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Introduction and aim: Hemangiosarcoma (HSA) is a malignant and very aggressive vascular tumour. 70% of patients show systemic metastases at the time of diagnosis. This tumour occurs frequently in older dogs with a strong predilection for the spleen and consists of transformed endothelial cells forming large and leaky vessel-like structures. A novel strategy to treat this malignancy could be anti-angiogenic therapy based on the inhibition of proliferation, migration and three-dimensional organization of transformed cells. Therefore the aim of our study was to establish an in vitro model of canine hemangiosarcoma to test the safety and efficacy of anti-angiogenic drugs.
Materials and methods: Tumours were collected from dogs during surgery or immediately after euthanasia. Isolation of cells was done from different areas of the tumours and by enzymatic digestion of the tissue. Cells were incubated in culture media with and without endothelial growth factors. In order to select specific subpopulations a new method using surgical threads as a guide rail for cells was developed. Cells were characterized by lectin histochemistry (DBA, UEA I; BS I). Cells were also immunostained with antibodies to von Willebrand factor (vWF), the receptor for angiopoietin I (Tie-2), CD31 and CD 51/61.
Results and conclusions: Different cultures of cells from canine hemangiosarcoma were successfully isolated and plated on culture dishes. Cells morphologically show characteristics of microvascular cells. Cultures incubated in growth factor medium showed features resembling in vitro models already established in our laboratory (Plendl et al., 2002, Kaessmeyer, 2009).
Interestingly, the cultured cells showed vascular development of two distinct mechanisms. In certain areas of the culture dishes, cells proliferated to clusters from which tube-like structures emanated. In the further progress, cell clusters were connected by long mono-cellular projections. During the following days, bridging endothelial cells proliferated, arranged linearly side by side in a vessel-like formation and three-dimensional multi-cellular bridges were built up. Glycohistochemistry using lectins demonstrated strong staining. Analysis of immunolabeling showed a nearly 60% positive reaction for vWF within the cells. 100% of the cells were immunopositive for Tie-2. 80% of the cells were immunopositive for CD31. CD 51/61 was detected with a reactivity of 100% of the cells. These results demonstrate that tumourous vascular structures of canine hemangiosarcoma might be formed by vasculogenesis and angiogenesis.