SRA

Epithelial-to-Mesenchymal transition (EMT) regulates tumor 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 tumor cells in skin SCCs, as well as decreased the number of metastasis and increased the sensitivity of tumor 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 tumor epithelial states. ShRNA knockdown (KD) of Netrin-1 and its receptor Unc5b in EPCAM+ tumor cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature promoting tumor 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 tumor appearance and size were detected by daily observation and palpation. Mice were euthanized when tumor size was reached or when mice presented signs of distress. To determine the effect of anti-Netrin-1 molecule (NP137) on primary tumor, 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 tumors from LKPR mice were dissected, rinsed and digested in collagenase type I (Sigma) at 3,5mg/ml in HBSS (Gibco) for 1h at 37°C on a rocking plate protected from the light. Collagenase was blocked by the addition of EDTA (5mM) and then the cells were rinsed 1 time in PBS supplemented with 10% FBS and the cell suspension were filtered through a 70-µm cell trainer (BD Bioscience). For the wash next and antibody incubation, cells were resuspended in PBS supplemented with 2% FBS (facs buffer). Cells were incubated with BV711- conjugated anti-EPCAM (rat, clone G8.8, BD Bioscience Cat#563134, dilution 1:100), PE-conjugated anti-CD45 (rat, clone 30-F11, BD, Cat#553081, dilution 1:100) and PE-conjugated anti-CD31 (rat, clone MEC 13.3, BD, Cat#553373, dilution 1:100) antibodies for 30 min at 4 °C protected from light. For cell sorting, cells were also filtered through a 40-µm cell trainer (BD Bioscience). Living single tumor cells were selected by forward and side scatter, doublet discrimination and Hoestch exclusion. Profile of the tumors was analyzed following viability and profile of YFP, Lin- (CD45/CD31) and EPCAM expression. Once screened all lived cells were sorted for single cell RNA sequencing experiment using FACSAria and FACSDiva software (BD Bioscience). After FACS isolation, lived cells were loaded onto each channel of the Chromium Single Cell 3' microfluidic chips (V2-chemistry, 10X Genomics) and individually barcoded with a 10X Chromium controller according to the manufacturer's recommendations (10X Genomics).