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 in vivo and the capacity of sorafenib to inhibit VM in vitro. VM was identified in situ in formalin-fixed paraffin-embedded CMT samples (n = 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 (P < 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 (n = 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) in vitro and evaluated the anti-tumoral effect of sorafenib. The CM9 cell line showed a half maximal inhibitory concentration (IC 50) of 2.61 µM, and the CM60 cell line showed an IC 50 of 1.34 µM. We performed a VM assay in vitro and treated each cell line with an IC 50 dose of sorafenib, which was able to inhibit VM in vitro. 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 in vitro.

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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 in vitro, and two cell lines showed in vivo 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.

Mammary neoplasms affect a population of uncastrated and elderly female dogs and most of these neoplasms are malignant. In order to study this disease cell culture presents itself as a promising preclinical model, creating the opportunity to deposit cell lines at a cell bank, allowing a great repetition of the assays and making the validation of the results more reliable. Including, in vitro experiments for vasculogenic mimicry (VM) evaluation. VM is related to cancer cells capable of generate vascular-like structures without endothelial cells, mimicking the vasculogenic process. The aim of this study was to establish and characterize ten cell lines from canine mammary gland tumour according to immunophenotype and tumorigenicity, and with its ability to form vasculogenic mimicry-like structures in vitro. Fifteen samples from canine mammary gland carcinoma were collected and cultured in vitro and ten cell lines were established and characterized. Cells were evaluated for morphology, phenotype, vascular mimicry and tumorigenicity. All cell lines presented spindle shape morphology and expressed concomitant pan-cytokeratin and CK8/18. Four cell lines had vasculogenic mimicry ability and two cell showed in vivo tumorigenic potential. Cell characterization of those lines will help to create a database for more knowledge of mammary gland carcinomas in dogs, including studies of tumor behavior and new therapeutic targets.

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