Extended Data Fig. 5: LEC-secreted CCL21, but not CCL19, stimulates proliferation of CCR7+ GSCs in vitro.
a, Cell counting was performed for GSC1552 0, 2, 4, and 6 days after adding either IgG (10 ng ml−1), CCL19 recombinant protein (10 ng ml−1), or CCL21 recombinant protein (10 ng ml−1). b, Quantification of spheres formation of GSC2907 (left), GSC20 (middle), or GSC1552 (right) when treated with either IgG, CCL19 recombinant protein, or CCL21 recombinant protein. Data are presented as mean ± SD from 6 independent experiments. P-values were calculated using two-tailed unpaired Student’s t-test. c, Representative images of spheres formed by GSCs when treated with either IgG, CCL19 recombinant protein, or CCL21 recombinant protein. Scale bar, 50 μm. Images are representative of six independent experiments. d, Cell counting was performed 0, 2, 4, 6 days after adding either IgG or CCL21 monoclonal antibody. P-values were calculated using two-tailed unpaired Student’s t-test. e, Statistics for spheroid formation of GSCs treated with CCL21 monoclonal antibody when co-cultured with LECs. Data are presented as mean ± SD from 6 independent experiments. P-values were calculated using two-tailed unpaired Student’s t-test. f, Cell counting was performed for GSC1552 cells transfected with either shCONT or shCCR7 and treated with CCL21(n = 6). g and h, Representative FACS dot plots showing CCR7+ and CD133+ CT2A cells (g) or GL261 cells (h). i, Schematic model that illustrates cell sorting strategy of CD133+CCR7+ murine glioma cell lines (GL261 and CT2A) and implantation into CCL21−/− mice. j, qPCR analysis showing mRNA expression of CCL21 following shRNA-mediated knockdown. Data are presented as mean ± SD from three independent experiments. P-values were calculated using two-tailed unpaired Student’s t-test. k, Cell counting for GSCs co-cultured with either LECs-shCONT, LECs-shCCL21.242, or LECs-shCCL21.214 was performed on Day 0, 2, 4, and 6. (a-b, d–f, k) Data are presented as mean ± SD from six independent experiments. P-values were calculated using one-way ANOVA test.
