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Fig. 2 | Molecular Cancer

Fig. 2

From: Integrative gene network and functional analyses identify a prognostically relevant key regulator of metastasis in Ewing sarcoma

Fig. 2

| High TCF7L1 expression inhibits metastasis in EwS through its DNA binding domain. a) Weighted Gene Correlation Network Analysis (WGCNA) depicting functional gene enrichment of down- or up-regulated genes in TCF7L1 re-expressing EwS cells. Network depicts signatures presenting P < 0,05, NES > 2. NES, normalized enrichment score. Arrows depict direction of gene regulation. b) Relative percentage of migrated cells in 6 h. TC-71 and SK-N-MC EwS cells containing a DOX-inducible re-expression construct for TCF7L1 where pre-treated with or without DOX for 72 h. n ≥ 7 biologically independent experiments. Two-sided Mann-Whitney test. c) Invasion and single cell migrated distance in 15 h. TC-71 and SK-N-MC EwS cells containing a DOX-inducible re-expression construct for TCF7L1 where pre-treated with or without DOX for 72 h and added to a microfluidic chamber containing a fibrin compartment. n ≥ 3 biologically independent experiments. Two-sided unpaired t-test. d) Schematic representation of the experimental design: TC-71 or SK-N-MC EwS cell lines containing a DOX-inducible re-expression construct for TCF7L1 were injected in the right tibia plateau. Animals were subsequently randomized and treated with or without DOX. At the end of the experiment, mice were evaluated ex vivo for presence of spontaneous metastases in inner organs. Pie charts depict percentage of metastasis-free organs (blue) in each condition, n represents total number of metastasis. Bottom pictures show representative images of metastatic organs in DOX(−) and DOX(+) conditions. n = 8 animals/group. e) Graph depicts relative area of histological metastatic spread of orthotopically injected SK-N-MC EwS cells containing a DOX-inducible re-expression construct for TCF7L1. n = 21 slides/group. Two-sided Mann Whitney test. Pictures show representative histological images of HE stainings from the evaluated organs. Scale bar is 50 μm. M, metastasis; N, normal tissue. f) Comparison of relative TCF7L1 expression of EwS primary tumors versus metastasis (n = 125), normalized using RPLP0 expression of the same samples. g) Analysis of relative TCF7L1 expression (normalized to RPLP0 expression of the same samples) in paired metastasis/primary samples from EwS patients 1–4. Independent one sample t-test. h) Relative colony number of colony-forming assays (CFAs) of TC-71 (left) and SK-N-MC (right) cells containing a DOX-inducible re-expression construct for TCF7L1, empty control, or one of the two deletion mutants for TCF7L1 (deletion mutant for the β-catenin binding domain, ∆CTNNB; deletion mutant for the DNA binding domain, ∆HMG). Cells were grown either with or without DOX. n = 4 biologically independent experiments. i) Relative percentage of migrated cells in 6 h. TC-71 and SK-N-MC EwS cells containing a DOX-inducible re-expression construct for TCF7L1 DNA binding domain (∆HMG) where pre-treated with or without DOX for 72 h. n ≥ 8 biologically independent experiments. j) Growth of EwS subcutaneous xenografts of TC-71 and SK-N-MC cells containing a DOX-inducible re-expression construct for TCF7L1 deletion mutant ∆HMG (arrow indicates start of DOX treatment). Data are represented as means (n = 7 animals/group). Two-sided Mann-Whitney test

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