Notch signaling is highly conserved in most animals and plays critical roles during neurogenesis as well as embryonic development. Synthetic Notch-based systems, modeled from Notch receptors, have been developed to sense and respond to a specific extracellular signal. Recent advancement of synNotch has shown promise for future use in cellular engineering to treat cancers. However, synNotch from Morsut et al. (2016) has a high level of ligandindependent activation, which limits its application. Here we show that adding an intracellular hydrophobic sequence (QHGQLWF, named as RAM7) present in native Notch, significantly reduced ligand-independent activation. Our enhanced synthetic Notch receptor (esNotch) demonstrates up to a 14.6-fold reduction in ligand-independent activation, without affecting its antigen-induced activation efficiency. Our work improves a previously reported transmembrane receptor and provides a powerful tool to develop better transmembrane signaling transduction modules for further advancement of eukaryotic synthetic biology.

Background Angiogenesis, a pivotal component in the tumor microenvironment (TME), boosts tumor growth and metastasis. Cancer-derived exosomes, which have been widely reported to play a crucial role in the establishment of TME can be effective angiogenic modulators. We aim to investigate the contribution of microRNA-21 (miR-21) for angiogenesis which was packaged in cancer-derived exosomes in esophageal squamous cell carcinoma (ESCC). Methods The co-cultivation model was constructed to mimic the tumor microenvironment based at a physical level to explore the effects of cancer-derived exosomes on angiogenesis of human umbilical vein endothelial cells (HUVECs). EdU assay, transwell assay and tube formation assay formation experiments were conducted for the evaluation of HUVECs proliferation, migration, and angiogenesis, respectively. In addition, Dual-luciferase reporter (DLR) assay was performed to validate the relationship between miR-21 and its target gene PTEN. Similarly, miR-21 inhibitors and LY294002 was applied to evaluate the regulation of miR-21 via pro-angiogenesis in recipient HUVECs by PTEN/Akt signaling pathway. Results After 24 h co-cultivation with EC9706 cells, miR-21 levels in recipient HUVECs was raised. The results from EdU assay, transwell assay and blood vessel formation experiment showed that exosomes which were secreted from EC9706 cells (EC9706-Exo) delivered miR-21 stimulated proliferation, migration and tube formation of HUVECs. DLR assay indicated that miR-21 could directly bind to the 3'-untranslated region (UTR) of PTEN genes, real-time PCR and western blot analysis for PTEN showed it was inhibited by EC9706-Exo shuttled miR-21. Meanwhile, phospho-Akt (p-Akt) (Ser473), one of the downstream genes of PTEN, was significantly increased in recipient HUVECs compared to the control group, while inhibiting miR-21 and PI3K/Akt pathway respectively both led to a sharp decrease in p-Akt levels, suggesting that exosomal miR-21 promote angiogenesis via activating PTEN/Akt signaling pathway. Conclusion Exosomal miR-21 acts as a driver of pro-angiogenesis by activating PTEN/Akt signaling pathway, it might serve as a blood-based biomarker for ESCC metastasis. Suppressing the expression or blocking the transmission of these exosome-derived miR-21 might be a novel antiangiogenic therapeutic strategy for ESCC.