Derivation of a murine Lymphatic Endothelial Cell line (LEC-C2) and analysis of chemokines expression profile.

S. Mancardi,PhD2, R. Inga2, E. Vecile PhD1, G. Stanta MD2, A. Dobrina MD1, O.R. Burrone PhD2

International Center of Genetic Engineering and Biotechnology ICGEB, Trieste, Trieste Italy 1 Dipartimento di Fisiologia e Patologia, Universita degli Studi di Trieste,Trieste Italy 2 Dipartimento di Morfologia Umana Normale, Universita degli Studi di Trieste,Trieste Italy

Aim: Freunds adjuvant induced peritoneal lymphangioma in mice represents a unique and convenient source of lymphatic endothelial cells able to propagate in vitro. We used these cultures to produce a stable lymphatic endothelial cell line in order to investigate the biological and functional properties of lymphatic endothelium. In particular we were interested in determining the role of chemokines expression in leukocytes recruitment and trafficking. Methods: Primary cultures of lymphatic endothelial cells from mice lymphangioma were infected and transformed by a supernatant containing middle T oncogene of polyoma virus. Selected clones were morphologically, histologically and molecularly analysed in comparison with lymphangioma derived cells. The C2 clone, which retains the expression of specific lymphatics markers, was further investigated for chemokines expression and the chemotactic properties of its supernatant evaluated on different cell populations. In vivo assays were performed in order to estimate the possible promotion of melanoma metastasis spreading through lymphatics. Results: The lymphatic endothelial cell C2 (LEC-C2) clone, express all the markers specific for lymphatics: vascular endothelium growth factor receptor 3 (VEGFR-3) podoplanin and desmoplakin, with the exception of the hyaluronan receptor LYVE-1, positive in lymphangioma cells. Expression of other markers, generally attributed to endothelial cells, was also identified. LEC-C2 is able of autonomous growth because of the autocrine production of VEGFC. Angiogenetic potential was evident in Matrigel. Lec-C2 grows in Balb/c mice and originates a tumour resembling Kaposis sarcoma. The profile of chemokines expression resembles the one described in lymphangioma and is confirmed by migration assay. Positive preliminary results about melanoma metastasis proliferation and migration are also produced in vivo. Conclusions: The LEC-C2 cell line is of lymphatic endothelial origin and express different chemokines. In addition it provides strong evidences for the establishment of the lymphatic origin of Kaposis sarcoma.

KEY WORDS: lymphangioma, endothelium, cell line, chemokines.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Carcinogenesis.