Tumor/Endothelium Crosstalk Attracts T Cells Figure 3. T lymphocytes that migrated to tumors in vivo fail to migrate to tumor cells in vitro. (A) T lymphocytes were placed in the upper wells of 4 mm transwell systems. Supernatants from primary medulloblastoma cells (MB1277 and MB1323), Daoy medulloblastoma cells, LCLs and unconditioned media were placed in the lower well. While LCL supernatants induced brisk T lymphocyte migration (p,0.001), their migration towards Daoy supernatants was comparable to that of media only. (B) Chemokine signatures of Daoy and 7 primary medulloblastoma culture supernatants. (C) Flowcytometry of CD4 and CD8 subsets to study the chemokine receptor expression pattern on T lymphocytes. No chemokine receptors corresponding to tumor-derived chemokines were detectable on the cell surface. This explained the failure of supernatants from tumor cells to attract T lymphocytes ex vivo. doi:10.1371/journal.pone.0020267.g003 (Figure 3C). Hence T lymphocytes lacked measurable chemokine receptors that are cognate for the chemokines the tumor cells produce, explaining the observed lack of in vitro migration. Tumor cells trigger the secretion of the potent chemo- attractant: Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) from the endothelium and induce avid T-cell migration in vitro The discrepancy between the in vivo and in vitro migration of T lymphocytes, led us to hypothesize that successful migration occurred in vivo because primary tumor cells triggered a secondary, tumor-stroma derived, chemokine signal that was successfully recognized by T lymphocytes. Growing evidence supports an essential and complex role for the vascular endothelium in cancer biology and progression. [25] In particular, crosstalk between endothelial and tumor cells was shown to regulate tumor progression and resistance to anti-cancer therapies in various manners depending on the tumor type. [25– 27] We therefore cocultured tumor cells with murine brain tumor endothelial cells (bEND.3) to more closely mimic our in vivo system. We characterized changes in the chemokine profile during the period of coculture. Supernatants from Daoy and bEND.3 cocultures contained a chemokine pattern that was different from those observed in either cell population alone. Distinctly, the coculture contained large amounts of RANTES, which were detectable after 48 hours and reached a plateau after 6 days (Figure 4A and Figure S2). This effect was not contact- PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20267