17-AAG inhibits vasculogenic mimicry of metastatic osteosarcoma cell line

Introduction: Tumour vasculature is derived from a variety of sources including vasculogenic mimicry (VM). Treatments based on Hsp90 (Heat-shock-protein-90) inhibition negatively influences several factors involved in VM regulation including VegfR1 and Hif1α. Aim of the study was to verify the ability of two canine osteosarcoma cell lines to generate VM in 3D culture, identify specific markers of endothelial-like cells and estabilish 17-AAG (17-N-allyloamino-17-demethoxygeldanamycin) activity on tubular-like structure formation in vitro and degradation of Hsp90 client proteins. Materials and Methods: D22 and D17 canine osteosarcoma cells were seeded on Collagen Rat Tail Type 1 and the presence of tubular-like structures was evaluated. After 4 weeks, sections of cultures were H&E stained or exposed to immunohistochemical evaluation of CD31, vWf, VegfR1 and HSp90. Cells were also treated with 17-AAG and principal VM features, cells migration and VegfR1 and Hif1α were quantified. Results: Only metastatic cells showed tubular-like structures in 3D culture. Histological investigation of D17 3D long term culture further demonstrated the presence of vessel-like channels showing cavities in serial transversal sections surrounded by endothelial-like cells expressing Hsp90 and VegfR1. Furthermore, 17-AAG induced a significant decrease of VM features in a time-dependent manner. Conclusions: 17-AAG activity on canine osteosarcoma 3D culture could offer new prospects for the development of therapeutic strategies based on the synergy between VM-blocking and anti-angiogenic property determined by hsp90 inhibition. This is the first study that highlight the presence of vessel-like structure in long term canine osteosarcoma 3D cultures, laying the technical groundwork to search specific tumour endothelial-like cell markers.