Publications by authors named "Renée Laufer Amorim"

48 Publications

Investigation of Prognostic Value of Claudin-5, PSMA, and Ki67 Expression in Canine Splenic Hemangiosarcoma.

Animals (Basel) 2021 Aug 14;11(8). Epub 2021 Aug 14.

Department of Veterinary Surgery and Animal Reproduction, Sao Paulo State University-UNESP, Botucatu 18618-681, Brazil.

Splenic hemangiosarcoma (HSA) is a malignant tumor of endothelial cells that affects middle-aged and elderly dogs and is characterized by the formation of new blood vessels, commonly associated with necrotic and hemorrhagic areas. Despite its importance in veterinary medicine, few studies have identified markers with prognostic value for canine HSA. Thus, this study aimed to associate the clinicopathological findings (prostate-specific membrane antigen [PSMA], Claudin-5, and Ki67 gene and protein expression) with overall survival in HSA-affected patients. Fifty-three formalin-fixed and paraffin-embedded canine splenic HSA samples, previously diagnosed by histopathological examination, were used in this study. Claudin-5, PSMA, and Ki67 protein expression levels were evaluated by immunohistochemistry, and gene expression was evaluated by quantitative polymerase chain reaction. Claudin-5 protein overexpression was observed in patients with metastasis ( = 0.0078) and with stage III tumors compared to those with stage I and II tumors ( = 0.0451). In patients treated with surgery alone, low PSMA gene and protein expression ( = 0.05 and = 0.0355, respectively) were associated with longer survival time. Longer survival time was observed in patients with a low Ki67 index ( = 0.0488). Our results indicate that Claudin-5 protein expression is associated with metastatic status, and PSMA gene and protein expression, and Ki67 index are associated with survival time.
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http://dx.doi.org/10.3390/ani11082406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388721PMC
August 2021

Editorial: Precision Medicine in Veterinary Oncology.

Front Vet Sci 2021 14;8:718891. Epub 2021 Jul 14.

Department of Veterinary Clinic, São Paulo State University-UNESP, Botucatu, Brazil.

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http://dx.doi.org/10.3389/fvets.2021.718891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316583PMC
July 2021

Expression and prognostic significance of vascular endothelial growth factor-A (VEGF-A) and its receptor in canine prostate cancer.

Prostate 2021 Oct 28;81(14):1021-1031. Epub 2021 Jul 28.

Department of Veterinary Surgery and Anesthesiology, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu, Sao Paulo, Brazil.

Background: Vascular endothelial growth factor-A (VEGF-A) and its receptor, VEGF receptor-2 (VEGFR-2), represent a complex family of angiogenic molecules consisting of different ligands and receptors. Due to the importance of VEGF-A/VEGFR-2 signaling in tumor proliferation and angiogenesis, this study aimed to evaluate the protein and gene expression levels of VEGF-A and VEGFR-2 in canine prostate cancer (PC).

Methods: We analyzed VEGF-A and VEGFR-2 expression in 87 PC samples by immunohistochemistry and quantitative-polymerase chain reaction. PC samples were graded according to the Gleason score and the immunohistochemical staining for VEGF-A and VEGFR-2 was quantified using a selected threshold from the ImageJ Software. Microvascular density was assessed by cluster of differentiation 31 staining and counting the number of positive vessels. Additionally, the homology of VEGF-A and VEGFR-2 between humans and dogs was assessed, followed by the construction of a protein structure homology model to compare the tertiary structures of these proteins in both species.

Results: Negative to weakly positive expression levels of VEGF-A and VEGFR-2 were observed in the epithelial cells of the normal prostate (NP) and prostatic hyperplasia samples. In contrast, the canine proliferative atrophy and PC samples exhibited higher VEGF-A (p < .0001) and VEGFR-2 (p < .0001) compared to NP. Moreover, positive correlations between the expression levels of VEGF-A and VEGFR-2 (Spearman's coefficient (r) = .68, p = .013) and the expression levels of VEGF-A and VEGFR-2 proteins (r = .8, p < .0001) were also observed in the NP samples. Additionally, the patients with PC exhibiting higher VEGFR-2 expression levels experienced a shorter survival period (p = .0372). Furthermore, we found an association between the microvascular density and overall survival. Dogs with a higher number of vessels showed a shorter survival time. We further demonstrated that the VEGF-A and VEGFR-2 exhibited high homology between humans and dogs, and identified their protein structures in both species.

Conclusions: In conclusion, VEGFR-2 appears to be an independent prognostic factor in animals with PC. VEGF-A and VEGFR-2 are highly conserved between humans and dogs, which can be investigated further in future cross-species studies to explore their therapeutic applications.
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http://dx.doi.org/10.1002/pros.24199DOI Listing
October 2021

International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Vet Pathol 2021 Sep 20;58(5):766-794. Epub 2021 Jul 20.

Deceased.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.
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http://dx.doi.org/10.1177/03009858211013712DOI Listing
September 2021

Aggrecan, IL-1β, IL-6, and TNF-α profiles in the Articular Cartilage of Miniature Horses with Chondrodysplastic Dwarfism.

J Equine Vet Sci 2021 08 30;103:103643. Epub 2021 Apr 30.

