Role of CLEVER-1 in breast cancer metastasis

The role that the novel lymphatic-associated adhesion molecule CLEVER-1 plays in breast cancer metastasis has been examined by assessing its expression in human breast tumour specimens and by conducting in vitro experiments to monitor its involvement in regulating cell adhesion to human umbilical vein endothelial cells (HUVEC) and hTERT immortalised lymphatic endothelial cells (LEC).

CLEVER-1 expression was examined in tonsil, lymph node and 148 formalin-fixed paraffin-embedded archival breast carcinoma specimens using standard immunohistochemistry protocols. In vitro CLEVER-1 expression was studied, in HUVEC and LEC, via fluorescence-activated cell sorting. Tumour cell (breast MCF-7 and MDA-MB-231, and melanoma SKMEL-30) adhesion and leukocyte adhesion to parental and CLEVER-1 siRNA knockdown endothelial cells was also examined.

The results show that, in tissue specimens, CLEVER-1 is present in blood and lymphatic vessels and in certain leukocyte sub-populations (macrophage or dendritic cells). Although expression, in tumours, is higher in blood vessels than in lymphatic vessels (62.4% versus 18.2%), only lymphatic expression is associated with lymph node metastasis (P = 0.027). CLEVER-1 expression in blood vessels and lymphatic vessels correlates with the density of inflammatory infiltrate (P < 0.001 and P = 0.004, respectively) and expression in macrophages (P < 0.001). In vitro results show that although CLEVER-1 is expressed intracellularly in both HUVEC and LEC, only LEC exhibit surface expression. Interestingly, adhesion assays show that tumour cells adhere preferentially to LEC with maximal adhesion exhibited at 30 to 40 minutes. Tumour cells adhere less to CLEVER-1 knockdown LEC than to control LEC. The role of CLEVER-1 in cellular adhesion is being further investigated, using tumour cells and different leukocyte populations, to determine its involvement in adhesion and migration of different cell types across lymphatics.