Alpha basic crystallin expression in canine mammary tumors

The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, αB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of αB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with αB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). αB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of αB-c and primary mammary gland tumors in canines.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

NLRP3炎症小体及其下游炎症因子在犬乳腺肿瘤组织中的表达

本研究旨在明确NLRP3炎症小体及下游炎症因子在犬乳腺肿瘤中的表达及临床意义.采用RT-qPCR方法检测45例犬乳腺肿瘤组织(15例良性肿瘤和30例恶性肿瘤)以及16例肿瘤旁正常乳腺组织中的NLRP3、Caspase-1、IL-1β和IL-18 mRNA的表达水平,并分析NLRP3与肿瘤临床病理学特征的关系(肿瘤大小、...     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

犬乳腺肿瘤的病理组织学观察及p63蛋白的表达

通过对35例犬乳腺肿瘤的临床病史调查、光学显微镜观察和免疫组化染色,探讨了犬乳腺肿瘤的临床病理形态学特点、诊断和鉴别诊断.结果显示34例发生在5岁以上,6岁～10岁为发病高峰期;51.4％的病例为恶性肿瘤,其中44.4％的乳腺癌发生转移.免疫组化染色结果显示,在正常乳腺和良性肿瘤组织中,p63蛋白100％表达,并且仅仅...     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

Expression of maspin in mammary gland tumors of the dog

Maspin is a serine protease inhibitor that inhibits tumor invasion and metastasis in human breast cancer and is consistently expressed by mammary myoepithelial cells (MECs). To analyze the value of maspin as a marker of the MEC layer of the normal and tumoral canine mammary gland, the immunohistochemical expression of maspin was studied in formalin-fixed tissues from 55 benign and malignant tumors (40 tumors also contained the surrounding normal mammary gland) using a commercially available monoclonal antibody. Periacinar and periductal MECs of all 40 normal mammary glands were stained by the anti-human maspin monoclonal antibody, and immunoreactivity was observed in the nucleus and cytoplasm of these cells. In addition, maspin was found in 53 (98%) of the tumors studied, reacting with the MECs in 100% of benign tumors and 93% of malignant tumors and to the epithelial cells of 16% of benign and 73% of malignant tumors. In the MEC compartment, immunoreactivity was observed in the cytoplasm of hypertrophic MECs, fusiform MECs, stellate MECs, rounded (myoepithelial) cells, and chondroblasts. In the epithelial cell compartment, immunoreactivity was observed in the cytoplasm of cells with and without squamous differentiation. Stromal myofibroblasts were unreactive. Maspin appears to be a very sensitive marker of the normal and neoplastic myoepithelium that, contrary to smooth muscle differentiation markers, does not stain stromal myofibroblasts. In addition, a subset of neoplastic epithelial cells reacted with the maspin antibody. The relationship between maspin expression in different cellular compartments of canine mammary carcinomas and the biologic aggressiveness of the disease remains to be elucidated.     

Decorin concentrations in canine normal and neoplastic mammary gland tissues

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.     

Expression and the molecular forms of neutrophil gelatinase‐associated lipocalin and matrix metalloproteinase 9 in canine mammary tumours

Neutrophil gelatinase‐associated lipocalin (NGAL) is a new biomarker for renal injury. It is also involved in tumorigenesis of different human cancer types. The oncogenic role of NGAL is related to its molecular forms, and heterodimer formation with matrix metalloproteinase 9 (MMP9) promotes human breast cancer (HBC) invasion and metastasis. To date, the levels of NGAL and NGAL/MMP9 complex have not yet been explored in canine mammary tumours (CMTs). Hence, this study aimed to investigate whether NGAL and its molecular forms could be the biomarker for CMT diagnosis. To this end, expression profile of NGAL and MMP9 in mammary epithelial cells as well as in urine samples were detected. By immunohistochemistry staining, NGAL was expressed at variable levels. Unlike HBC, a significant reduction in NGAL expression was demonstrated in benign and malignant CMTs as compared with normal controls. Additionally, NGAL expression was significantly reduced in dogs with metastatic CMTs. By contrast, the mean score of MMP9 expression in ascending order was normal groups, benign, and malignant CMTs. Interestingly, analysis of the molecular form revealed the NGAL/MMP9 complex presents in most mammary tissues and urine of dogs with benign or malignant CMTs, whereas the complex was absent in samples from dogs without CMTs. In conclusion, NGAL and MMP9 are ubiquitously expressed in canine mammary epithelial cells in normal and cancerous status. However, the NGAL/MMP9 complex exclusively presents in mammary tissues and urine of dogs with tumours.     

Expression of a tumor-associated antigen, RCAS1, in canine mammary tumors

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), one of novel cancer cell-surface antigens, is strongly expressed in invasive cancers. RCAS1 inhibits the in vitro growth of lymphocytes such as T cells and natural killer (NK) cells, and induces apoptotic cell death. We investigated the expression of RCAS1 in canine mammary tumor cell lines and tumor cells by immunohistochemistry, and also in situ deoxyribonucleic acid (DNA) fragmentation in tumor-infiltrating lymphocytes (TILs) by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. All canine mammary tumor cell lines expressed RCAS1 at both the messenger ribonucleic acid (mRNA) and protein level. Immunohistochemically, RCAS1 was negative in 100% of normal mammary glands, but was expressed in 100% of malignant tumors examined. In most malignant mammary tumors, RCAS1 was localized in the cytoplasm with no polarity of expression. In benign mammary tumors, it was detected on the luminal surface of the tumor cell. RCAS1 expression or localization was significantly correlated with malignancy. In situ DNA fragmentation of CD3-positive TILs was observed in RCAS1-expressing tumors. RCAS1-expressing tumors, indicating a possible induction of apoptotic cell death in TILs through RCAS1 expression. These observations suggest that RCAS1 probably plays an important role in tumor progression and escape from immune surveillance in canine mammary tumors.     