São Paulo State University (UNESP), School of Veterinary Medicine and Animal Science, Department of Veterinary Clinical Science, Botucatu 18618-681, Brazil. Electronic address:

Dwarfism is a skeletal disorder that causes abnormal growth. In Miniature horses, dwarfism can occur as chondrodysplastic dwarfism, an autosomal recessive disorder associated with five mutations (D1, D2, D3*, D4 and c.6465A > T variant) in the aggrecan (ACAN) gene. The aim of this study was to evaluate the expression of aggrecan (at the gene and protein level) and specific cytokines (IL-1β, IL-6, and TNF-α) in the articular cartilage of Miniature horses with chondrodysplastic dwarfism (D4/c.6465A > T genotype). Metatarsal bone samples from eight dwarf Miniature horses were collected for histopathological analysis, and articular cartilage was collected to detect and quantify aggrecan levels through Western blotting and determine the relative expression levels of ACAN, IL-1β, IL-6, and TNF-α through qPCR. All affected animals presented chondrodysplasia-like lesions with disorganization of the chondrocyte layers and reduced the amount of an extracellular matrix. No significant difference in aggrecan expression levels in uncleaved samples from the dwarf and control groups (composed of phenotypically normal animals of similar age and breed (P = .7143)) was found using Western blotting. qPCR revealed that ACAN gene expression was higher in the affected animals than in normal animals (P = .0119). No significant difference in cytokine levels was detected between the groups. Mutant aggrecan may interfere with normal cellular function, leading to chondrodysplasia and the observed phenotypic findings.
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http://dx.doi.org/10.1016/j.jevs.2021.103643DOI Listing
August 2021

Effects of Lapatinib on HER2-Positive and HER2-Negative Canine Mammary Carcinoma Cells Cultured In Vitro.

Pharmaceutics 2021 Jun 17;13(6). Epub 2021 Jun 17.

Department of Veterinary Clinic, Sao Paulo State University-UNESP, Botucatu 18618-681, Brazil.

HER2 is a prognostic and predictive marker widely used in breast cancer. Lapatinib is a tyrosine kinase inhibitor that works by blocking the phosphorylation of the receptor HER2. Its use is related to relatively good results in the treatment of women with HER2+ breast cancer. Thus, this study aimed to verify the effects of lapatinib on four canine primary mammary gland carcinoma cell cultures and two paired metastatic cell cultures. Cultures were treated with lapatinib at concentrations of 100, 500, 1000 and 3000 nM for 24 h and the 50% inhibitory concentration (IC) for each cell culture was determined. In addition, a transwell assay was performed to assess the ability of lapatinib to inhibit cell migration. Furthermore, we verified expression by RT-qPCR analysis of cell cultures and formalin-fixed paraffin-embedded tissues from samples corresponding to those used in cell culture. Lapatinib was able to inhibit cell proliferation in all cell cultures, but it was not able to inhibit migration in all cell cultures. The higher the expression of in a culture, the more sensitive the culture was to treatment. This relationship may be an indication that the expression of may be a predictive factor and opens a new perspective for the treatment of primary and metastatic mammary gland cancer.
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http://dx.doi.org/10.3390/pharmaceutics13060897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8235449PMC
June 2021

Morphological and Molecular Characterization of Proliferative Inflammatory Atrophy in Canine Prostatic Samples.

Cancers (Basel) 2021 Apr 14;13(8). Epub 2021 Apr 14.

School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu 18618-681, Brazil.

Proliferative inflammatory atrophy (PIA) is an atrophic lesion of the prostate gland that occurs in men and dogs and is associated with a chronic inflammatory infiltrate. In this study, we retrospectively reviewed canine prostatic samples from intact dogs, identifying 50 normal prostates, 140 cases of prostatic hyperplasia, 171 cases of PIA, 84 with prostate cancer (PC), 14 with prostatic intraepithelial neoplasia (PIN) and 10 with bacterial prostatitis. PIA samples were then selected and classified according to the human classification. The presence of PIA lesions surrounding neoplastic areas was then evaluated to establish a morphological transition from normal to preneoplastic and neoplastic tissue. In addition, the expression of PTEN, P53, MDM2 and nuclear androgen receptor (AR) were analyzed in 20 normal samples and 20 PIA lesions by immunohistochemistry and qPCR. All PIA lesions showed variable degrees of mononuclear cell infiltration around the glands and simple atrophy was the most common histopathological feature. PIA was identified between normal glands and PC in 51 (61%) out of the 84 PC samples. PIA lesions were diffusely positive for molecular weight cytokeratin (HMWC). Decreased PTEN and AR gene and protein expression was found in PIA compared to normal samples. Overall, our results strongly suggest that PIA is a frequent lesion associated with PC. Additionally, this finding corroborates the hypothesis that in dogs, as is the case in humans, PIA is a pre neoplastic lesion that has the potential to progress into PC, indicating an alternative mechanism of prostate cancer development in dogs.
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http://dx.doi.org/10.3390/cancers13081887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071022PMC
April 2021

A Comparative in Silico Analysis of CD24's Prognostic Value in Human and Canine Prostate Cancer.

J Pers Med 2021 Mar 23;11(3). Epub 2021 Mar 23.

Department of Veterinary Surgery and Animal Reproduction, Sao Paulo State University-UNESP, Botucatu 18618-681, Brazil.

CD24 is a cell surface molecule anchored by glycosyl-phosphatidyl-inositol and expressed by different human cancers, including prostate cancer (PC). Some studies have demonstrated that CD24 expression is associated with poor patient outcome; however, few studies have investigated CD24 expression in spontaneous animal models of human PC, such as canine PC. This study aimed to evaluate the expression of CD24 in human PC using the in silico analysis of the data obtained from The Cancer Genome Atlas (TCGA) and comparing it with the previously published prostatic canine transcriptome data. In addition, CD24 expression was confirmed by immunohistochemistry in an independent cohort of canine prostatic samples and its prognostic significance assessed. The systematic review identified 10 publications fitting with the inclusion criteria of this study. Of the 10 manuscripts, 5 demonstrated a direct correlation between CD24 overexpression and patient prognoses. CD24 expression was also associated with PSA relapse (2/5) and tumor progression (1/5). However, the in silico analysis did not validate CD24 as a prognostic factor of human PC. Regarding canine PC, 10 out of 30 normal prostates and 27 out of 40 PC samples were positive for CD24. As in humans, there was no association with overall survival. Overall, our results demonstrated a significant CD24 overexpression in human and canine prostate cancer, although its prognostic value may be questionable. However, tumors overexpressing CD24 may be a reliable model for new target therapies and dogs could be used of a unique preclinical model for these studies.
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http://dx.doi.org/10.3390/jpm11030232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004660PMC
March 2021

Structural and Ultrastructural Morphological Evaluation of Giant Anteater () Prostate Gland.

