intratumoral injection of MDT-NPs and real-time fluorescence imaging before and after fractionated photothermal therapy was performed to study the intratumoral fate of MDT-NPs. Gross tumor and histological changes were made comparing MDT-NP treated and control tumor-bearing mice. RESULTS: The dual dye-loaded mesoporous NPs (ie, MDT-NPs; circa 100 nm) retained both their NIR absorbing and NIR fluorescent capabilities after photoactivation. In vivo MDT-NPs remained localized in the intratumoral position after photothermal ablation. With fractionated photothermal therapy, there was significant treatment effect observed macroscopically (P = 0.026) in experimental tumor-bearing mice compared to control treated tumor-bearing mice. CONCLUSION: Fractionated photothermal therapy for cancer represents a new therapeutic paradigm enabled by the application of novel functional nanomaterials. MDT-NPs may advance clinical treatment of cancer by enabling fractionated real-time image guided photothermal therapy.

Preliminary report on the expression of leptin and leptin receptor (ObR) in normal, hyperplastic and neoplastic canine mammary tissues.

Leptin and its receptor (ObR) expression were investigated by immunohistochemistry in normal, hyperplastic and neoplastic canine mammary tissues and related to clinical-pathological features. Leptin expression was detected in healthy mammary tissues, adenosis and in benign mammary tumours and was lower in ductal hyperplasias and malignant tumours. A high percentage of ObR-positive cells were present in adenosis, benign tumours and in complex carcinomas, while ObR expression was lower in healthy mammary tissues, in ductal hyperplasias and in a large part of invasive mammary carcinomas. Our data demonstrated that cancer cells expressed at low level leptin and ObR in canine mammary tumours with a more aggressive behaviour, as well as in lymph node metastases. Consequently, leptin and ObR expressions in this species resulted to be not associated with a reduced overall survival.

Overexpression of α-enolase correlates with poor survival in canine mammary carcinoma.

BACKGROUND: α-Enolase (ENO1) is a key glycolytic enzyme implicated in the development of many human cancers including breast cancer. Increased expression of ENO1 has recently been reported in estrogen (ER)-positive human breast cancer patients. The present study examined the expression of ENO1 and assessed its significance in canine mammary carcinoma. RESULTS: Immunohistochemical staining was employed to investigate the expression of ENO1 in 82 cases of canine mammary tumor (32 benign tumors and 50 carcinomas). Quantification of immunohistochemistry was carried out using Quick score and the results showed cytoplasmic ENO1 overexpression in 9 of the 50 carcinomas (18%). Overexpression of ENO1 correlated significantly with shorter cause-specific survival (P = 0.019), but was not associated with ER positivity in canine mammary carcinoma. CONCLUSIONS: Our findings suggest that overexpression of ENO1 may be used as a prognostic marker for poor outcome in canine mammary carcinoma.