Biological and molecular characterization of a canine hemangiosarcoma-derived cell line

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Abstract

Canine hemangiosarcoma (HSA) is a devastating disease. Investigation of novel therapies has been limited by the limited availability of canine HSA-derived cell lines. We report the development of a canine HSA-derived cell line, DEN-HSA, which recapitulates features of angiogenic endothelium. DEN-HSA cells were derived from a spontaneous HSA arising in the kidney of a dog. DEN-HSA displayed surface molecules distinctive of endothelial histogenesis, including factor VIII-related antigen, ICAM-1 and αvβ3 integrin. In vitro, DEN-HSA formed microvascular tube-like structures on Matrigel®, and proliferated in response to a variety of angiogenic growth factors. The cells expressed mRNA and protein specific for bFGF and its receptors, and VEGF and its receptors, among others. DEN-HSA conditioned medium evoked a marked angiogenic response in a murine corneal pocket assay, indicating potent proangiogenic activity of substances secreted by this cell line. This research confirms the DEN-HSA cell line as endothelial in origin, suggests the presence of angiogenic growth factor autocrine loops, and offers the potential to utilize DEN-HSA cells for the study of novel therapies that modulate endothelial proliferation.

Introduction

Altered endothelial cell proliferation and survival is of prime importance in many disease conditions, including neoplasia, wound healing, and chronic inflammatory disease. Perhaps the most dramatic example of dysregulated angiogenesis is seen in malignancy derived from vascular endothelium. Investigation of the mechanisms responsible for tumourigenesis in these neoplasms has the potential to shed light onto basic concepts of angiogenesis, and potentially identify novel strategies for its modulation.

Despite the fact that vascular tissue is extremely abundant, malignant tumours derived from the vascular endothelium are rare in humans, accounting for only 2% of soft-tissue sarcomas in one evaluation (Fata et al., 1999). When encountered, haemangiosarcomas of the human breast, liver, spleen and scalp behave aggressively, metastasizing commonly and yielding very poor long-term survival rates (Falk et al., 1979, Holden et al., 1987, Neuhauser et al., 2000, Silverman et al., 1994).

Haemangiosarcoma (HSA) is a relatively common, spontaneous tumour in dogs, accounting for approximately 20% of all soft-tissue sarcomas (Dorn et al., 1968), and up to 5% of all malignant canine neoplasms (Bastianello, 1983, MacVean et al., 1978). Canine HSA can occur in any site, however the spleen, skin, right atrium, and liver are the most common primary sites (MacEwen, 2001). Canine HSA is typified by very aggressive biological behavior, with wide and rapid metastasis being common. Doxorubicin-based chemotherapy has the potential to modestly improve survival time following surgery, however, one-year survival rates remain less than 10% (Sorenmo et al., 2000, Sorenmo et al., 1993, Vail et al., 1995).

Canine HSA has potential utility as a spontaneously occurring model of human endothelial tumours, such as angiosarcoma/hemangiosarcoma, hemangioma of infancy, and Kaposi’s sarcoma, a malignant tumour characterized by the dysregulated proliferation of endothelial cells. Moreover, canine HSA cells may also be useful as a tool for the study of endothelial cell biology in malignancy. For these purposes, we sought to establish a continuous cell line derived from a canine HSA, and to characterize this line with regard to its in vitro biological behavior, cell surface phenotype, and growth factor/growth factor receptor expression.

Section snippets

Cell lines

The DEN-HSA cell line was established from a spontaneous renal HSA arising in an 11-year-old castrated male golden retriever. The primary tumour was mechanically and enzymatically dissociated (collagenase III 50 U/mL, DNAse 100 U/mL, hyaluronidase 200 U/mL, Sigma, St. Louis, MO) (Kemmer et al., 1987), and passaged in tissue culture flasks in complete minimal essential medium (Cellgro Mediatech, Herndon, VA) supplemented with 5% fetal bovine serum, 5% newborn bovine serum, 100 U/mL penicillin, 100 

Morphology

The DEN-HSA cell line has been morphologically stable in culture through >150 passages. When subconfluent, DEN-HSA cells appeared elongated and spindleoid, but adopted a more rounded, “cobblestone-like” morphology characteristic of EC from many species, including dogs (Gerhart et al., 1988) when confluent (Fig. 1(A)). Upon plating on Matrigel®, DEN-HSA formed microvascular tube-like structures (Fig. 1(B)). However, after 24 h these tube-like structures disappeared, and the cells appeared to form

Discussion

There are few endothelial-derived malignant tumour cell lines available for study. Those available include the Eoma murine endothelioma cell line (Obeso et al., 1990), and several artificially transformed murine endothelial cell lines, with in vivo characteristics similar to that of hemangioma or angiosarcoma (Arbiser et al., 2000, Arbiser et al., 1997, Montesano et al., 1990, RayChaudhury et al., 1994). More recently, cell line derived from spontaneous human and canine angiosarcoma have been

Acknowledgments

The authors dedicate this manuscript to their esteemed colleague and co-author, the late Dr. E. Gregory MacEwen, and to acknowledge the expert technical assistance of A.K. Marr, J.M. Schmidt, B. Charles and B. Shinners. This work was supported by the University of Wisconsin Animal Cancer Treatment Fund, and by a generous donation from Mr. A. Rolfe.

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    1

    Present address: Georgia Tech/IBB, 315 Forest Drive, Box 160, Atlanta, GA 30332, USA.

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    Present address: Primate Research Center, University of Wisconsin-Madison, 425 Henry Mall, Madison, WI 53706, USA.

    MacEwen is deceased.

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