Fig. 4

Melanoma metastases differ in vascular density. Intratumoral EC express continuous EC-associated marker proteins. A Images of tile scans of immunofluorescence stainings for Lyve-1, Emcn and TRP-2 of liver metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Representative images are displayed. Scale bars = 100 µm, n ≥ 5. Detail magnifications of corresponding images show the border of hepatic metastasis to adjacent hepatic tissue. Scale bars = 100 µm. B Immunofluorescences for CD31, CD32b and DAPI of hepatic metastasis of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma at the border to adjacent liver. White dotted lines present the border of hepatic metastases to adjacent liver tissue. Scale bars = 100 µm, n ≥ 5. C Quantification of intratumoral blood vessels. Emcn⁺ area in relation to total tumor area is presented. A Dunn’s test was used (p = 0.0200 for B16F10 vs. WT31; p = 0.0105 for B16F10 vs. D4M; p = 0.0400 for RET vs. D4M; p = 0.0130 for WT31 vs. HCmel12; p < 0.0001 for WT31 vs. D4M; p = 0.0020 for HCmel12 vs. D4M). A box and whisker plot is presented with distribution of values from minimum to maximum. D Intratumoral EC were analyzed for Emcn or Lyve-1 marker expression. Corresponding marker expression was set in relation to total intratumoral vessel area (100%). Proportion of Lyve⁺, Lyve⁺  + Emcn⁺ as well as Emcn⁺ area is displayed. A Dunn’s test was applied. Red bars present the statistical comparisons of Emcn expression, blue bars of Lyve-1 and Emcn expression and green bars demonstrate statistical differences in Lyve-1 expression (Emcn: p = 0.0140 for B16F10 vs. HCmel12; p = 0.0020 for WT31 vs. HCmel12; p = 0.0010 for HCmel12 vs. D4M; Lyve-1 and Emcn: p = 0.0120 for B16F10 vs. D4M; p = 0.0440 for RET vs. D4M; p = 0.0170 for WT31 vs. HCmel12; p < 0.0001 for HCmel12 vs. D4M; Lyve-1: p = 0.0140 for B16F10 vs. HCmel12; p = 0.0080 for WT31 vs. HCmel12; p < 0.0001 for HCmel12 vs. D4M). Number of animals analyzed = 6 (B16F10 luc2), 3 (RET), 7 (WT31), 3 (HCmel12), 4 (D4M). Number of metastases analyzed = 6 (B16F10 luc2), 4 (RET), 10 (WT31), 10 (HCmel12), 25 (D4M). E. Expression of CD31 and CD32b of intratumoral vessels was analyzed (see E for IF images) and was set in relation to total intratumoral vessel area (100%). Proportion of CD31⁺, CD31⁺  + CD32b⁺ as well as CD32b⁺ area is displayed. A Dunn’s test was applied. Red bars present statistical comparison of CD31 expression, blue bars of CD31 and CD32b expression and whereas green bars demonstrate statistical differences in CD32b expression (CD31: p = 0.0140 for B16F10 vs. HCmel12; p = 0.0020 for WT31 vs. HCmel12; p = 0.0020 for B16F10 vs. D4M, p = 0.0210 for RET vs. D4M, p < 0.0001 for WT31 vs. D4M,; CD31 + CD32b: p < 0.0001 for B16F10 vs. D4M; p = 0.0080 for RET vs. D4M; p < 0.0001 for WT31 vs. D4M; p < 0.0001 for HCmel12 vs. D4M; CD32b: p = 0.0280 for WT31 vs. HCmel12). Number of animals analyzed = 4 (B16F10 luc2), 3 (RET), 5 (WT31), 4 (HCmel12), 3 (D4M). Number of metastases analyzed = 6 (B16F10 luc2), 4 (RET), 16 (WT31), 6 (HCmel12), 12 (D4M). F A Spearman’s correlation of the mean vascular density and the mean size of hepatic melanoma metastases was calculated among all melanoma cell lines (r = 0.9). A one-sided t-test was performed (p = 0.0417). Data information: *P < 0.05, **P < 0.01, *** P < 0.0001, n.s. = not significant.