Biology (Basel) 2021 Mar 17;10(3). Epub 2021 Mar 17.

Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu 18618-81, Brazil.

The giant anteater () is a vulnerable species from Central and South America, and is considered possibly extinct in Belize, Guatemala, El Salvador, and Uruguay. Due to the species' conservation and reproductive importance, this research aimed to characterize the morphology, histochemical, immunohistochemical, and ultrastructural feature of the giant anteater prostate gland. For this, we collected 11 giant anteater prostate glands and performed macroscopic, morphological, histochemical, immunohistochemical, and ultrastructural analysis. Nine prostate glands from an adult subject and two from young subjects were studied. Grossly, the adult giant anteater prostate gland is divided in two distinct zones; the central zones (composed mainly of ducts) and the peripheral zones (of acini formed by secretory cells). The secretory cells showed positive periodic acid-Schiff staining. Furthermore, the immunohistochemical characterization revealed a similar human prostate pattern, with p63 staining basal cells, uroplakin III (UPIII) superficial cells of prostatic urethra, androgen receptor (AR) expressing nucleus of secretory and stromal cells, and prostatic specific antigen (PSA) staining prostatic epithelial cells. Overall, our research provided an in-depth morphological description of the giant anteater's prostate gland, providing valuable information for futures studies focused on giant anteater conservation.
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http://dx.doi.org/10.3390/biology10030231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002480PMC
March 2021

Proteomics Approach of Rapamycin Anti-Tumoral Effect on Primary and Metastatic Canine Mammary Tumor Cells In Vitro.

Molecules 2021 Feb 25;26(5). Epub 2021 Feb 25.

Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu 18618-681, Brazil.

Rapamycin is an antifungal drug with antitumor activity and acts inhibiting the mTOR complex. Due to drug antitumor potential, the aim of this study was to evaluate its effect on a preclinical model of primary mammary gland tumors and their metastases from female dogs. Four cell lines from our cell bank, two from primary canine mammary tumors (UNESP-CM1, UNESP-CM60) and two metastases (UNESP-MM1, and UNESP-MM4) were cultured in vitro and investigated for rapamycin IC. Then, cell lines were treated with rapamycin IC dose and mRNA and protein were extracted in treated and non-treated cells to perform AKT, mTOR, PTEN and 4EBP1 gene expression and global proteomics by mass spectrometry. MTT assay demonstrated rapamycin IC dose for all different tumor cells between 2 and 10 μM. RT-qPCR from cultured cells, control versus treated group and primary tumor cells versus metastatic tumor cells, did not shown statistical differences. In proteomics were found 273 proteins in all groups, and after data normalization 49 and 92 proteins were used for statistical analysis for comparisons between control versus rapamycin treatment groups, and metastasis versus primary tumor versus metastasis rapamycin versus primary tumor rapamycin, respectively. Considering the two statistical analysis, four proteins, phosphoglycerate mutase, malate dehydrogenase, l-lactate dehydrogenase and nucleolin were found in decreased abundance in the rapamycin group and they are related with cellular metabolic processes and enhanced tumor malignant behavior. Two proteins, dihydrolipoamide dehydrogenase and superoxide dismutase, also related with metabolic processes, were found in higher abundance in rapamycin group and are associated with apoptosis. The results suggested that rapamycin was able to inhibit cell growth of mammary gland tumor and metastatic tumors cells in vitro, however, concentrations needed to reach the IC were higher when compared to other studies.
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http://dx.doi.org/10.3390/molecules26051213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956669PMC
February 2021

Transcriptome of Two Canine Prostate Cancer Cells Treated With Toceranib Phosphate Reveals Distinct Antitumor Profiles Associated With the PDGFR Pathway.

Front Vet Sci 2020 26;7:561212. Epub 2020 Nov 26.

Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu, Brazil.

Canine prostate cancer (PC) presents a poor antitumor response, usually late diagnosis and prognosis. Toceranib phosphate (TP) is a nonspecific inhibitor of receptor tyrosine kinases (RTKs), including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and c-KIT. This study aimed to evaluate VEGFR2, PDGFR-β, and c-KIT protein expression in two established canine PC cell lines (PC1 and PC2) and the transcriptome profile of the cells after treatment with TP. Immunofluorescence (IF) analysis revealed VEGFR2 and PDGFR-β protein expression and the absence of c-KIT protein expression in both cell lines. After TP treatment, only the viability of PC1 cells decreased in a dose-dependent manner. Transcriptome and enrichment analyses of treated PC1 cells revealed 181 upregulated genes, which were related to decreased angiogenesis and cell proliferation. In addition, we found upregulated β, and expression in PC1 cells, and the upregulation of β was also observed in treated PC1 cells by qPCR. PC2 cells had fewer protein-protein interactions (PPIs), with 18 upregulated and 22 downregulated genes; the upregulated genes were involved in the regulation of parallel pathways and mechanisms related to proliferation, which could be associated with the resistance observed after treatment. The canine PC1 cell line but not the PC2 cell line showed decreased viability after treatment with TP, although both cell lines expressed PDGFR and VEGFR receptors. Further studies could explain the mechanism of resistance in PC2 cells and provide a basis for personalized treatment for dogs with PC.
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http://dx.doi.org/10.3389/fvets.2020.561212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726326PMC
November 2020

Mechanisms of Resistance to Chemotherapy in Breast Cancer and Possible Targets in Drug Delivery Systems.

Pharmaceutics 2020 Dec 9;12(12). Epub 2020 Dec 9.

Department of Veterinary Surgery and Animal Reproduction, Sao Paulo State University-UNESP, Botucatu-SP 18618-681, Brazil.

Breast cancer (BC) is one of the most important cancers worldwide, and usually, chemotherapy can be used in an integrative approach. Usually, chemotherapy treatment is performed in association with surgery, radiation or hormone therapy, providing an increased outcome to patients. However, tumors can develop resistance to different drugs, progressing for a more aggressive phenotype. In this scenario, the use of nanocarriers could help to defeat tumor cell resistance, providing a new therapeutic perspective for patients. Thus, this systematic review aims to bring the molecular mechanisms involved in BC chemoresistance and extract from the previous literature information regarding the use of nanoparticles as potential treatment for chemoresistant breast cancer.
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http://dx.doi.org/10.3390/pharmaceutics12121193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763855PMC
December 2020

A Comparative Meta-Analysis and Analysis of Differentially Expressed Genes and Proteins in Canine and Human Bladder Cancer.

