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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2003 as doi:10.1096/fj.02-0560fje. |
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Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada
2Correspondence: Charles H. Best Institute, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada. E-mail: chi.hung.siu{at}utoronto.ca
SPECIFIC AIM
Migration of tumor cells is an important step in the process of tumor metastasis. Tumor cells must traverse the endothelial lining of the lymphatic and vascular systems in order to metastasize to distant organs. We test the hypothesis that endothelial cells secrete chemoattractants that induce chemotaxis of tumor cells across the endothelium.
PRINCIPAL FINDINGS
Conditioned endothelial medium was tested for its chemoattractant activity on metastatic melanoma cells in under-agarose migration and transwell migration assays. Interleukin (IL) -8 was identified as the major endothelium-secreted chemokine for metastatic melanoma cells. Antibody neutralization studies indicated that IL-8 elicits chemotactic response through the chemokine receptor CXCR1 (IL-8RA) on melanoma cells. Migration of melanoma cells across the endothelium was inhibited by antibodies against IL-8 or CXCR1, suggesting a role for the endothelium-secreted IL-8 at the transendothelial migration stage of cancer metastasis.
1. Endothelial cells secrete melanoma chemoattractants
To determine whether endothelial cells secrete factors that can induce melanoma cell chemotaxis, the metastatic melanoma cell line WM239 was examined in an under-agarose chemotaxis assay using either fresh or conditioned endothelial medium. Conditioned medium derived from passage 6 to 8 endothelial cells stimulated a migratory response in WM239 cells. The chemotactic migration of WM239 cells was time dependent. Cells migrated toward the conditioned medium at a rate of 4–5 µm/h during the first 48 h, then increased to 
8 µm/h between 48 and 72 h.
2. IL-8 is the major melanoma chemoattractant secreted by endothelial cells
Heat treatment and dialysis studies suggested that the chemoattractant secreted by endothelial cells was a protein of Mr<10,000. To identify the chemotactic agent, components in the endothelial medium were separated on an anion exchange column. Column fractions were separated into different pools and assayed for their ability to stimulate melanoma cell migration. Protein blots of these fractions were probed with antibodies directed against several chemokines secreted by endothelial cells. The CXC chemokine IL-8 was present in all fractions that elicited melanoma chemotaxis, suggesting it may be the major chemoattractant for melanoma cells. As IL-8 is known to be secreted in two major isoforms, an endothelial-derived IL-877 and a more common IL-872, we tested both IL-8 isoforms as well as other chemokines known to be secreted by endothelial cells, including MCP-1 and RANTES, in the under-agarose assay. IL-877 was the most potent chemoattractant, whether on its own or added to fresh endothelial medium. In contrast, GRO
, MCP-1 and RANTES failed to elicit any response from WM239 cells.
3. Effects of IL-8 depletion from the conditioned medium on WM239 cell chemotaxis
To confirm the role of IL-877 as a chemoattractant for melanoma cells, different amounts of antibodies were used to precipitate IL-8 from the conditioned endothelial medium. Immunoblot analysis showed that complete removal of IL-8 from the medium was achieved with 10 µg/mL of the antibody. These samples were tested in the under-agarose assay and a dose-dependent effect was observed (Fig. 1A
). The chemotactic response of WM239 cells was abrogated at 10 µg/mL of anti-IL-8 antibody. Immunoprecipitation with the similar amounts of anti-GRO antibody resulted in only a small reduction of the chemotactic response. If the loss in chemoattractant activity was due to the removal of IL-8, the addition of purified IL-877 to the depleted medium should restore its activity. Indeed, when the depleted medium was reconstituted with IL-877, the chemotactic response of WM239 cells was restored to 75% of the control (Fig. 1B
).
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4. IL-8-induced chemotaxis is mediated by the chemokine receptor CXCR1
To identify the IL-8 receptor involved in the IL-8-stimulated chemotactic response of melanoma cells, WM239 cells were incubated with function blocking antibodies against CXCR1 or CXCR2, then subjected to the under-agarose assay. The inhibitory effect of the anti-CXCR1 antibody was dose dependent and the chemotactic response of WM239 cells was abrogated at 100 µg/mL of the antibody. In contrast, antibody against CXCR2 and control antibody did not have significant effects, suggesting that IL-8 elicited chemotaxis through binding of CXCR1 and not CXCR2. CXCR1 belongs to the seven-transmembrane receptor family, which is coupled to heterotrimeric G-proteins. This would predict sensitivity to pertussis toxin treatment. Indeed, pertussis toxin inhibited chemotactic migration of WM239 cells. These results confirm the involvement of CXCR1 and pertussis toxin-sensitive G-proteins.
