Abstract
Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1,2,3,4,5,6,7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line—which undergoes EMT following TGFβ1 administration8,9—with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.
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Data availability
All raw sequence data for mouse RNA-seq, single cell RNA-seq and 10x Visium have been deposited in the Gene Expression Omnibus under the accession number GSE234267. Source data are provided with this paper.
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Acknowledgements
The authors thank the ULB animal facility; ULB genomic core facility (F. Libert and A. Lefort) for bulk RNA-seq and scRNA-seq; the Gilles Thomas bioinformatic platform; Centre de Recherche en Cancérologie de Lyon, Fondation Synergie Lyon cancer for the spatial transcriptomic sequencing; S. Bottieau for technical assistance; F. Lavial for Unc5b shRNA; and R. Derynck for the A549 cell line. J.L. is supported by NETRIS Pharma. I.P. is supported by FNRS and WELBIO. S.V. is supported by a PhD fellowship for Strategic Basic Research (1S93320N) from the Research Foundation Flanders (FWO). C. Decaestecker is a senior Research Associate with Fond National de la Recherche Scientifique (FNRS, Brussels, Belgium). DIAPath and the Department of Pathology are supported by the Fonds Yvonne Boël. The CMMI is supported by the European Regional Development Fund and the Walloon region (Wallonia-biomed; grant no. 411132–957270; project “CMMI-ULB” support the Center for Microscopy and Molecular Imaging and its DIAPath department). C.B. is supported by WELBIO, FNRS, TELEVIE, Fondation Contre le Cancer, ULB Foundation, Foundation Baillet Latour, FNRS/FWO EOS (40007513) and the European Research Council (AdvGrant 885093). This work was also supported by institutional grants from CNRS, University of Lyon1, Centre Léon Bérard and from the Ligue Contre le Cancer, INCA, ARC Sign’it and ANR (nos. ANR-10-LABX- 0061, ANR-17-CONV-0002 and ANR-18-RHUS-0009).
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Contributions
J.L., I.P., S.V. and C.B. designed the experiments and performed data analysis. J.L. and I.P. performed most of the biological experiments. S.V. performed most of bioinformatic analysis for single-cell sequencing. N.R., Y.S. and A.S. helped with bioinformatic analysis. J.V.H. helped with 10x single-cell sequencing. R.M.S. helped with RNAscope analysis. V.M., A. Boinet., S.S., S.L., S.G. and S.B. helped with cell culture experiments, immunostaining, blocking antibody injection and follow-up with the mice. I.S., J.A., E.Z., C. Decaestecker. and A.C. performed immunostaining and quantification of EMT in human cancer samples. B.D., M.B. and N.B. performed biological in vivo and in vitro experiments on Ishikawa endometrial cell lines. C.S. and D.V. performed bioanalysis from TCGA. C. Dubois performed FACS sorting. T.V. helped and supervised the single-cell data analysis. P.M. and A. Bernet helped with the design of the experiments, data analysis and provided NP137 antibody. All authors read and approved the final manuscript.
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A. Bernet and P.M. declare a conflict of interest as founders and shareholders of NETRIS Pharma. J.L., P.M., B.D., M.B. and N.B. declare a conflict of interest as employees of NETRIS Pharma. A. Bernet. and N.R. declare a conflict of interest as consultants for NETRIS. T.V. is co-inventor on licensed patents WO/2011/157846 (Methods for Haplotyping Single Cells), WO/2014/053664 (High-Throughput Genotyping by Sequencing Low Amounts of Genetic Material) and WO/2015/028576 (Haplotyping and Copy Number Typing Using Polymorphic Variant Allelic Frequencies).
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Extended data figures and tables
Extended Data Fig. 1 Strategy to study the impact of Netrin-1 on EMT in mouse skin SCCs.
a, Mouse model of skin SCC allowing the expression of KrasG12D, YFP, p53 deletion and overexpression of human NETRIN-1 in hair follicle stem cells and their progeny using Lgr5CreER. b, Relative mRNA expression of Ntn1 in EPCAM- control LKPR (n = 5) and LKPR-NTN1 (n = 8) skin SCC defined by qRT-PCR (data are normalized to Tbp gene, mean ± s.e.m., two tailed Mann-Whitney U test). c, Western blot analysis of Netrin-1 expression in EPCAM- control LKPR and LKPR-NTN1 skin SCC TCs. d, FACS plots showing the gating strategy used to FACS-isolate or to analyse the proportion of YFP+/EPCAM+ and EPCAM− tumour cells. e, Drawing showing the experimental strategy of NP137 administration after Tamoxifen induction in Lgr5CreER/KrasLSL-G12D/p53fl/fl/Rosa26-YFP+/+ mice. IP, intraperitoneal.
Extended Data Fig. 2 Single cell analysis of the cellular composition of control and NP137-treated skin SCCs.
a,b Uniform Manifold Approximation and Projection (UMAP) plot for control (a) and NP137 -treated skin SCC (b) coloured by the identified cell types. c,d, UMAP plot for control (c) and NP137-treated skin SCC (d) coloured by the sample of origin for each cell. CAFs, cancer-associated fibroblasts.
