Figure 3

Migration of Lewis lung carcinoma cells toward lymphatic endothelial cells depends on lymphatic heparan sulfate. A. Schematic representation of a modified transwell chemotaxis assay, wherein liquid medium separates migrating LLC tumor cells that initiate in the upper well and migrate toward lower wells that contain either no cells or hLEC monolayers treated under various conditions. B and C. Transwell migration of tumor cells into lower wells plated with either no cells as a negative control ("NC") or hLEC monolayers treated as indicated was quantified, and plotted for each condition as the mean -fold response over the value for NC (and normalized to the NC value). Conditions in B refer to the addition of specific blocking antibodies or pre-treatment of the hLEC with heparinase (H'ase); and conditions in C refer to treatment of the hLEC with siRNA targeting the indicated HS biosynthetic enzymes. *P < 0.01, ^#P < 0.05, as compared to hLEC group in B and siDS group in C. D. Graphs quantifying transwell migration of either LLC (upper graph) or H1650 (lower graph) lung carcinoma cells into lower wells plated with either no cells (NC), control hLEC (siDS), or siXylT2 targeted hLEC. The condition "siXylT2+HS" (far right) refers to addition of heparan sulfate purified from cultured control hLEC into the lower-well medium during tumor cell migration toward siXylT2 targeted hLEC. Fractions (0.04 and 0.2) of the total HS needed for rescue were used in separate wells to test dose-response. ^#P < 0.05, *P < 0.01, **P < 0.001 for histogram comparisons indicated by horizontal bars.