Many cancers, including breast, colon, prostate, and skin cancers, spread and metastasize preferentially through the lymphatic system (lymphogenous metastasis). Despite the prevalence of lymphogenous metastasis and its dire consequences for patient survival, little is known about how tumor cells locate and migrate to lymphatic vessels, enter (intravastate) into the vessels, and then travel to the lymph nodes. Using a novel toolkit consisting of models for the tumor microenvironment, lymphatic vessels, and the lymph nodes, we are asking questions about the mechanisms and molecules involved in each step of this process.

To examine the tumor microenvironment, we have developed a number of in vitro models that incorporate interstitial flow, a 3-D matrix, and/or other cell types. We have employed a 3-D transwell assay to demonstrate that breast cancer cells migrate in the direction of interstitial flow, and that this migration is dependent on the chemokine receptor CCR7. We are also using 3-D radial flow chambers to examine autologous chemotaxis of cancer cells and tumor cell-fibroblast interactions. To study tumor-lymphatic endothelial cell (LEC) interactions, we have used the same transwell assay (by seeding LECs on the opposite side of the membrane) as well as a 3-D segregated co-culture model, where tumor cells and LECs are cultured in separate but contiguous collagen gels. In this system we have shown that tumor cells secreting VEGF-C can promote migration towards LECs through autocrine and paracrine signaling. Once tumor cells reach the lymphatics, they must enter the vessel and then travel downstream to the lymph nodes. Using a 3-D model of the tissue space that includes interstitial flow and an in vitro engineered lymphatic vessel that collects the draining fluid, we are asking questions about how tumor cells intravasate into lymphatics, and specifically what adhesion molecules are involved in this process.

To model the behavior of tumor cells once they are inside the lymphatic vessel, tumor cells are flowed over a monolayer of LECs to quantify behaviors such as rolling, attachment, and adhesion. Taken together, this continuum of models addresses many aspects of lymphogenous metastasis: the tumor microenvironment (and the role played by other cell types), homing to lymphatic vessels, and subsequent intravasation and trafficking to lymph node. Throughout, we emphasize the importance of interstitial fluid flow as a biophysical phenomenon that has a profound effect on cancer cell migration, invasion, and metastasis.

Manuscripts:

• J.D. Shields, M.E. Fleury, C. Yong, A.A. Tomei, G.J. Randolph, and M.A. Swartz (2007). Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling. Cancer Cell 11:526-538. (comment: J. Cell Biol., June 18 2007 doi:10.1083/jcb.1781rr2).