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

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 IC50. Then, cell lines were treated with rapamycin IC50 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 IC50 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 IC50 were higher when compared to other studies.

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

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-ß, 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-ß 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 PDGFR-A, PDGFR-ß, and PDGF-D expression in PC1 cells, and the upregulation of PDGFR-ß 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.

Primary diffuse mammary hemangiosarcoma in a female dog

In veterinary medicine, primary mammary hemangiosarcoma is a very rare disease and there was no previous report describing the disease in dogs. Herein, we describe necropsy findings of a female dog, which presented a diffuse mammary hemangiosarcoma affecting the mammary gland. A diffuse irregular plaque was found in all mammary gland, involving the whole mammary gland tissue. The histopathological evaluation revealed neoplastic cells with an ovoid to spindle shaped basophilic and numerous atypical neoplastic tumor cells forming vascular structures. There was no glandular proliferation and no changes in the superficial dermis and no neoplastic emboli in the lymph vessels. Immunohistochemistry for vimentin, pan-cytokeratin, CK8/18 and CD31 was conducted. Positive expression was found to the pan-cytokeratin and CK8/18 by remaining epithelial cells confirmed the luminal mammary origin. Vimentin and CD31 positive expression by neoplastic cells confirmed the endothelial origin of the neoplasia. The histopathological and immunohistochemical findings supported the diagnosis of a primary mammary hemangiosarcoma.

Primary diffuse mammary hemangiosarcoma in a female dog

In veterinary medicine, primary mammary hemangiosarcoma is a very rare disease and there was no previous report describing the disease in dogs. Herein, we describe necropsy findings of a female dog, which presented a diffuse mammary hemangiosarcoma affecting the mammary gland. A diffuse irregular plaque was found in all mammary gland, involving the whole mammary gland tissue. The histopathological evaluation revealed neoplastic cells with an ovoid to spindle shaped basophilic and numerous atypical neoplastic tumor cells forming vascular structures. There was no glandular proliferation and no changes in the superficial dermis and no neoplastic emboli in the lymph vessels. Immunohistochemistry for vimentin, pan-cytokeratin, CK8/18 and CD31 was conducted. Positive expression was found to the pan-cytokeratin and CK8/18 by remaining epithelial cells confirmed the luminal mammary origin. Vimentin and CD31 positive expression by neoplastic cells confirmed the endothelial origin of the neoplasia. The histopathological and immunohistochemical findings supported the diagnosis of a primary mammary hemangiosarcoma.(AU)