Additional file 1 of Angiogenic and molecular diversity determine hepatic melanoma metastasis and response to anti-angiogenic treatment
Creators: name: Wohlfeil, Sebastian A.
name: Häfele, Verena
name: Dietsch, Bianca
name: Weller, Céline
name: Sticht, Carsten
name: Jauch, Anna Sophia
name: Winkler, Manuel
name: Schmid, Christian David
name: Irkens, Anna Lena
name: Olsavszky, Ana
name: Schledzewski, Kai
name: Reiners-Koch, Philipp-Sebastian
name: Goerdt, Sergij
name: Géraud, Cyrill
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Evolutionary Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Plant Biology

description: Additional file 1: Figure S1. Morphological characterization of melanoma cell lines. A. A selection of representative pictures of hepatic metastases of WT31, B16F10 luc2, RET, D4M and HCmel12 melanoma after spleen injection. Besides, liver metastases of WT31 melanoma after i.v. injection are shown. Scale bar = 1 cm. B. Images of cell pellets of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. C. Representative pictures of lungs, brain, tibia, kidneys and spleen with metastases after intravenous injection of WT31 melanoma. Scale bar = 1 cm. Figure S2. Sirius red and PAS stainings of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M. A. Images of Sirius red (SR) stainings of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bar = 200 µm, n ≥ 5. B. Pictures of liver metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma stained by PAS. Scale bar = 200 µm, n ≥ 5. Figure S3. Heat map of top25 up- and downregulated genes and heat map of melanocytic differentiation genes. A. A heatmap of the 25 most up- and downregulated genes of HIM-melanoma in comparison to LM-melanoma is shown. Please also refer to Fig. 3A, B for definition of this gene set. B. A heat map of common melanocytic differentiation genes is presented. A red color code presents an upregulation of genes, whereas the green color code shows downregulation. Clustering of genes is presented on the left side. Figure S4. Heat map GOBP—cell migration. A heat map of the GOBP cell migration gene set is presented for B16F10, RET, WT31, HCmel12 and D4M melanoma cells. The heat map was split in two parts to fit the page. A red color code presents an upregulation of genes, whereas the green color code shows downregulation. Clustering of genes is presented on the left side. Figure S5. Heat map GOBP—angiogenesis. A heat map of the GOBP angiogenesis gene set is presented for B16F10, RET, WT31, HCmel12 and D4M melanoma cells. A red color code presents an upregulation of genes, whereas the green color code shows downregulation. Clustering of genes is presented on the left side. Figure S6. A. Additional pictures of immunofluorescences for CD31, CD32b and DAPI of hepatic metastasis of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma at the border to adjacent liver. Scale bars = 100 µm, n ≥ 5. B. Additional pictures of immunofluorescences for Collagen IV, CD31 and DAPI of hepatic metastasis of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma at the border to adjacent liver. Scale bars = 100 µm, n ≥ 5. Figure S7. Analysis of peri- and intratumoral Stab2 expression as well as extracellular Collagen I and Collagen III matrix deposition in hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. A. Immunofluorescence images of Stab2 and DAPI of B16F10 luc2, RET, WT31, D4M and HCmel12 melanoma liver metastases at the metastatic border. White dotted lines present the border of hepatic metastases to adjacent liver tissue. Scale bars = 100 µm, n ≥ 5. B. Immunofluorescences of Collagen I of hepatic metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bar = 100 µm, n ≥ 5. C. Immunofluorescences of Collagen III of liver metastases of B16F10 luc2, RET, WT31, HCmel12 and D4M melanoma. Scale bar = 100 µm, n ≥ 5. Figure S8. Treatment with Sorafenib leads to loss intratumoral vessels and cystic degeneration of hepatic melanoma metastases. A. 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/solvent controls. At day 19 mice were sacrificed. Macroscopic visible metastases were quantified. A Mann-Whitney Utest was performed (WT31: p = 0.0043; B16F10 luc2: p = 0.0745). Representative images are shown. Scale bars = 1 cm. Number of animals analyzed = 8 (WT31, Sorafenib), 9 (WT31, Vehicle), 6 (B16F10 luc2, Sorafenib), 6 (B16F10, Vehicle). B. The size of hepatic metastases of WT31 (left graph) or B16F10 luc2 melanoma (right panel) in mice treated with Sorafenib or vehicle was measured. Mann-Whitney U-tests were performed (WT31: p = 0.5207; B16F10: p = 0.0326). Number of animals analyzed = 6 (WT31, Sorafenib), 6 (WT31, Vehicle), 5 (B16F10 luc2, Sorafenib), 4 (B16F10, Vehicle). Number of metastases analyzed = 48 (WT31, Sorafenib), 32 (WT31, Vehicle), 57 (B16F10 luc2, Sorafenib), 28 (B16F10, Vehicle). C. Comparison of the percentage of pseudocystic WT31 or B16F10 luc2 melanoma metastases after treatment with Sorafenib. MannWhitney U-tests were performed (p = 0.1970). Number of animals analyzed = 6 (WT31, Sorafenib), 5 (B16F10 luc2, Sorafenib). Data information: *P &lt; 0.05, **P &lt; 0.01, *** P &lt; 0.0001, n.s. = not significant.