Haemangiosarcoma is a highly malignant neoplasm arising from vascular endothelial cells. The neoplasm occurs more commonly in the dog than in any other species and is very invasive and metastasises widely. Human angiosarcomas have been cultured, but no canine haemangiosarcoma cell line had been established in vitro at the time of commencement of this study.
A detailed review of the literature on canine haemangiosarcomas was undertaken, as well as on the techniques used to establish and characterise vascular endothelial cells and vascular neoplasms in vitro.
Canine aortic endothelial cells and fibroblasts were established in tissue culture and nine attempts were made to establish canine haemangiosarcoma cells in culture. A cell line designated K9-HSA was established from tissue explants of a splenic haemangiosarcoma. The resultant pleomorphic cell population grew in confluent layers that were morphologically consistent with atypical endothelial cells. These cells were passaged 88 times, proliferated independent of growth factors, and did not display contact-inhibited growth. When established on a Matrigel® substrate, K9-HSA cells adopted the reticular pattern of growth characteristic of endothelial cells. The cells soon invaded the matrix and formed inter-anastomosing arrays. A comparison of cell proliferation rates on differing substrates and in different culture media was made.
Transmission and scanning electron microscopy of K9-HSA revealed no features that distinguished them from non-transformed endothelial cells. An immunoperoxidase technique was optimised for von Willebrand factor staining of freshly collected canine aortic endothelial cells and this was used as the basis for immunostaining cultured K9-HSA cells. Only multinucleate cell forms displayed significant staining with von Willebrand factor antibody.
An immunoperoxidase technique for von Willebrand factor staining of paraffin-embedded formalin-fixed tissue was also optimised. The staining pattern resembled that for the cultured K9-HSA cells with very occasional, and typically multinucleate, cells being the only ones displaying positive staining. The immunoreacfivity of the K9-HSA cells and of the primary neoplasm for a range of other antibodies was the same and this staining was consistent with a poorly differentiated haemangiosarcoma.
The K9-HSA cells demonstrated an ability to take up and metabolise Dil-Ac-LDL at a rate significantly higher than fibroblasts and with a fluorescence pattern consistent with cells of endothelial origin.
The isolation of the K9-HSA cell line from a malignant vascular neoplasm, along with the pattern of growth, ultrastructural morphology, immunocytochemical profile, and pattern of uptake of Di-I-Ac-LDL, all indicated that this is a poorly differentiated endothelial cell line. The ability to manipulate this cell line in vitro would allow for an investigation into the metastatic mechanisms used by these tumours. Preliminary SDS-PAGE zymography revealed that cultured K9-HSA cells inherently release matrix metalloproteinase-2. A more detailed comparison with the release of metalloproteinases by well-differentiated endothelial cells is required. The interaction of neoplastic cells with the adjacent basement membrane has also been highlighted as a potential point of change associated with transformation. This cell line offers opportunities for identifying the molecular genetic determinants of malignancy.