Figure 2.

The B16-R2L variant shows (A) no difference in tumor size but (B) an increase in lymphatic metastasis to sentinel lymph nodes. B16FI and B16-R2L cells were injected intradermally (1x10⁶ in the backskin of C57Bl6) and tumor size and LN metastasis was measured/evaluated at day 14 after injection. Statistical analysis was performed using the Mann-Whitney test. Data represent means ± SEM. ^*P<0.05; ^**P<0.01. (C–E) Increased peritumoral lymphangiogenesis. Staining for LYVE-1 and computer-assisted evaluation (Image Pro software) reveal (C) increased peritumoral number of lymphatic vessels and (D) vessel size for the B16-R2L variant in comparison to the parental cell line (B16F1) while (E) average size was not altered. Statistical analysis was performed using the unpaired Student’s t-test. Data represent means ± SEM. ^*P<0.05; ^**P<0.01. (F–I) Increased sentinel lymph node lymphangiogenesis in B16 variants. Lymph node lymphangiogenesis was evaluated at day 17 after intradermal injection of 1×10⁶ cells in C57Bl6 mice (8–10 weeks). Staining revealed an increased sentinel lymph node lymphangiogenesis indicated by LYVE-1 (green)/CD31 (red) immunofluorescence stain. Nuclei, blue (Hoechst); magnification, ×5.