5. Chemotactic response of WM35 cells is dependent on the expression of CXCR1
We found that the nonmetastatic melanoma cell line WM35 failed to respond chemotactically to IL-8. It was therefore of interest to determine whether WM35 cells expressed CXCR1. Consistent with the role for CXCR1 in melanoma chemotaxis, immunoblot analysis revealed a very low level of CXCR1 in WM35 cells vs. WM239 cells. In contrast, similar levels of CXCR2 were detected in both cell lines (Fig. 2A
).
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We previously found that TNF- can stimulate WM35 cells to undergo transendothelial migration, so we tested the effect of TNF- on CXCR1 expression. Treatment of WM35 cells with TNF- stimulated a rapid increase in the level of CXCR1 (Fig. 2B
). The cellular level of CXCR1 increased >10-fold. When tested in the under-agarose assay, TNF--treated WM35 cells responded chemotactically to endothelial cell-conditioned medium (Fig. 2C
). Similar results were obtained using the transwell assay (Fig. 2D
). Prior treatment of cells with function blocking antibodies against CXCR1 inhibited the chemotactic response but antibodies against CXCR2 did not. These results thus confirm the role of CXCR1 in melanoma cell chemotaxis and implicate its participation in the transendothelial migration process.
6. Role of IL-877 in WM239 transendothelial migration
To assess the role of IL-8 in diapedesis, the effect of IL-877 on transendothelial migration of WM239 cells was examined in an in vitro transendothelial migration assay. DiI-labeled melanoma cells were seeded on top of a monolayer of endothelial cells cultured on Matrigel. The coculture was fixed at 5 h and melanoma cells that showed a spreading morphology under the endothelium were scored as transmigrated cells. When assayed in the presence of rabbit anti-IL-8 antibody or mouse anti-CXCR1 mAb, transendothelial migration of WM239 cells was reduced by 30% in each case. Impregnation of the Matrigel with IL-877 enhances the transmigration of WM239 cells but not WM35 cells. These results implicate a role for IL-8 and the CXCR1 receptor in the extravasation of melanoma cells during metastasis.
CONCLUSION AND SIGNIFICANCE
In the past few years, there is increasing evidence that IL-8 plays a key role in cancer metastasis. The constitutive expression of IL-8 by human melanoma cells has been correlated with their lung colonization efficiency in nude mice. Although IL-8 is known to stimulate the secretion of metalloproteinases and thus promote the migration and invasion of the extracellular matrix by metastatic cancer cells, these activities cannot account for the directional movement of cancer cells during diapedesis. Directional movement would suggest the presence of a chemoattractant from a separate source. Our studies have highlighted the importance of the endothelial IL-877 as a chemoattractant during transendothelial migration of melanoma cells. Results from our in vitro transendothelial migration studies are also consistent with the involvement of IL-877 in the extravasation of melanoma cells. The organ specificity of certain tumor metastases might depend on the types of chemokines secreted by the host endothelium and the expression pattern of chemokine receptors on the cancer cell. Recent studies have demonstrated that chemokines and their receptors play a critical role in determining the metastatic target of breast cancer cells.
Studies of metastatic and nonmetastatic melanoma cell lines are consistent with a role for chemotaxis in the transendothelial migration process. Our results suggest that IL-877 functions through CXCR1 and not the more promiscuous CXCR2. Potentially, a chemotactic gradient could arise via secretion on the basolateral surface as depicted in our model in Fig. 3
. IL-877 can be trapped in the extracellular matrix through protein–protein interactions and through interactions with proteoglycans, generating a gradient in the paracellular region. After the initial attachment of melanoma cells on the endothelium, IL-877 immobilized on the lumenal surface of endothelial cells can bind CXCR1 and induce melanoma cells to undergo directional migration and augment their ability to invade the endothelium.
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Our studies show that treatment of cells with antibodies against IL-8 and/or CXCR1 results in only partial inhibition of transendothelial migration. The partial inhibition of transmigration implicates the involvement of cellular processes in addition to chemotaxis. We have previously demonstrated that the cell adhesion molecules vß3 integrin and N-cadherin promote heterotypic cell–cell binding and constitute a favorable substrate for the transendothelial migration of melanoma cells. Future studies of the coordination of chemotactic response and adhesive interactions, as well as their signaling pathways, should lead to a better understanding of the transendothelial migration process during cancer metastasis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0560fje; doi: 10.1096/fj.02-0560fje 
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) antibody. After removal of the immunoprecipitates, the chemokine-depleted medium was assayed for WM239 melanoma cell chemotaxis in the under-agarose assay. The inset shows the immunoblots of depleted medium and immunoprecipitates obtained at 10 µg/mL of anti-IL-8 antibody. Lane a, IL-8-depleted medium; lane b, conditioned medium; lane c, immunoprecipitates. B) IL-877 was added to the IL-8-depleted conditioned medium at 10 µg/mL and the reconstituted medium was assayed in the under-agarose assay. Data represent mean ± SD (n=12).
