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

Maria Carolina Mangini Prado; Sofia de Almeida Losant Macedo; Giulia Gumiero Guiraldelli; Patricia de Faria Lainetti; Antonio Fernando Leis-Filho; Priscila Emiko Kobayashi; Renee Laufer-Amorim; Carlos Eduardo Fonseca-Alves

doi:10.3389/fonc.2019.01445

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

Front Oncol. 2019 Dec 19:9:1445. doi: 10.3389/fonc.2019.01445. eCollection 2019.

Authors

Maria Carolina Mangini Prado¹, Sofia de Almeida Losant Macedo¹, Giulia Gumiero Guiraldelli², Patricia de Faria Lainetti¹, Antonio Fernando Leis-Filho¹, Priscila Emiko Kobayashi², Renee Laufer-Amorim², Carlos Eduardo Fonseca-Alves^{1

3}

Affiliations

¹ Department of Veterinary Surgery and Anesthesiology, São Paulo State University-UNESP, Botucatu, Brazil.
² Department of Veterinary Clinic, São Paulo State University-UNESP, Botucatu, Brazil.
³ Institute of Health Sciences, Universidade Paulista-UNIP, Bauru, Brazil.

Abstract

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 (IC₅₀) of 2.61 μM, and the CM60 cell line showed an IC₅₀ of 1.34 μM. We performed a VM assay in vitro and treated each cell line with an IC₅₀ 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.

Keywords: angiogenesis; antiangiogenic drugs; breast cancer; dog; tubular assay.