Front Vet Sci 2020 16;7:558978. Epub 2020 Nov 16.

Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu, Brazil.

Canine and human bladder cancer present similar anatomical, morphological, and molecular characteristics, and dogs can be considered a model for human bladder cancer. However, the veterinary literature lacks information regarding cross-validation analysis between human and canine large-scale data. Therefore, this research aimed to perform a meta-analysis of the canine literature on bladder cancer, identifying genes and proteins previously evaluated in these studies. In addition, we also performed a cross-validation of the canine transcriptome data and the human data from The Cancer Genome Atlas (TCGA) to identify potential markers for both species. The meta-analysis was performed using the following indexing terms: "bladder" AND "carcinoma" AND "dog" in different international databases, and 385 manuscripts were identified in our initial search. Then, several inclusion criteria were applied, and only 25 studies met these criteria. Among these studies, five presented transcriptome data, and 20 evaluated only isolated genes or proteins. Regarding the studies involving isolated protein analysis, the HER-2 protein was the most studied (3/20), followed by TAG-72 (2/20), COX-2 (2/20), survivin (2/20), and CK7 (2/20), and the remaining nine studies evaluated one isolated protein each. Regarding the cross-validation analysis of human and canine transcriptome data, we identified 35 dysregulated genes, including , and . Our results demonstrate that the canine literature on bladder cancer previously focused on the evaluation of isolated markers with no association with patient survival. This limitation may be related to the lack of a homogenous protocol for treating patients and the lack of follow-up during treatment. In addition, the lack of information regarding tumor muscle invasion can be considered an important limitation when comparing human and canine bladder tumors. Our analysis involving canine and human transcriptome data provided several genes with the potential to be markers for both human and canine bladder tumors, and these genes should be considered for future studies on canine bladder cancer.
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http://dx.doi.org/10.3389/fvets.2020.558978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7701042PMC
November 2020

Establishment and Characterization of Canine Mammary Gland Carcinoma Cell Lines With Vasculogenic Mimicry Ability and .

Front Vet Sci 2020 27;7:583874. Epub 2020 Oct 27.

School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu, Brazil.

Mammary tumors affect intact and elderly female dogs, and almost 50% of these cases are malignant. Cell culture offers a promising preclinical model to study this disease and creates the opportunity to deposit cell lines at a cell bank to allow greater assay reproducibility and more reliable validation of the results. Another important aspect is the possibility of establishing models and improving our understanding of tumor characteristics, such as vasculogenic mimicry. Because of the importance of cancer cell lines in preclinical models, the present study established and characterized primary cell lines from canine mammary gland tumors. Cell cultures were evaluated for morphology, phenotype, vasculogenic mimicry (VM), and tumorigenicity abilities. We collected 17 primary mammary carcinoma and three metastases and obtained satisfactory results from 10 samples. The cells were transplanted to a xenograft model. All cell lines exhibited a spindle-shaped or polygonal morphology and expressed concomitant pancytokeratin and cytokeratin 8/18. Four cell lines had vasculogenic mimicry ability , and two cell lines showed tumorigenicity and VM in the xenotransplanted tumor. Cellular characterization will help create a database to increase our knowledge of mammary carcinomas in dogs, including studies of tumor behavior and the identification of new therapeutic targets.
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http://dx.doi.org/10.3389/fvets.2020.583874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655132PMC
October 2020

Evaluation of a new variant in the aggrecan gene potentially associated with chondrodysplastic dwarfism in Miniature horses.

Sci Rep 2020 09 17;10(1):15238. Epub 2020 Sep 17.

School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp), Botucatu, 18618-681, Brazil.

Chondrodysplastic dwarfism in Miniature horses is an autosomal recessive disorder previously associated with four mutations (D1, D2, D3*, and D4) in the aggrecan (ACAN) gene. The aim of this study was to identify additional variants in the candidate ACAN gene associated with chondrodysplastic dwarfism in Miniature horses. Fifteen dwarf Miniature horses were found to possess only one of the dwarfism-causing variants, and two possessed none of the variants. The ACAN exons (EquCab3.0) of seven dwarf Miniature horses were sequenced. A missense SNP in coding exon 11 (g.95271115A > T, c.6465A > T-RefSeq XM_005602799.2), which resulted in the amino acid substitution p.Leu2155Phe (RefSeq XP_005602856.2), was initially associated with the dwarf phenotype. The variant was tested and found present in 14 dwarf foals as well as one parent of each, and both parents of a dwarf possessing two copies. Genetic testing of 347 phenotypically normal Miniature horses demonstrated that none had more than one of the dwarf alleles or c.6465A > T. However, a study of large breeds revealed the presence of c.6465A > T, which was present in homozygosis in two Mangalarga Marchador horses. We suggest that c.6465A > T as a marker of disequilibrium or complex interactions in the Miniature horse genome could contribute to the associated dwarfism.
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http://dx.doi.org/10.1038/s41598-020-72192-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499210PMC
September 2020

Investigation of the Prognostic Significance of Vasculogenic Mimicry and Its Inhibition by Sorafenib in Canine Mammary Gland Tumors.

Front Oncol 2019 19;9:1445. Epub 2019 Dec 19.

Department of Veterinary Surgery and Anesthesiology, São Paulo State University-UNESP, Botucatu, Brazil.

