Fig. 6
Treatment with Sorafenib leads to pseudocystic hemorrhagic degeneration and loss of intratumoral vessels of hepatic melanoma metastases. WT31 melanoma cells (0.3 × 105 cells) or B16F10 luc2 melanoma cells (1.5 × 105 cells) were injected intrasplenically. From day 1 to 18 mice received daily i.p. injections of Sorafenib (60 mg/kg KG) or vehicle controls (solvent controls). At day 19 mice were sacrificed. Number of animals/ group = 6 (WT31, Sorafenib), 7 (WT31, Vehicle), 6 (B16F10 luc2, Sorafenib), 6 (B16F10 luc2, Vehicle). A Pictures of H&E stainigs of metastases of WT31 (left panels) or B16F10 luc2 melanoma (right panels) of mice treated with Sorafenib or vehicle. Scale bar = 200 µm, n ≥ 5. B The percentage of pseudocystic metastases in relation to the total number of metastases was quantified for WT31 melanoma treated with Sorafenib or vehicle. Analysis is shown per animal. A Mann–Whitney U-test was performed (p = 0.0022). Number of animals analyzed = 6 (WT31, Sorafenib), 6 (WT31, Vehicle). Number of metastases analyzed = 48 (WT31, Sorafenib), 32 (WT31, Vehicle). Besides, the tumor cell area of WT31 metastases in mice treated with Sorafenib or vehicle was measured. A Mann–Whitney U-test was performed (p < 0.0001). Number of animals analyzed = 6 (WT31, Sorafenib), 6 (WT31, Vehicle). Number of metastases analyzed = 41 (WT31, Sorafenib), 32 (WT31, Vehicle). C The percentage of pseudocystic metastases in relation to the total number of metastases was quantified for B16F10 luc2 melanoma treated with Sorafenib or vehicle. Analysis is shown per animal. A Mann–Whitney U-test was performed (p = 0.0159). Number of animals analyzed = 5 (B16F10 luc2, Sorafenib), 4 (B16F10 luc2, Vehicle). Number of metastases analyzed = 39 (B16F10 luc2, Sorafenib), 28 (B16F10 luc2, Vehicle). Moreover, the tumor cell area of B16F10 luc2 metastases in mice treated with Sorafenib or vehicle was measured. A Mann–Whitney U-test was performed (p = 0.0002). Number of animals analyzed = 5 (B16F10 luc2, Sorafenib), 4 (B16F10 luc2, Vehicle). Number of metastases analyzed = 39 (B16F10 luc2, Sorafenib), 28 (B16F10 luc2, Vehicle). D Immunofluorescences of Lyve-1, Emcn, TRP-2 and DAPI of livers with hepatic melanoma metastases of WT31 (left panels) or B16F10 luc2 melanoma (right panels) treated either with Sorafenib or vehicle. White dotted lines present the border of hepatic metastases to adjacent liver tissue. Scale bars = 100 µm, n ≥ 5. E The pseudocystic area was set in relation to the total size of WT31 or B16F10 luc2 metastases that were treated with Sorafenib. A Mann–Whitney U-test was performed (p = 0.0004). Number of animals analyzed = 6 (WT31, Sorafenib), 5 (B16F10 luc2, Sorafenib). Number of metastases analyzed = 40 (WT31, Sorafenib), 30 (B16F10 luc2, Vehicle). F The response to Sorafenib was determined as percentage of tumor cell area in the Sorafenib group and normalized to the percentage of tumor cell area of vehicle controls. Normalization needs to be performed because of variable size of metastases, necrotic or cystic areas in the vehicle control group. A Mann–Whitney U-test was performed (p < 0.0001). Number of animals analyzed = 6 (WT31, Sorafenib), 5 (B16F10 luc2, Sorafenib). Number of metastases analyzed = 40 (WT31, Sorafenib), 30 (B16F10 luc2, Vehicle). Data information: *P < 0.05, **P < 0.01, *** P < 0.0001, n.s. = not significant