Publications by authors named "Antonio Fernando Leis-Filho"

9 Publications

  • Page 1 of 1

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

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

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

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

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

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

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

Tumor-infiltrating CD4 and CD8 lymphocytes and macrophages are associated with prognostic factors in triple-negative canine mammary complex type carcinoma.

Res Vet Sci 2019 Oct 13;126:29-36. Epub 2019 Aug 13.

Department of Veterinary Surgery and Anesthesiology, School of Veterinary Medicine and Animal Science São Paulo State University - UNESP, Botucatu, SP, Brazil; Universidade Paulista - UNIP, Bauru, SP, Brazil. Electronic address:

This study aimed to evaluate the association of CD3, CD4, and CD8 T cells and tumor-infiltrating macrophages (TIMs) with the clinical parameters of female dogs harboring mammary gland tumors. Thirty female dogs affected with mammary carcinomas were used, and all tumors were histologically classified as complex carcinoma and were triple-negative phenotype determined by immunohistochemistry. Freshly frozen sections were used to determine CD3, CD4 and CD8 T cells by immunohistochemistry, and TIMs were determined by immunofluorescence assays. Ten out of the 30 dogs showed lymph node metastasis at diagnosis. Fifteen dogs had a tumor of grade I (15/30), nine (9/30) had a tumor of grade II and six (6/30) had a tumor of grade III. The mean overall survival was 680.5 days (± 200.4). Dogs with sentinel lymph node positivity (10/30) (P = .0035) and dogs that developed metastasis (P = .0001) showed a shorter survival time. In addition, dogs with a high level of inflammatory infiltrate in tumor tissues presented a shorter survival time (P = .0001) than that of other dogs. Dogs with tumors containing higher numbers of CD3+ T cells (P = .001), CD4+ T cells (P = .001), or TIM cells (P < .0001) showed a shorter survival time than that of other dogs. Our results suggested that characteristics of immune cell infiltrates, including CD3 T cells, CD4 T cells, and TIMs, can be used as potential prognostic indicators for predicting clinical outcomes in dogs with mammary gland tumors, particularly tumors with a complex histological subtype and triple-negative phenotype.
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http://dx.doi.org/10.1016/j.rvsc.2019.08.021DOI Listing
October 2019

Deregulation of VEGFR-2 and PDGFR Expression and Microvascular Density in a Triple-Negative Model of Canine Malignant Mammary Tumors with Lymph Node or Lung Metastasis.

Vet Sci 2019 Jan 9;6(1). Epub 2019 Jan 9.

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

Canine mammary tumors (CMT) represent the most common cancer in noncastrated female dogs. Interestingly, triple-negative tumors are the most common molecular subtype in female dogs. In this study, we proposed to evaluate the expression of vascular endothelial growth factor receptor 2 (VEGFR-2), Platelet-derived growth factor receptor (PDGFR), and microvascular density (MVD) in a group of metastatic and nonmetastatic triple-negative CMT and compare the expression based on clinical parameters. Twenty-six female dogs with triple-negative mammary tumors were divided into three groups: nonmetastatic tumors (NMT) ( = 11), tumors with lymph node metastasis (LNM) ( = 10), and tumors with lung metastasis (LM) ( = 5). We observed increased VEGFR-2 expression in LNM compared with NMT and a positive correlation between tumor grade and VEGFR-2 expression. A positive correlation was noted between VEGFR-2 and PDGFR expression. Regarding microvascular density (MVD), we identified a higher number of vessels in primary tumors with lymph node metastasis and lung metastasis compared with tumors with no metastasis. The primary tumors with lung metastasis exhibited an increased MVD compared with carcinoma with lymph node metastasis. Overall, our results suggest a deregulation of VEGFR-2 and PDGFR and high MVD in metastatic tumors, indicating a role for angiogenesis in tumor progression.
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http://dx.doi.org/10.3390/vetsci6010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466043PMC
January 2019
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