Differential Effects of Cobalt Chloride Preconditioning on Vasculogenic Mimicry‐Like Structure Formation in Two Canine Mammary Tumour Cell Lines

ABSTRACT

Vasculogenic mimicry (VM) is a tumour cell‐lined vessel‐like structure associated with poor prognosis, metastasis, and treatment resistance in human breast cancer. Furthermore, it has been described in canine mammary tumours (CMTs). Hypoxia is an established driving factor for VM formation in tumours with this capability. This study aimed to compare VM‐like structure formation between two CMT carcinoma cell lines, CF41.Mg and CMT‐U27. VM‐like tubular structure formation was evaluated using a Matrigel‐based 3D cell culture system under normal conditions and after CoCl ₂ preconditioning. VM‐like tubular structures were observed only in CF41.Mg cells, and CoCl ₂ preconditioning increased the number of meshes and segments without a time‐dependent difference between 24 and 48 h. In contrast, CMT‐U27 cells did not form VM‐like structures under the tested conditions. Subsequently, RNA sequencing, quantitative polymerase chain reaction, enzyme‐linked immunosorbent assay, and Western blot analyses were conducted. Exploratory RNA sequencing of untreated cells showed differences in the expression patterns of selected genes involved in angiogenesis, epithelial‐to‐mesenchymal transition, cell migration, hypoxia‐associated responses, and extracellular matrix organization between the two cell lines. CoCl ₂ treatment was associated with VM‐related transcriptional and protein‐level changes in CF41.Mg cells. In contrast, CoCl ₂ treatment was associated with E‐cadherin upregulation and no significant changes in MMP2 or CDH2 expression in CMT‐U27 cells. Taken together, under the tested conditions, CoCl ₂ preconditioning enhanced VM‐like structure formation in CF41.Mg cells but did not induce VM‐like tubular structures in CMT‐U27 cells.