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

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 (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.

From Conventional to Precision Therapy in Canine Mammary Cancer: A Comprehensive Review

“…On the other hand, sorafenib, a VEGFR, PDGFR, and rapid accelerated fibrosarcoma-1 (Raf-1) kinase inhibitor, has antitumor effects in vitro and inhibits neovascularization in xenograft models of HBC ( 165 ). Sorafenib has been assessed in veterinary medicine, and it has shown a promising ability to inhibit VM in CMC cell lines in vitro ( 166 ). Other TKIs that target VEGFR-2 and that have been proven to significantly diminish the level of active (phosphorylated) VEGFR2, reduce cell proliferation and migration, and increase apoptosis in in vitro studies against CMT cell lines are rivoceranib (apatinib) ( 167 ) and vandetanib ( 168 ).…”

“…VM has been associated with the spread and metastasis of human ( 177 ) and canine mammary tumors ( 178 ). VM was found in 33% of canine mammary tumors, and its presence was correlated with histological grade of malignancy and shorter survival times ( 166 ).…”

Establishment of a New Cell Line of Canine Mammary Tumor CMT-1026

“…Developed CMT cell lines include CMT-1 to CMT-6 (46), CMT12, CMT27 (47), CMT-U27, CMT-U309, IPC-366 (48)(49)(50), and CMT-U229 (51). The canine TNBC cell line shares many pathological features with those of the illness in humans and thus offers an excellent animal model for human disease study (52)(53)(54). Nevertheless, only one triple negative canine mammary tumor cell line is available: CMT-7364, developed by Zhang et al (33).…”

Longitudinal assessment of B-RAF V595E levels in the peripheral cell-free tumor DNA of a 10-year-old spayed female Korean Jindo dog with unresectable metastatic urethral transitional cell carcinoma for monitoring the treatment response to a RAF inhibitor (sorafenib)

“…5,28 Further, it inhibited vasculogenic mimicry in canine mammary gland tumor cancer cells in vitro. 29 Based on this spectrum of activities, sorafenib was selected for treatment in the present case. B-RAF mutation analysis is a new and effective method for the confirmational diagnosis of TCC in dogs.…”