Figure 6. Anti-netrin-1 therapy inhibits EMT in human cancer cells.

a, Scatter plots of NTN1 and UNC5B expression versus Hallmark EMT signatures are shown for LUSCC (n = 484 primary tumours) and LUAD (n = 510 primary tumours) cancer types from the TCGA. Spearman correlations are shown at the top of each graph. b, Bar plots showing Spearman correlations between NTN1, UNC5B expression and three EMT signature scores¹¹,³⁸,⁴⁰ acrossLUSCC (n = 484 primary tumours), LUAD (n = 510 primary tumours) and SKCM (n = 443 tumours including 76 primary tumours and 367 metastases) cancer types from TCGA, with 95% confidence intervals (clipped at 0 for low correlations). To obtain EMT scores, the Hallmark signature was computed using single-sample Gene Set Enrichment Analysis⁴¹ (ssGSEA) on genes from the HALLMARK_EPITHELIAL_ MESENCHYMAL_TRANSITION signature, from MSigDB³⁸; the Thiery signature was computed similarly using genes from ref. 11; the Mak signature was calculated from the gene sets in ref. 40 as the difference of two signatures: a mesenchymal signature defined as the mean of mesenchymal gene expression and an epithelial signature defined as the mean of epithelial gene expression. c, Microscopic appearance of A549 NSCLC cells, following no treatment or after 3 days of TGFβ1 treatment (n = 3). Scale bars, 20 μm. d, Western blot analysis of netrin-1, E-cadherin (CDH1) and vimentin expression in the A549 NSCLC cell line in control conditions or following 3 days of TGFβ1 treatment. e, Co-immunostaining of E-cadherin and pan-cytokeratin on tumours arising from subcutaneous grafting into immunodeficient mice of A549 cells pre-treated with TGFβ1 in vitro for 6 days. The mice were treated with physiologic serum or NP137 for 25 days and the tumours were collected for histological analysis. Scale bars, 20 μm. f, Tumour cells expressing high levels of E-cadherin as a percentage of pan-cytokeratin-positive tumour cells (each dot represents the mean of E-cadherin-high cells in 4 representative areas from each tumour; n = 6 control tumours and n = 6 NP137-treated tumours). Data are mean ± s.e.m. Two-tailed t-test.