The Study On L-PGDS Modified Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes Inhibiting Cancer Progression

Objective:Cancer is an important cause of disease-related death,and its incidence and mortality are increasing.Despite the advancement of treatment methods in past decade,the clinical prognosis of cancer is still poor.Exosomes are important vehicles in intercellular communication.And exosomes are main paracrine components of mesenchymal stem cells?MSCs?.As therapeutic vehicles,exosomes play important roles in the treatment of many diseases,including cancer.The aim of this study was to product human umbilical cord MSCs derived-exosomes overexpressing lipocalin-type prostaglandin D synthase?L-PGDS?and investigate the inhibiting cancer effects of L-PGDS modified exosomes in cancer therapy.Methods:Adherence method was used to isolate and culture umbilical cord MSCs.Adenovirus encoding L-PGDS?Ad-L-PGDS?and Vector?Ad-Vector?was transfected into MSCs in good growth state.The transfection efficiency was observed by fluorescence microscope.The expression of L-PGDS in MSCs was detected by Western blot to verify the transfection effect.The supernatant of MSCs was collected and exosomes were separated by ExoQuick-TC^TM extraction kit,resulting in exosomes overexpressing L-PGDS?EX-L-PGDS?and empty vector exosomes?EX-Vector?.The expression of exosomes surface markers CD9,CD63 and CD81 was detected by Western blot.The morphology and size of exosomes were observed by transmission electron microscopy.The diameter distribution of exosomes was measured by Nanosight visible nanoparticle analyzer.Western blot was used to detect the expression of L-PGDS in exosomes.Vybrant CM-DIL dye labeled exosomes,confocal microscopy was used to detect the uptake of exosomes by tumor cells,RT-PCR and Western blot were used to detect the expression of L-PGDS in tumor cells after exosomes treatment.To explore the inhibitory effects of L-PGDS modified exosomes on tumor cells in vitro,exosomes were used to treat SGC-7901 and SMMC-7721.Migration and invasion experiments were used to detect the migration and invasion ability of cancer cells.Flow cytometry was used to detect the apoptotic level of cancer cells,and Colony-forming assay was used to detect the proliferation ability of cancer cells.SGC-7901subcutaneous tumor-bearing mice model was established in vivo.PBS and EX-Vector were used as controls to observe the inhibitory effect of EX-L-PGDS on tumor growth.Tumor growth rate,tumor weight and HE staining were used to evaluate the inhibitory effect of EX-L-PGDS on tumor growth.The expression of proliferating cell nuclear antigen?PCNA?was detected by immunohistochemical staining,L-PGDS by immunofluorescence staining,apoptosis by TUNEL and Bax,Bcl2 and L-PGDS by Western blot.Results:Human umbilical cord MSCs were successfully isolated.We transfected MSCs with Ad-L-PGDS and Ad-Vector adenovirus at good cell growth status.The results of fluorescence microscopy showed that adenovirus transfected umbilical cord MSCs had desired transfection efficiency 24 hours later.Western blot demonstrated that the expression of L-PGDS in MSCs transfected with Ad-L-PGDS increased significantly.The supernatant of MSCs was collected and exosomes were separated by ExoQuick-TC^TM extraction kit.Overexpression of L-PGDS and empty vector exosomes?EX-L-PGDS and EX-Vector?presented typical disc shape with a diameter of about 100 nm by transmission electron microscopy and Nanosight visible nanoparticle analyzer,respectively.Western blot demonstrated that both exosomes expressed specific exosomes markers CD9,CD63 and CD81.Compared with EX-Vector,the expression of L-PGDS in EX-L-PGDS increased significantly.When exosomes were co-incubated with SGC-7901,exosomes could be absorbed into the cell.Compared with EX-Vector,EX-L-PGDS significantly increased the expression of L-PGDS in SGC-7901.Transwell migration and invasion experiments indicated that EX-L-PGDS further inhibited the migration and invasion ability of SGC-7901 and SMMCC-7721 cells compared with PBS and EX-Vector.Flow cytometry showed that apoptotic cells of SGC-7901 and SMMCC-7721 were significantly increased after EX-L-PGDS treatment.Colony-forming assay demonstrated that EX-L-PGDS inhibited the cloning ability of SGC-7901 and SMMCC-7721 cells.Nude mice subcutaneous tumor-bearing experiment revealed that SGC-7901 treated with EX-L-PGDS grew slower and formed smaller tumors.HE staining indicated that the tumors in EX-L-PGDS group were more porous and had less angiogenesis than those in PBS and EX-Vector groups.PCNA expression was detected by immunohistochemical staining.PCNA expression was lower in EX-L-PGDS group,and more apoptotic cells were detected by TUNEL.Immunofluorescence staining showed more L-PGDS expression in EX-L-PGDS group.Western blot analysis demonstrated that expression of Bax and L-PGDS were significantly increased in EX-L-PGDS group.Conclusion:We successfully establish L-PGDS modified MSCs and obtain EX-L-PGDS.Our results indicate that EX-L-PGDS can increase the expression of L-PGDS in tumor cells and inhibit the migration,invasion,colony formation,and promote apoptosis of tumor cells in vitro,and inhibit the growth of tumors in vivo.Our findings provide a novel idea for the treatment of exosomes-based cancer therapy.