Canine mammary gland tumor (CMT) is one of the most important tumors in intact female dogs, and due its similarity to human breast cancer (BC), it is considered a model in comparative oncology. A subset of mammary gland tumors can show aggressive behavior, and a recurrent histological finding is the presence of vasculogenic mimicry (VM). VM is a process in which highly aggressive cancer cells fuse, forming fluid-conducting channels without endothelial cells. Although, VM has been described in canine inflammatory carcinoma, no previous studies have investigated the prognostic and predictive significance of VM in CMT. Thus, this research aimed to investigate the prognostic significance of VM and the capacity of sorafenib to inhibit VM . VM was identified in formalin-fixed paraffin-embedded CMT samples ( = 248) using CD31/PAS double staining. VM was identified in 33% of tumors (82/248). The presence of VM was more strongly related to tumor grade than to histological subtype. Patients with positive VM experienced shorter survival times than dogs without VM ( < 0.0001). Due to the importance of the VEGF-A/VEGFR-2 autocrine feed-forward loop in epithelial tumors, we investigated the association between VEGF-A and VEGFR-2 expression by neoplastic tumor cells and the associations of VEGF-A or VEGFR-2 expression with VM. Among the VM-positive samples, all ( = 82) showed high scores (3 or 4) for VEGF-A and VEGFR-2, indicating that VM was a common finding in tumors overexpressing VEGF-A and VEGFR-2. Thus, we cultured two CMT primary cell lines with VM abilities (CM9 and CM60) and evaluated the anti-tumoral effect of sorafenib. The CM9 cell line showed a half maximal inhibitory concentration (IC) of 2.61 μM, and the CM60 cell line showed an IC of 1.34 μM. We performed a VM assay and treated each cell line with an IC dose of sorafenib, which was able to inhibit VM . Overall, our results indicated that VM was a prognostic factor for dogs bearing CMT and that sorafenib had an inhibitory effect on VM in CMT cancer cells .
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http://dx.doi.org/10.3389/fonc.2019.01445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930929PMC
December 2019

Description of the D4/D4 genotype in Miniature horses with dwarfism.

J Vet Diagn Invest 2020 Jan 6;32(1):99-102. Epub 2020 Jan 6.

São Paulo State University (Unesp), School of Veterinary Medicine and Animal Science, Department of Veterinary Clinical Science, Botucatu, São Paulo, Brazil.

Four causative mutations (D1, D2, D3*, and D4) of chondrodysplastic dwarfism have been described in the equine () gene. Homozygotes for one of these mutations and heterozygotes for any combination of these mutations exhibit the disproportionate dwarfism phenotype. However, no case description of homozygotes for D4 (D4/D4) has been reported in the literature, to our knowledge. We report 2 Miniature horses with the genotype D4/D4 in the gene. Clinically, the 2 dwarfs had a domed head that was large compared to the rest of the body, mandibular prognathism, and short and bowed limbs, mainly in the proximal region of the metatarsal bones. Radiographic examination revealed contour irregularities of the subchondral bone in the long bones and confirmed mandibular prognathism; histopathology revealed irregular chondrocyte organization. To determine the genotypes of the horses, we performed DNA extraction from white blood cells, PCR, and Sanger sequencing. Genotyping demonstrated that these 2 animals had the D4/D4 genotype in the gene. The D4/D4 dwarfs were clinically similar to animals with the other genotypes reported for this disease. Identification of heterozygous animals makes mating selection possible and is the most important control measure to minimize economic losses and casualties.
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http://dx.doi.org/10.1177/1040638719898164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003212PMC
January 2020

E-Cadherin Downregulation is Mediated by Promoter Methylation in Canine Prostate Cancer.

Front Genet 2019 29;10:1242. Epub 2019 Nov 29.

Department of Veterinary Surgery and Anesthesiology, School of Veterinary Medicine and Animal Science, Sao Paulo State University-UNESP, Botucatu, Brazil.

E-cadherin is a transmembrane glycoprotein responsible for cell-to-cell adhesion, and its loss has been associated with metastasis development. Although E-cadherin downregulation was previously reported in canine prostate cancer (PC), the mechanism involved in this process is unclear. It is well established that dogs, besides humans, spontaneously develop PC with high frequency; therefore, canine PC is an interesting model to study human PC. In human PC, methylation has been associated with E-cadherin downregulation. However, no previous studies have described the methylation pattern of promoter in canine PC. Herein, we evaluated the E-cadherin protein and gene expression in canine PC compared to normal tissues. DNA methylation pattern was investigated as a regulatory mechanism of silencing. Our cohort is composed of 20 normal prostates, 20 proliferative inflammatory atrophy (PIA) lesions, 20 PC, and 11 metastases from 60 dogs. The E-cadherin protein expression was assessed by immunohistochemistry and western blotting and gene expression by qPCR. Bisulfite- pyrosequencing assay was performed to investigate the promoter methylation pattern. Membranous E-cadherin expression was observed in all prostatic tissues. A higher number of E-cadherin negative cells was detected more frequently in PC compared to normal and PIA samples. High-grade PC showed a diffuse membranous positive immunostaining. Furthermore, PC patients with a higher number of E-cadherin negative cells presented shorter survival time and higher Gleason scores. Western blotting and qPCR assays confirmed the immunohistochemical results, showing lower E-cadherin protein and gene expression levels in PC compared to normal samples. We identified promoter hypermethylation in PIA and PC samples. An in vitro assay with two canine prostate cancer cells (PC1 and PC2 cell lines) was performed to confirm the methylation as a regulatory mechanism of E-cadherin expression. PC1 cell line presented hypermethylation and after 5-Aza-dC treatment, a decreased methylation and increased gene expression levels were observed. Positive E-cadherin cells were massively found in metastases (mean of 90.6%). In conclusion, low levels of E-cadherin protein, gene downregulation and hypermethylation was detected in canine PC. However, in metastatic foci occur E-cadherin re-expression confirming its relevance in these processes.
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http://dx.doi.org/10.3389/fgene.2019.01242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895247PMC
November 2019

Comprehensive Genomic Profiling of Androgen-Receptor-Negative Canine Prostate Cancer.

Int J Mol Sci 2019 Mar 28;20(7). Epub 2019 Mar 28.

Department of Clinical Genetics, Vejle Hospital, Institute of Regional Health Research, University of Southern Denmark, 7100 Vejle, Denmark.

