Additional file 1 of Hepatic passaging of NRAS-mutant melanoma influences adhesive properties and metastatic pattern
Creators: name: Dietsch, Bianca
name: Weller, Céline
name: Sticht, Carsten
name: de la Torre, Carolina
name: Kramer, Martin
name: Goerdt, Sergij
name: Géraud, Cyrill
name: Wohlfeil, Sebastian A.
Publisher: name: figshare
Year: 2023
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplementary Figures. Suppl. Fig. 1. Verification of cell purity and identity by immunofluorescence staining and PCR. A. Melanoma cells from liver metastases were excised and expanded in cell culture. To verify cell purity every subline was stained for Melan A, CD68, αSMA, Desmin, CD31, CD45 or pan cytokeratin before i.v. injection. Representative immunofluorescence images of passaged WT31 after isolation from liver metastases and expansion in cell culture are shown. Melanoma cells were stained with MelanA (green). Besides CD68 (hepatic macrophages), CD31 (endothelial cells), alphaSMA (fibroblasts), CD45 (hematopoietic cells), Desmin (myogenic differentiated cells, myofibroblasts) or pan-cytokeratin (hepatocytes) were stained in red respectively, Scale bars = 200µm B. PCR of human NRAS of WT31 and sublines P1, P2, P3, P4 and P5 (WT31_P5IV). H2O and B16F10 luc2 melanoma serve as negative controls. Product length: 499 bp. C. The number of organs colonized by WT31 melanoma and its sublines WT31_P1, WT31_P2, WT31_P3, WT31_P4 and WT31_P5 is shown. The number of lung metastases for each subline is presented (P = 0.0193 for WT31_P1IV vs. WT31_P2IV, P = 0.0107 for WT31_P2IV vs. WT31_P5IV). The number of liver metastases are displayed (P = 0.0028 for WT31_P2IV vs. WT31_P4IV, P = 0.0189 for WT31_P2IV vs. WT31_P5IV) A Dunn’s test was performed respectively. Number of animals analyzed = 5 (WT31_P1IV), 7 (WT31_P2IV), 6 (WT31_P3IV), 6 (WT31_P4IV), 4 (WT31_P5IV). Suppl. Fig. 2. H&amp;E, Sirius Red, EvG and PAS staining of liver metastases of WT31 and WT31_P5IV. Images of H&amp;E (A), Sirius Red (B), EvG (C) and PAS (D) staining of hepatic metastases of WT31 and WT31_P5IV melanoma. Scale bars = 200µm, n=5, black dotted lines show border of metastases to normal liver tissue. Suppl. Fig. 3. Analysis of extracellular matrix deposits by immunofluorescence staining for Collagen IV. A. Images of immunofluorescence staining for CD31 and Collagen IV of liver metastases of WT31 and WT31_P5IV. Representative images are displayed. Scale bars = 200µm, n=5. White dotted lines present the border of hepatic metastasis of WT31 and WT31_P5IV to the adjacent liver. The Collagen IV+ area was quantified in relation to the tumoral area (P = 0.4858, t-test). Suppl. Fig. 4. Over-representation analysis (ORA) of gene ontology biological processes (GOBP) or HALLMARK pathways. RNA sequencing of cultured, sub-confluent WT31 and WT31_P5IV melanoma cells was performed and the gene sets were compared by an over-representation analysis of gene ontology biological processes (GOBP) (A) or HALLMARK pathways (B). Dot plots display both the number of regulated genes by count and gene ratio and the adjusted P value of corresponding pathways. C. Heatmaps of HALLMARK oxidative phosphorylation and glycolysis of WT31 and WT31_P5IV melanoma are shown. The red colored bars present upregulated genes, whereas blue colored bars show downregulation. Genes symbols are written on the right side. Genes were filtered by an adjusted P value &lt; 0.05 and logFC ≥ 1 or ≤ -1. Genes are also listed in Suppl. Table 5 and 6. D. A heatmap of HALLMARK mitotic spindle of WT31 and WT31_P5IV melanoma is shown. The red colored bars present upregulated genes, whereas blue colored bars show downregulation. Genes symbols are written on the right side. Genes were filtered by an adjusted P value &lt; 0.05 and logFC ≥ 1 or ≤ -1. Genes are also listed in Suppl. Table 7. E. A Heatmap of HALLMARK DNA repair of WT31 and WT31_P5IV melanoma is shown. The red colored bars present upregulated genes, whereas blue colored bars show downregulation. Genes symbols are written on the right side. Genes were filtered by an adjusted P value &lt; 0.05 and logFC ≥ 1 or ≤ -1. Genes are also listed in Suppl. Table 8. F. A Heatmap of HALLMARK PI3K/AKT/mTOR-signaling of WT31 and WT31_P5IV melanoma is shown. The red colored bars present upregulated genes, whereas blue colored bars show downregulation. Genes symbols are written on the right side. Genes were filtered by an adjusted P value &lt; 0.05 and logFC ≥ 1 or ≤ -1. Genes are also listed in Suppl. Table 9. Suppl. Fig. 5. Mito