Establishment of three‐dimensional canine osteosarcoma cell lines showing vasculogenic mimicry and evaluation of biological properties after treatment with 17‐AAG

Abstract

Vasculogenic mimicry (VM) is an alternative type of blood perfusion characterized by formation of non‐endothelial cell‐lined microcirculatory channels and is responsible for aggressive tumour biology and increased tumour‐related mortality. VM‐correlated genes are associated with vascular endothelial grown factor receptor 1 (VEGFR1), and hypoxia‐related (hypoxia inducible factor 1 α—HIF1α) signalling pathways, whose molecules are client proteins of Hsp90 (heat shock protein 90) and are potential therapeutic targets. This pilot study was aimed to investigate vasculogenic mimicry in a three‐dimensional (3D) cell culture system of two aggressive canine osteosarcoma (OSA) cell lines (D22 and D17), and to evaluate the response of these cells to 17‐AAG (17‐N‐allylamino‐17‐demethoxygeldanamycin) treatment in relation to tubular‐like structure formation in vitro. Only D17 cell line formed hollow matrix channels in long‐term 3D cultures and assumed endothelial morphology, with cells expressing both Hsp90 and VEGFR1, but lacking expression of endothelial marker CD31. 17‐AAG treatment inhibited migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of HIF1α transcript and protein in this cell line. Taken together, these preliminary data indicate that the biological effects of 17‐AAG on D17 3D culture and on HIF1α regulation can provide interesting information to translate these findings from the basic research to clinical approach for the treatment of canine OSA as a model in comparative oncology.

In vitro three-dimensional cell cultures for bone sarcomas

“…The generation of a hypoxia compartment induced the production of VEGF factor by tumour cells promoting proliferation and differentiation of HUVEC to produce vascular tubule-like structures. Using dog OS cell lines (D22 and D17) and 3D collagen gels, Massimini et al demonstrated that a non-human OS model was associated with induced vasculogenic mimicry and that 17-AAg drug abolished tumour progression and micro vascular channel formation [110] .…”

The Osteosarcoma Microenvironment: A Complex but Targetable Ecosystem

“…OS tumors appear to develop such an endothelial-free tumor-derived vasculogenic network, as vascular mimicry is found in 22.7% of osteoblastic-type OS samples and associated with unfavorable prognostis [104,105]. Human OS cell lines (U2OS [106], MG-63 [105]) and aggressive canine cell lines [107] are able to form vessel-like structures in three-dimensional cultures. Vascular mimicry mechanisms remain largely unknown, often being assessed via inappropriate and biased in vitro assays [108].…”

An Update on Molecular Pathways Regulating Vasculogenic Mimicry in Human Osteosarcoma and Their Role in Canine Oncology

“…As far as VEGF/VEGFR axis in veterinary oncology is concerned, VEGF family members were identified in several canine cancers ( 76 ), as well as OSA tissue, serum, and cultured cells ( 77 – 79 ). A relation between VM and VERGFR was found in D17 canine OSA cells cultured on type I collagen where malignant cancer cells with endothelial morphology express VEGFR1 ( 14 ). Correlation between VM and VEGF axis has been firstly investigated in dogs with mammary tumors ( 7 ).…”

“…Moreover, canine inflammatory mammary carcinomas were analyzed for the presence of VM by transmission and scanning electron microscopy ( 13 ). In addition, as far as canine OSA is concerned, the presence of vessel-like structures in a long-term canine D17 OSA cell cultured on type I collagen has been recently described ( 14 ). As well, treatment with the heat shock protein 90 (Hsp90) inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) inhibited the migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of hypoxia-inducible factor 1α (HIF1α) transcript and protein expression ( 14 ).…”

“…In addition, as far as canine OSA is concerned, the presence of vessel-like structures in a long-term canine D17 OSA cell cultured on type I collagen has been recently described ( 14 ). As well, treatment with the heat shock protein 90 (Hsp90) inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) inhibited the migration of D17 OSA cells, also decreasing VM markers in vitro and inducing a reduction of hypoxia-inducible factor 1α (HIF1α) transcript and protein expression ( 14 ). Notwithstanding this, information regarding VM formation, molecular features, and prognostic implications in canine oncology is still limited.…”