Figure 4

CCL21/CCR7 axis has lymphangiogenic potential in vivo . (A) Macroscopic digital images of angioreactors collected with surrounding tissues. (B) Fluorescence assay analysis of LYVE1, Prox1, and CD31 markers as expressed by mouse lymphatic endothelial cells and blood vascular endothelial cells recruited into the angioreactors. Data are presented as mean relative fluorescent units ± SEM. (*) indicates significant difference (p < 0.005). (C) Quantitative real-time PCR analysis of LYVE1 and CD31 mRNA expression in the cellular contents of angioreactors. Expression levels are normalized to actin (ACTB). (*) indicates significant difference (p < 0.05) (n = 4). (D to G). Representative images of immunofluorescence localization of CD31 (red), LYVE1 (green), Podoplanin (green), and Prox1 (green) in serial sections of angioreactors containing MCF-7 mock vs. CCL21 KI MCF-7 cells (D), (E) and MDA-MB-231 mock vs. CCR7 KD MDA-MB-231 cells (F), (G). Nuclei are stained with DAPI (blue). Scale bar equals 50 μm. (H) Representative images of immunofluorescence localization of CD31 (red), LYVE1 (green), Podoplanin (green), and Prox1 (green) in serial sections of angioreactors containing CCR7 KD MDA-MB-231 cells and recombinant human VEGF-C (30 ng/μl). Nuclei are stained with DAPI (blue). Scale bar equals 50 μm. (I) Quantification of MVD and LVD. “Hot spot” scores for CD31-LYVE1, CD31-Podoplanin, and CD31-Prox1 were calculated by means of Image J (40× magnification). Data are presented as mean of “hot spot” ± SEM.