Functional Heterogeneity of Canine Osteosarcoma Cell Lines and Differential Expression of miR-27b-3p and IGF2BP3

Canine osteosarcoma (OSA) is a highly aggressive primary bone tumor and a valuable model in comparative oncology. Nevertheless, commonly used canine in vitro models remain incompletely and inconsistently characterized, while exhibiting substantial biological heterogeneity affecting experimental outcomes. This study aimed to comparatively characterize three canine osteosarcoma cell lines (OSCA8, OSCA29, and D17) in reference to canine hTERT fibroblasts, and with a focus on functional properties and selected molecular features, namely including miR-27b-3p and IGF2BP3 expression. The cytophysiological profile of the cells was evaluated in relation proliferation and migratory capacity. In turn gene expression was determined with RT-qPCR, and proteins detected with Western blotting. The D17 cell line showed the highest metabolic activity and the largest fraction of S-phase cells, whereas OSCA8 cells demonstrated the greatest clonogenic potential and the highest migratory activity in the wound healing assay. OSCA29 cells displayed an intermediate functional profile, while all OSA cell lines exhibited comparable migratory capacity in transwell assay. At the molecular level, miR-27b-3p expression was significantly higher in OSCA8 and D17 cells than in OSCA29 cells. In turn, IGF2BP3 transcript levels were lower in OSCA29 cells, whereas protein analysis revealed distinct immunoreactive forms. Together, these findings highlight the functional heterogeneity of commonly used canine osteosarcoma cell lines and broaden their current characterization.