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. 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).

上皮间质转化（Epithelial-to-Mesenchymal transition, EMT）调控肿瘤的发生、进展、转移以及对抗癌治疗的耐药性。尽管近年来在解析癌症中EMT的调控作用与机制方面已取得长足进展，但迄今尚未开发出以EMT为药理学靶点的治疗策略。本研究发现，在出现自发性EMT的皮肤鳞状细胞癌（skin squamous cell carcinoma, SCCs）原代小鼠模型中，Netrin-1表达上调。通过给予NP137——一种目前已进入人类癌症临床试验的抗Netrin-1阻断型单克隆抗体——进行药理学抑制Netrin-1，可降低皮肤SCCs中EMT阳性肿瘤细胞的比例，同时减少转移灶数量，并增强肿瘤细胞对化疗的敏感性。单细胞RNA测序（single-cell RNA-seq, scRNA-seq）结果显示，对照组SCCs中存在多种EMT状态，包括上皮型、早期及晚期混合EMT状态，以及完全间质型EMT状态。与之相反，NP137给药可阻断癌细胞向晚期EMT状态进展，并维持肿瘤的上皮表型。在基质细胞缺失的体外环境中，通过短发夹RNA（short hairpin RNA, ShRNA）敲低EPCAM+肿瘤细胞中的Netrin-1及其受体Unc5b，可抑制EMT进程，并调控一套共同的基因特征，以维持肿瘤上皮状态、限制EMT发生。为验证上述发现对人类癌症的适用性，我们对移植了A549人类癌细胞系的小鼠进行了NP137给药处理，该细胞系在转化生长因子-β1（transforming growth factor-β1, TGF-β1）诱导下可发生EMT。体内实验证实，Netrin-1抑制可降低A549细胞的EMT水平。综上，本研究鉴定出一种全新的靶向EMT的药理学治疗策略，为抗癌治疗开辟了新的干预途径。

为在Lgr5CreER/ KrasLSL-G12D/p53fl/fl/Rosa26-YFP+/+（LKPR）小鼠中诱导肿瘤发生，将他莫昔芬以25 mg/ml的浓度稀释于含10%乙醇的葵花籽油（Sigma）中。于小鼠出生后第28天（P28），每日对LKPR小鼠腹腔注射2.5 mg他莫昔芬，连续给药4天。他莫昔芬给药后7至9周，通过每日观察与触诊检测肿瘤的发生情况与体积大小。当肿瘤体积达到既定阈值或小鼠出现痛苦窘迫症状时，对其实施安乐死。

为评估抗Netrin-1分子NP137对原代肿瘤的干预效果，于他莫昔芬给药后4周起，每隔两日对LKPR小鼠腹腔注射10 mg/kg的NP137，直至小鼠自然死亡。解剖分离LKPR小鼠的对照组与NP137处理组皮肤肿瘤，其中部分肿瘤样本留存用于免疫组织化学检测。肿瘤组织先于室温下用4%多聚甲醛（PFA）固定2小时，经磷酸盐缓冲液（PBS）漂洗后，进行福尔马林固定石蜡包埋（FFPE）。

为分析对照组与抗Netrin-1治疗的皮肤SCCs肿瘤中，此前通过scRNA-seq鉴定的不同EMT肿瘤状态的空间分布与定位，我们采用VISIUM空间转录组测序技术（10X Genomics）开展分析。将FFPE组织切片放置于Visium玻片上，并严格按照10X Genomics官方实验流程进行样本制备。完成H&E染色、成像及脱交联步骤后，将组织切片与靶向10551个基因的人类特异性探针（10X Genomics，Visium小鼠转录组探针套装v1.0）进行孵育。