Canine carcinomas have been considered natural models for human diseases; however, the genomic profile of canine prostate cancers (PCs) has not been explored. In this study, 14 PC androgen-receptor-negative cases, 4 proliferative inflammatory atrophies (PIA), and 5 normal prostate tissues were investigated by array-based comparative genomic hybridization (aCGH). Copy number alterations (CNAs) were assessed using the Canine Genome CGH Microarray 4 × 44K (Agilent Technologies). Genes covered by recurrent CNAs were submitted to enrichment and cross-validation analysis. In addition, the expression levels of , and were evaluated in an independent set of cases by qPCR. PC cases presented genomic complexity, while PIA samples had a small number of CNAs. Recurrent losses covering well-known tumor suppressor genes, such as , , , and , were found in PC. The in silico functional analysis showed several cancer-related genes associated with canonical pathways and interaction networks previously described in human PC. The , , and copy number alterations were translated into altered expression levels. A cross-validation analysis using The Cancer Genome Atlas (TCGA) database for human PC uncovered similarities between canine and human PCs. Androgen-receptor-negative canine PC is a complex disease characterized by high genomic instability, showing a set of genes with similar alterations to human cancer.
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http://dx.doi.org/10.3390/ijms20071555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480132PMC
March 2019

Immunopathological findings in a cat with auricular chondritis.

Acta Vet Hung 2019 03;67(1):81-86

1 Laboratory of Veterinary Pathology , 70330-050 Brasília, DF , Brazil.

At clinical examination, a 5-year-old male domestic short-haired cat exhibited painful swelling and erythema of the pinnae of both ears. Microscopically, the lesions on both pinnae were composed of diffuse granulomatous chondritis with degeneration and necrosis of the pinnal cartilage. Numerous mast cells were also observed within and surrounding the inflammatory lesion. Immunohistochemistry showed a mixed inflammatory infiltrate characterised by the predominance of macrophages (CD68+, MAC 387+ and Lysozyme+), T lymphocytes (CD3+), some B lymphocytes (CD79α+) and neutrophils. Immunopathological characterisation of the lesion showed a granulomatous inflammation profile and suggests that the morphological changes and immunopathogenesis of auricular chondritis in cats presents a similarity with relapsing polychondritis in humans.
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http://dx.doi.org/10.1556/004.2019.009DOI Listing
March 2019

Characterization of Collagen Fibers (I, III, IV) and Elastin of Normal and Neoplastic Canine Prostatic Tissues.

Vet Sci 2019 Mar 2;6(1). Epub 2019 Mar 2.

Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp), Botucatu, São Paulo 18618-681, Brazil.

This study aimed to investigate collagen (Coll-I, III, IV) and elastin in canine normal prostate and prostate cancer (PC) using Picrosirius red (PSR) and Immunohistochemical (IHC) analysis. Eight normal prostates and 10 PC from formalin-fixed, paraffin-embedded samples were used. Collagen fibers area was analyzed with ImageJ software. The distribution of Coll-I and Coll-III was approximately 80% around prostatic ducts and acini, 15% among smooth muscle, and 5% surrounding blood vessels, in both normal prostate and PC. There was a higher median area of Coll-III in PC when compared to normal prostatic tissue ( = 0.001 for PSR and = 0.05 for IHC). Immunostaining for Coll-IV was observed in the basal membrane of prostate acini, smooth muscle, blood vessels, and nerve fibers of normal and PC samples. Although there was no difference in Coll-IV area between normal tissue and PC, tumors with Gleason score 10 showed absence of Coll-IV, when compared to scores 6 and 8 ( = 0.0095). Elastic fibers were found in the septa dividing the lobules and around the prostatic acini of normal samples and were statistically higher in PC compared to normal tissue ( = 0.00229). Investigation of ECM components brings new information and should be correlated with prognosis in future studies.
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http://dx.doi.org/10.3390/vetsci6010022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466295PMC
March 2019

Metastatic immune infiltrates correlate with those of the primary tumour in canine osteosarcoma.

Vet Comp Oncol 2019 Sep 8;17(3):242-252. Epub 2019 Apr 8.

The Comparative Oncology Laboratory and Center for Companion Animal Health, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, California.

Our lack of understanding of the immune microenvironment in canine osteosarcoma (cOSA) has limited the identification of potential immunotherapeutic targets. In particular, our ability to utilize readily available tissue from a dog's primary tumour to predict the type and extent of immune response in their pulmonary metastatic lesions is unknown. We, therefore, collected 21 matched pairs of primary tumours and pulmonary metastatic lesions from dogs with OSA and performed immunohistochemistry to quantify T-lymphocyte (CD3), FOXP3+ cell, B-lymphocyte (Pax-5), and CD204+ macrophage infiltration. We found that T-lymphocytes and FOXP3+ infiltrates in primary tumours positively correlated with that of metastatic lesions (ρ = 0.512, P = 0.038 and ρ = 0.698, P = 0.007, respectively), while a strong trend existed for CD204+ infiltrates (ρ = 0.404, P = 0.087). We also observed T- and B-lymphocytes, and CD204+ macrophages to be significantly higher in a dog's pulmonary metastasis compared to their primary tumour (P = 0.018, P = 0.018, P = 0.016, respectively), while FOXP3+ cells were only significantly higher in metastases when all primary tumour and metastasis lesions were compared without pairing (P = 0.036). Together, these findings suggest that the metastatic immune microenvironment may be influenced by that of the primary cOSA, and that primary tumour immune biomarkers could potentially be applied to predict immunotherapeutic responses in gross metastatic disease. We, therefore, provide a rationale for the treatment of cOSA pulmonary metastases with immunotherapeutics that enhance the anti-tumour activity of these immune cells, particularly in dogs with moderate to high immune cell infiltration in their primary tumours.
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http://dx.doi.org/10.1111/vco.12459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658355PMC
September 2019

Characterization of OCT3/4, Nestin, NANOG, CD44 and CD24 as stem cell markers in canine prostate cancer.