ATP production rate (P = 0.4145) and glyco ATP production rate (P = 0.1908) of WT31 and WT31_P5IV measured by Seahorse XF analysis. A. Mito ATP production rate and glyco ATP production rate in WT31 and WT31_P5IV were measured by a Seahorse XF analysis 24 h after seeding by consecutive injections of oligomycin (1µM) and antimycin A / rotenone (0.5 µM). The experiment was repeated three times. A pooled analysis of the mean values for either the mito ATP production rate and the glycoATP production rate is presented for WT31 and WT31_P5IV. Besides, representative graphs of OCR (pmol/min/cells) and ECAR (mpH/min/cells) are shown. Red line = WT31_P5IV; blue line = WT31. Suppl. Fig. 6. Quantitative real-time PCRs (qRT-PCR) of selective genes in parental WT31 in comparison to WT31_P5IV. A.Selected genes of Top 25 strongly up- or downregulated genes in WT31_P5IV are shown in comparison to parental WT31. Upregulated genes: Capg (P &lt; 0.0001, t-test), Gm773 (P = 0.0079, Mann-Whitney test), Gpm6a (P = 0.0244, t-test), Pcdhgb8 (P &lt; 0.0001, t-test); downregulated gene: Igfbp3 (P &lt; 0.0001, t-test). The normalized expression values were calculated using the Pfaffl method. Two reference genes (Gak and Srp72) were used for normalization. Suppl. Table 1 lists TOP 25 up- and downregulated genes in WT31 and WT31_P5IV whereas Suppl. Table 10 lists the Primers which were used for qRT‑PCRs. B.Selected up- and downregulated genes of HALLMARK mitotic spindle in WT31_P5IV are shown in comparison to parental WT31. Upregulated genes: Notch2 (P &lt; 0.0001, t-test), Sun2 (P = 0.0001, t-test); downregulated genes: Arhgef3 (P &lt; 0.0001, t-test), Sorbs2 (P &lt; 0.0001, t‑test). The normalized expression values were calculated using the Pfaffl method. Two reference genes (Gak and Srp72) were used for normalization. Suppl. Table 10 lists the primers which were used for qRT‑PCRs. C. Selected up- and downregulated genes of GOBP cell adhesion as well as selected integrin and disintegrin-like proteins in WT31_P5IV are shown in comparison to parental WT31. Upregulated genes: Itga2b (P = 0.0079, Mann-Whitney test), Itga4 (P &lt; 0.0001, t-test); downregulated genes: Adam23 (P &lt; 0.0001, t-test), Itgb5 (P &lt; 0.0001, t-test), Adamts18(P = 0.0002, t-test). The normalized expression values were calculated using the Pfaffl method. Two reference genes (Gak and Srp72) were used for normalization. Suppl. Table 10 lists the Primers which were used for qRT‑PCRs. Suppl. Fig. 7. Western blot for total AKT (AKT) and phosphorylated AKT (pAKT) in WT31 and WT31_P5IV. A. A western blot of total AKT and GAPDH in WT31 and WT31_P5IV is shown. The original marker bands are shown at the left side. The experiment was repeated two times. Besides, a quantification of AKT in correlation to GAPDH by densitometry was performed with ImageJ and is presented on the right side of the western blots. B. A western blot of phosphorylated AKT (pAKT) and GAPDH in WT31 and WT31_P5IV is shown. The original marker bands are shown at the left side. The experiment was repeated two times. Besides, a quantification of pAKT in correlation to GAPDH by densitometry was performed by ImageJ and is presented on the right side of the western blots. C. The ratio of normalized pAKT/normalized AKT of each individual sample was calculated and is shown for WT31 in comparison to WT31_P5IV (P = 0,0291, t-test). Pooled data of two individually performed experiments are presented. Suppl. Fig. 8. Protein-protein interaction network by STRING analysis of GOBP: cell adhesion. A. A STRING analysis of GOBP: celladhesion displays possible protein-protein interactions. Up- and downregulated genes with an adjusted P &lt; 0.05 were included into the analysis. Light blue line = known interactions from curated databases, violet line = known interactions experimentally determined, green line = textmining, black line = co-expression, pale violet line = protein homology, blue line = predicted interactions in gene co-occurrence. Suppl. Fig. 9. Initial tumor cell adhesion and retention to the liver vasculature was studied ex vivo for WT31 and WT31_P5IV after i.v. injection. A. Ex vivo fluorescence imaging of livers 90 min after i.v. injection of DIL labelled WT31 or WT31_P5IV. Quantification of adherent WT31 or WT31_P5IV melanoma cells. Livers were set as regions of interest and fluorescence signal was quantified respectively (P = 0.5031, Mann-Whitney test). N = 4/group, the experiment was repeated three times. Scale: min = 5.04e8 p/sec/cm2/sr; max = 6.01e9/sec/cm2/sr.