Extended Data Fig. 3 Annotation of the cell types found by single cell RNA-seq in control and NP137-treated skin SCCs.
a, UMAP plots coloured by normalized Yfp and Epcam expression in the control tumours. Gene expression values are visualized as colour gradient with grey indicating no expression and red indicating the maximum expression. b, UMAP plots coloured by normalized Yfp and Epcam in NP137-treated samples. c, UMAP plots coloured by the activity of modules containing the mouse-specific marker genes of the different cell types including CAFs, Macrophages, Neutrophils, Endothelial cells and T cells obtained from the PanglaoDB database in control samples (left) and anti-Netrin-1 treated samples (right). Module activity visualized as a colour gradient with blue indicating no expression and yellow indicating maximum activity. d, UMAP plots coloured by normalized Pdgfra, Acta2, Pecam1, Cd3d, Ptprc, Itgam, Cd86 and Cxcr2 expression in the control samples (left) and NP137-treated samples (right). CAFs, cancer-associated fibroblasts. e, UMAP plot coloured by normalized Ntn1 expression in control condition.
Extended Data Fig. 4 Impact of anti-Netrin antibody administration on the cellular composition of skin SCCs.
a,b, Uniform Manifold Approximation and Projection (UMAP) plots coloured by the cell type labels obtained from the analysis of the microenvironment for the integration of all the samples in total (a) and split per sample (b), respectively. c, Boxplot depicting the proportions of the different cell types for the 4 samples, split by their condition. The boxplots are coloured by their condition, and the individual measurements are visualized as red dots. The centre line, top and bottom of the boxplots represent respectively the median, 25th and 75th percentile and whiskers are 1.5 × IQR. Significant proportion changes are indicated by FDR < 0.2. d, barplot depicting the relative log fold change of the relative abundance of the different cell types after NP137-treated samples compared to the pericytes. Bars are coloured according to their cell type. e,f, UMAP plot of the CAFS subclustering, coloured by the identified seven subclusters and the sample the cell originated from, respectively. g, Boxplot depicting the proportions of the different CAF subclusters for the 4 samples, split by their condition. The boxplots are coloured by their condition, and the individual measurements are visualized as red dots. The centre line, top and bottom of the boxplots represent respectively the median, 25th and 75th percentile and whiskers are 1.5 × IQR. h. barplot depicting the relative log fold change of the relative abundance of the different CAF subclusters after NP137 treatment compared to the glycolysis CAFs subcluster. i, Co-immunostaining of YFP and Vimentin in control (top) (n = 5 tumours) and NP137- treated skin SCC (bottom) (n = 5 tumours) that defines YFP-/VIM+ CAFs as cells (Scale bars, 20 μm).
Extended Data Fig. 5 Expression of markers of the different EMT states in control and NP137-treated skin SCCs.
a, b, UMAP plots coloured by normalized gene expression values for the indicated genes in the control (a) and treated samples (b). Gene expression values are visualized as colour gradient with grey indicating no expression and red indicating the maximum expression. Circles represent TCs groups with a different degree of EMT based on the expression of Epcam, Krt14, Krt8, Vim, Pdgfra (green: Epcam+/Krt14+/Vim− as epithelial state; orange: Epcam−/Krt14+/Vim+ as early hybrid EMT state; red: Epcam−/Krt14−/Krt8+/Vim+ as late hybrid EMT state; dark red: Epcam−/Krt14−/Krt8−/Vim+ as late full EMT state expressing Pdgfra and Aqp1). c, Barplot depicting the relative log fold change of the relative abundance of the different EMT states after NP137-treatment compared to the early hybrid state. Significant proportion changes are indicated by FDR < 0.2.
Extended Data Fig. 6 Histological analysis of the control and NP137-treated tumors.
a–d, Haematoxylin and Eosin staining showing the control (n = 1) (a,b) or NP137-treated (n = 1) (c,d) tumour skin SCC analysed in Visium spatial transcriptomic method. The annotated areas represent the EMT states previously defined by the expression of Epcam, Krt14, Krt8 and Vim (1: epithelial, 2: early hybrid, 3, late hybrid, 4: full late EMT) (scale bars in a, c, 500 μm, scale bars in b, 20 μm).
Extended Data Fig. 7 Analysis of NP137 treatment on tumour growth, EMT and migration in endometrial human cancer cell line.
a, Tumor growth quantification of human Ishikawa endometrial carcinoma cells grafted in nude mice treated with either control (n = 9) or NP137 (n = 9) (mean ± s.e.m., 2-way ANOVA). b, Relative mRNA expression of epithelial markers CDH1, MUC1 and HOOK1 by qRT-PCR in Ishikawa human cells grafted in nude mice treated with control (n = 7) or NP137 (n = 8) (data are normalized to HPRT gene, mean +/− s.e.m., two tailed Mann-Whitney U test). c, Percentage of migrated Ishikawa cells treated with NP137 relative to the migration of control condition through serum deprived culture medium complemented with 2.5% Matrigel between 5 and 24 h of invasion. (n = 3) (mean ± s.e.m, two tailed t test).
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Lengrand, J., Pastushenko, I., Vanuytven, S. et al. Pharmacological targeting of netrin-1 inhibits EMT in cancer. Nature 620, 402–408 (2023). https://doi.org/10.1038/s41586-023-06372-2
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DOI: https://doi.org/10.1038/s41586-023-06372-2
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