Int J Biochem Cell Biol 2019 03 8;108:21-28. Epub 2019 Jan 8.

São Paulo State University - UNESP, Department of Veterinary Surgery and Anaesthesiology, School of Veterinary Medicine and Animal Science, Botucatu, Sao Paulo, Brazil. Electronic address:

The cancer cell population is heterogeneous, and cancer stem cells (CSCs) are important for tumor growth and maintenance. The CSC population is associated with different neoplastic characteristics, such as cell migration, resistance to apoptosis, radiation therapy and chemotherapy. To increase the knowledge of CSCs in canine prostate cancer (PC), we characterized CSC markers in canine PC tissues and tumorspheres. We performed immunohistochemistry of OCT3/4, Nestin, NANOG, CD44 and CD24 in 10 normal canine prostatic tissue samples, 10 prostatic hyperplastic (PH) tissue samples and 28 PC tissue samples. Then, we established two canine prostate cancer cell cultures and characterized the CSC profile of tumorspheres grown from these cultures. Normal and PH tissues were positive for Nestin, NANOG, CD44 and CD24 only in the basal cell layer. OCT3/4 was expressed in the luminal cells of normal and PH tissues. There was no significant difference in Nestin expression among the prostatic tissues. However, we found higher expression of NANOG and CD44 in canine PC tissues than that in normal and PH tissues. Tumorspheres from canine prostate cancer cells express OCT3/4, Nestin, NANOG and CD44, indicating that these markers may be potential cancer stem cell markers in canine PC. The results obtained can be useful to better characterize the stem cell population in canine prostatic cancer and to guide future studies in comparative oncology.
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http://dx.doi.org/10.1016/j.biocel.2019.01.002DOI Listing
March 2019

An immunohistochemical study of T and B lymphocyte density in prostatic hyperplasia and prostate carcinoma in dogs.

Res Vet Sci 2019 Feb 27;122:189-192. Epub 2018 Nov 27.

School of Veterinary Science, The University of Queensland, Gatton Campus, 4343 Gatton, Queensland, Australia.

The aim of this study was to characterise T and B lymphocyte density in 6 normal prostates, 15 benign prostatic hyperplasia (BPH) and 24 prostate carcinomas (PCs) in dogs by immunohistochemistry. Results revealed a statistically significant increase of T and B cells in PC compared to normal specimens and BPH. Regarding PC histological variants, lower number of CD3 and CD79 lymphocytes were observed in the most undifferentiated (solid) type. CD3 cell density was positively correlated with survival time. These results may help in understanding the immunological mechanisms regulating BPH and PC development and progression, as well as providing background data for future immunotherapeutic trials.
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http://dx.doi.org/10.1016/j.rvsc.2018.11.022DOI Listing
February 2019

p-mTOR, p-4EBP-1 and eIF4E expression in canine prostatic carcinoma.

Res Vet Sci 2019 Feb 16;122:86-92. Epub 2018 Nov 16.

Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, São Paulo State University (Unesp), Botucatu, São Paulo, Brazil. Electronic address:

The mTOR/4E-BP1/eIF4E pathway plays important roles in the neoplastic transformation process and in tumour growth. In men, the mTOR/4E-BP1/eIF4E pathway was described as altered in different tumours, including prostate cancer (PC). Apart from humans, the dog is the only species that develops PC with high frequency and is considered a good model for comparative oncology initiatives. Due to limited information on this pathway in canine tumours, this study aimed to investigate mTOR, 4E-BP1 and eIF4E gene and protein expression in canine PC, as well as in metastatic and normal prostatic tissues, and to evaluate the correlations between gene/protein expression and Gleason score (GS) in PC. A total of 35 formalin-fixed paraffin-embedded (FFPE) samples, including 13 of normal prostatic tissue, 17 PC samples and 5 metastasis samples, were evaluated by immunohistochemistry and qPCR. mTOR gene mutation in the kinase domain was also investigated. We identified higher p-mTOR and eIF4E protein levels in canine PC with higher GS values (≥ 8) and a significant positive correlation in expression between these proteins. eIF4E overexpression was observed in metastasis relative to expression in normal samples. Our data suggest that p-mTOR and eIF4E expression is positively correlated with GS in canine PC, similar to the pattern in humans. More studies of the mTOR/4EBP1/eIF4E pathway should be performed to identify possible correlations of the proteins involved with clinical and pathologic findings in canine PC and the roles of these proteins as therapeutic targets for the treatment of canine PC.
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http://dx.doi.org/10.1016/j.rvsc.2018.11.006DOI Listing
February 2019

Association of macrophage and lymphocyte infiltration with outcome in canine osteosarcoma.

Vet Comp Oncol 2019 Mar 19;17(1):49-60. Epub 2018 Sep 19.

The Comparative Oncology Laboratory and Center for Companion Animal Health, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California-Davis, Davis, California.

Immunotherapeutic strategies have shown promise for the treatment of canine osteosarcoma (cOSA). Very little is known about the immune microenvironment within cOSA, however, limiting our ability to identify potential immune targets and biomarkers of therapeutic response. We therefore prospectively assessed the disease-free interval (DFI) and overall survival time (ST) of 30 dogs with cOSA treated with amputation and six doses of adjuvant carboplatin. We then quantified lymphocytic (CD3+, FOXP3+) and macrophage (CD204+) infiltrates within the primary tumours of this cohort using immunohistochemistry, and evaluated their association with outcome. Overall, the median DFI and ST were 392 and 455 days, respectively. The median number of CD3+ and FOXP3+ infiltrates were 45.8 cells/mm (4.6-607.6 cells/mm ) and 8.5 mm (0-163.1 cells/mm ), respectively. The median area of CD204+ macrophages was 4.7% (1.3%-23.3%), and dogs with tumours containing greater than 4.7% CD204+ macrophages experienced a significantly longer DFI (P = 0.016). Interestingly, a significantly lower percentage of CD204+ macrophages was detected in cOSA arising from the proximal humerus compared to other appendicular bone locations (P = 0.016). Lymphocytic infiltrates did not appear to correlate with outcome in cOSA. Overall, our findings suggest that macrophages may play a role in inhibiting cOSA progression, as has been suggested in human osteosarcoma.
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http://dx.doi.org/10.1111/vco.12444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363827PMC
March 2019

Partial ablation of endometrial glands in dogs after exposure to progestin during the neonatal period.

