Pharmacological targeting Netrin-1 inhibits EMT in cancer [Visium]

Epithelial-to-Mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy. Whereas great progress had recently been made in understanding the role and mechanisms that regulate EMT in cancer, no therapeutic strategy to pharmacologically target EMT had been identified so far. Here, we found that Netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCCs) presenting spontaneous EMT. Pharmacological inhibition of Netrin-1 by administrating NP137, an anti-Netrin-1 blocking monoclonal antibody currently used in clinical trials in human cancer, decreased the proportion EMT tumour cells in skin SCCs, as well as decreased the number of metastasis and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA-seq revealed the presence of different EMT states including epithelial, early and late hybrid EMT as well as fully EMT states in control SCCs. In contrast, administration of NP137 prevents the progression of cancer cells towards a late EMT state and sustains tumour epithelial states. ShRNA knockdown (KD) 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 promoting tumour epithelial state and restricting EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with A549 human cancer cell line that undergoes EMT following TGF-b1 administration with NP137. Netrin-1 inhibition decreased EMT in A549 cells in vivo. Altogether, our results identify a new pharmacological strategy to target EMT in cancer opening novel therapeutic interventions for anti-cancer therapy. Overall design: To induce tumorigenesis on Lgr5CreER/ KrasLSL-G12D/p53fl/fl/Rosa26-YFP+/+ (LKPR), Tamoxifen was diluted at 25 mg/ml in sunflower seed oil, 10% EtOH (Sigma). Four daily intraperitoneal (IP) injections of 2.5 mg tamoxifen were administered at P28 to LKPR mice. After 7-9 week after Tamoxifen injection tumour appearance and size were detected by daily observation and palpation. Mice were euthanized when tumour size was reached or when mice presented signs of distress. To determine the effect of anti-Netrin-1 molecule (NP137) on primary tumour, LKPR mice were treated intraperitoneally at 10 mg/kg every two days from 4 weeks after Tamoxifen injection and until the death of animal. Control and NP137-treated skin tumours from LKPR mice were dissected, rinsed one part of the tumors has been kept for immunochemistry. Tumour tissues were pre-fixed in 4% PFA for 2 h at room temperature, rinsed in PBS, before to be embedded in FFPE. To analyze the spatial distribution and localization of different EMT tumors states previously described by scRNA-seq in Control and anti-Netrin-1 treated skin SCC tumors, we used VISIUM technology for spatial transcriptomic analysis (10X Genomics). FFPE tissue section were places on Visium slides and prepared according to 10X Genomics protocols. After H&E staining, imaging and decrosslinking steps, tissue sections were incubated with human specific probes targeting 10,551 genes (10X genomics, Visium Mouse Transcriptome probe Set v1.0).