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

p63: a novel myoepithelial cell marker in canine mammary tissues

Several immunohistochemical markers have been used to demonstrate the presence of myoepithelial cells in order to determine their role in the histogenesis of mammary tumors. p63, a recently characterized p53 homologue, is consistently expressed in myoepithelial cells of the human breast; however, no assessment of its immunoreactivity has been reported so far in canine mammary tissues. We investigated p63 immunohistochemical expression, as a novel myoepithelial cell nuclear marker, in 81 samples of normal (n = 2), hyperplastic (n = 11), and neoplastic (n = 68) canine mammary tissues. Myoepithelial phenotype was confirmed by using complementary monoclonal antibodies: alpha-smooth muscle actin, cytokeratin 14, cytokeratin AE1/AE3, and vimentin. p63 expression was observed in 91.4% (74/81) of the samples evaluated. Normal mammary glands, mammary hyperplasias, and benign tumors showed 100% immunoreactivity, with p63 expression restricted to myoepithelial cell nuclei. In general, benign mixed tumors showed a basal cell compartment immunoreactive to p63, with a gradual decrease of its expression during myoepithelial transformation. p63 expression was found in 72% of malignant tumors, allowing myoepithelial or basal cell identification in spindle-cell carcinomas (2/2), tubulopapillary carcinomas (8/9), solid carcinomas (7/10), and carcinosarcomas (1/3). The osteosarcoma analyzed was p63 negative. In our series, stromal components were consistently nonreactive to p63. In conclusion, the present study reveals p63 as a sensitive and highly specific marker of myoepithelial cells in canine mammary tissues, and the authors suggest p63 as an additional marker for defining myoepithelial histogenesis.     

Mast cells in canine mammary gland tumour: number, distribution and EPOR positivity

Erythropoietin (EPO)-mediated mitogenic and anti-apoptotic effects involve all the cells expressing functional receptors for EPO (EPOR), as demonstrated by in vitro and in vivo studies. EPO shows pleiotropic effects and acts as an endogenous mediator of adaptive tissue response to metabolic stress protecting tissues from different injuries. Recently, the EPO/EPOR complex has been identified in several neoplastic cell lines and solid tumours. In this study, the authors investigated the mast cells (MCs) number, distribution and their immunoreactivity for EPOR in normal, dysplastic and neoplastic canine mammary gland. The results showed that MCs were more numerous in displastic glands compared with normal and neoplastic glands. As far as the EPOR immunoreactivity is concerned, we did not observe MCs reaction on cancer, in contrast with previously published data where epithelium of neoplastic gland showed an increase in EPOR expression along with the neoplastic progression. Overall, our results might be suggestive for MCs role in oncogenesis and offer new insight regarding to the expression of EPOR in mammary gland cancer in dog.     

Immunohistochemical staining for S100 protein in the diagnosis of canine amelanotic melanoma

Formalin-fixed, paraffin-embedded tissue samples of canine amelanotic melanomas and normal canine tissues were studied immunohistochemically for the presence of S100 protein. Use of the avidin-biotin complex procedure demonstrated variable amounts of S100 protein in the tumor cell cytoplasm and nuclei in 26 of 31 tumors. S100 protein was not observed in some other common canine skin tumors stained by the avidin-biotin complex technique. These were a mast cell tumor, fibrosarcoma, mammary gland adenocarcinoma, histiocytoma, transmissible venereal tumor, and a thyroid gland adenocarcinoma. Among normal tissues the presence of S100 protein was demonstrated in chondrocytes in the trachea, myoepithelial cells in the breast, melanocytes in the skin, some sweat glands and ducts in the skin, stellate cells in the pituitary, and interdigitating reticulum cells in the lymph node and in Peyer's patches. These results indicate that the avidin-biotin complex procedure for demonstrating S100 protein is a useful diagnostic tool in the diagnosis of canine amelanotic melanoma.     

血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析

本研究旨在深入探讨血小板凝血酶蛋白1(THBS1)对犬乳腺肿瘤细胞CHMp的影响,并阐明其影响犬乳腺肿瘤发生发展的作用机制。通过构建THBS1慢病毒稳定过表达和THBS1沉默表达的犬乳腺肿瘤细胞CHMp细胞系,采用CCK-8试验、划痕试验、Transwell试验、原位荧光检测和流式细胞术检测THBS1对CHMp细胞增殖、迁移、侵袭、细胞凋亡和细胞周期情况的影响;通过qRT-PCR和Western blot检测THBS1对CHMp细胞凋亡相关因子(p53、Bcl-2、Bax)表达情况的影响,验证THBS1对CHMp细胞凋亡通路的影响。结果显示,过表达THBS1能有效增强犬乳腺肿瘤细胞CHMp的增殖、迁移和侵袭能力,并且减少CHMp细胞的凋亡数量,而沉默THBS1后结果与之相反;流式细胞术得出THBS1能够影响CHMp细胞的细胞周期分布;经qRT-PCR和Western blot检测发现,在THBS1过表达后,p53和Bcl-2的表达量均明显增高,Bax的表达量明显减少,在沉默THBS1后结果与之相反。结果表明,THBS1的异常表达能够影响犬乳腺肿瘤细胞CHMp的增殖、迁移、侵袭以及细胞周期;此外,THBS1能够通过影响细胞凋亡因子p53、Bcl-2和Bax的表达来影响CHMp细胞凋亡相关通路,进而影响犬乳腺肿瘤的发生发展。     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.