Anim Reprod 2018 Aug 16;15(1):45-50. Epub 2018 Aug 16.

Escola de Veterinária, , , .

Bitches with uteri devoid of endometrial glands should be sterile, and consequently could contribute to the population control of dogs. Considering that an inadequate exposure of the female reproductive system to steroids can lead to the formation of the uterine gland knock-out (UGKO) phenotype in some species, the aim of this study was to evaluate the effect of serial applications of medroxyprogesterone acetate (MPA) from birth until the age of six months on the development of endometrial glands in bitches. For this purpose, 16 female mongrel dogs from different litters were distributed into either an MPA group (n = 8), animals treated with 10 mg kg sc (Promone-E, Pfizer, Brasil) at 3-week intervals, from day one after birth until the age of six months, or a control group (n = 8), composed of animals that only received a 0.9% NaCl solution in place of MPA. At six months of age, ovariohysterectomy was performed and uterine horn samples were collected for histological and immunohistochemical examinations. The bitches from the MPA-treated group presented a 35% decrease in the number of endometrial glands, a larger diameter of the endometrial glands, a greater epithelial height, as well as a greater thickness of the uterine wall, endometrium, and myometrium. However, no significant differences were observed between the two groups in the expression of ER-α, ER-β, and PR on the surface epithelium and endometrial stroma. Therefore, the serial application of MPA from birth until the age of 6 months do not completely ablate the development of the endometrial glands in bitches, but impair it by 35%.
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http://dx.doi.org/10.21451/1984-3143-2018-0040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746220PMC
August 2018

Immunohistochemical panel to characterize canine prostate carcinomas according to aberrant p63 expression.

PLoS One 2018 12;13(6):e0199173. Epub 2018 Jun 12.

Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, São Paulo State University-UNESP, Botucatu, SP, Brazil.

An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin-HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199173PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997330PMC
January 2019

Characteristics of the Epithelial-Mesenchymal Transition in Primary and Paired Metastatic Canine Mammary Carcinomas.

Vet Pathol 2018 09 22;55(5):622-633. Epub 2018 May 22.

4 Penn Vet Cancer Center, University of Pennsylvania, Philadelphia, PA, USA.

The epithelial-mesenchymal transition (EMT) is a dynamic process linked to metastasis in many tumor types, including mammary tumors. In this study, we evaluated E-cadherin and vimentin immunolocalization in primary canine mammary carcinomas (20 cases) and their respective metastases, as well as their relationship with the core regulators SNAIL/SLUG. To assess the number of cells undergoing the process of EMT, we quantitated double-positive (E-cadherin/vimentin) cells using immunofluorescence, via cell counting and image analysis. In addition, SNAIL/SLUG expression was evaluated by established immunohistochemical methods. Primary tumors had significantly more E-cadherin/vimentin co-expression than their paired respective lymph node or distant metastasis, respectively. Furthermore, the percentage of E-cadherin/vimentin cells in grade II and III carcinomas was significantly higher than in grade I tumors. Primary tumors had significantly higher SNAIL/SLUG expression when analyzed based on the percentage of positive cells compared with their respective distant metastases in pairwise comparisons. An inverse correlation was noted between SNAIL/SLUG immunoreactivity and percentage of E-cadherin/vimentin immunopositive cells in primary tumor samples when SNAIL/SLUG immunoreactivity was grouped into 2 categories (high versus low) based on percentage-positive staining. These results show a positive correlation between E-cadherin/vimentin cells and higher tumor grade, establish differences between primary tumor and their respective metastases, and provide further support that EMT plays a critical role in the metastasis of canine mammary carcinoma. Furthermore, these data suggest that modulation of this process could provide greater therapeutic control and provide support for further research to determine if E-cadherin/vimentin co-immunoreactivity imparts predictive value in the clinical outcome of patients with canine mammary carcinomas.
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http://dx.doi.org/10.1177/0300985818776054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7993492PMC
September 2018

Allogeneic mesenchymal stem cell transplantation in healthy equine superficial digital flexor tendon: A study of the local inflammatory response.

Res Vet Sci 2018 Jun 21;118:423-430. Epub 2018 Mar 21.

Department of Veterinary Surgery and Anesthesiology, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Distrito de Rubião Júnior s/n, Botucatu, SP 18618-9070, Brazil. Electronic address:

The superficial digital flexor tendon (SDFT) is a structure frequently affected by injuries in high-performance athletic horses, and there are limited therapeutic options. Regenerative medicine has evolved significantly in treating different illnesses. However, understanding the cellular behaviour during mesenchymal stem cell (MSC) transplantation in healthy tissues is not fully known yet. To address the inflammatory response induced by allogeneic MSC transplantation, this study evaluated the local inflammatory response after the application of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) in the equine tendon compared to an autologous transplant and the control group. Eighteen thoracic limbs (TL) in nine animals were divided into three groups and subjected to the application of AT-MSCs in the healthy tendon. In the allogeneic group (Gallog), the animals received an allogeneic AT-MSC application in the TL. The autologous group (Gauto) received an application of autologous cells in the TL, and in the control group (Gcont), phosphate-buffered saline (PBS) was applied. There were no significant differences among the evaluated groups in the physical, morphological, thermography, and ultrasonography analyses. A higher number of CD3-positive lymphocytes was observed in the Gauto group compared to the control (P < 0.05). Additionally, we did not observe different expressions of CD172 and microvascular density among the groups. The allogeneic transplantation of AT-MSCs did not result in an adverse or inflammatory reaction that compromised the use of these cells in this experiment. Their behaviour was similar to that of autologous transplantation.
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http://dx.doi.org/10.1016/j.rvsc.2018.03.012DOI Listing
June 2018
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