Experimental Study Of MDSCs-derived Exosomes Inducing Epithelia-mesenchymal Transformation In Mouse Colorectal Cance Cells

ObjectiveTo investigate the regulatory effect of MDSC-exosomes（MDSC-exo）on epithelial-mesenchymal transformation（EMT）of tumor cells and the effect on tumor metastasis,We prepared exosomes derived from myeloid-derived suppressor cells（MDSCs）and tried to explore the possible molecular mechanisms.Methods（1）First,we established mouse model of colorectal cancer transplantation,and then we injected subcutaneously 1×10⁶ colorectal cancer CT26 cells into Balb/c mice.MDSCs from spleen of tumor-bearing mice were isolated by immunomagnetic bead method.Cell purity and cell activity was determined by flow cytometry（FCM）.To obtain exo-depleted CS,we removed exosomes from MDSC-CS by hypervelocity centrifugation（100000×g,16h）.The MDSCs obtained above were cultured in RPMI-1640 medium containing 10%exo-free FBS for 20h,and the MDSCs culture supernatant（MDSC-CS）was collected.MDSC-CS depleted of exosomes（exo-depleted CS）were obtained by supercentrifugation（100000×g,16h）.MDSC-CS and exo-depleted CS were added into the CT26 cell culture system for 48h.CT26 cell migration ability was evaluated through transwell migration and wound healing test.EMT-related protein expression of CT26 cells was detected by western blot（WB）,andβ-catenin expression and intracellular distribution were detected by immunofluorescence（IF）.（2）Exosomes in MDSC-CS were extracted by combined hypervelocity centrifugal method and Exoquick reagent method,namely MDSC-exo.Transmission electron microscope（TEM）,nanoparticle tracking analysis（NTA）and WB was used to observe the morphology of MDSC-exo particles,to detect the diameter distribution of MDSC-exo particles,and to identy exosome signature proteins.（3）CT26 cells were co-cultured with Di I-labeled MDSC-exo for 24h,and the internalization of MDSC-exo in CT26 cells was observed by laser confocal microscopy.MDSC-exo was added into the CT26 cell culture system for 72h,and WB and quantitative real-time PCR（q RT-PCR）were used to detect the expression of EMT-related proteins and EMT-inducing transcription factors（EMT-TFs）in CT26 cells.CT26 cell proliferation and apoptosis were detected by FCM,cell activity was detected by CCK8 assay.（4）ROS levels in MDSCs and CT26 cells were detected.MDSC-exo was added into CT26 cell culture system and cultured for 72h.The changes of intracellular ROS levels in CT26 cells were detected.ROS scavenging agent NAC was added to the MDSCs culture system for 20h,and the supernatant was collected to prepare MDSC-exo(MDSC^NAC-exo).The levels of ROS in MDSCs,MDSC-exo and MDSC^NAC-exo cells and CT26 cells treated with MDSC^NAC-exo were detected.MDSC^NAC-exo and MDSC-exo were added into CT26 cell culture system respectively for 72h,and the differences in the effects of MDSC-exo and MDSC-exo on the expression of EMT-related proteins,cell migration and invasion ability of CT26 cells were compared.（5）We established mouse models of lung metastasis from colorectal cancer.After MDSC^NAC-exo and MDSC-exo were added into CT26 cell culture system respectively for 72h.A total of 1×10⁵ CT26 cells were resuspended in 100μL PBS and injected into mice through caudal vein.The mice were sacrificed after 21 days,and the lung metastases of colorectal cancer were observed by counting lung tumor nodules,calculating organ index and HE staining.Result（1）The purity of MDSCs obtained by immunomagnetic bead method was greater than90%,and the cell activity of MDSCs was greater than 95%.Wound healing test showed that the cell mobility in MDSC-CS group was 0.15±0.02%higher than that of control group 0.08±0.02%（P<0.01）.Transwell migration assay showed that the number of cell migration in MDSC-CS group was 241.7±18.62 higher than that in control group148.8±12.45（P<0.001）.WB results showed that the expression of E-cadherin and Claudin-1 in MDSC-CS group was lower than that in control group（P<0.001）,N-cadherin and Snail1 expressions were higher than those in the control group（P<0.001）,Vimentin expression was not statistically significant（P>0.05）;E-cadherin expression in exo-depleted CS group was higher than that in MDSC-CS group（P<0.01）,the protein expression of N-cadherin was lower than that of MDSC-CS group（P<0.001）,and there was no significant difference in Vimentin and Snail1 expression（P>0.05）.IF results showed that the expression ofβ-catenin in CT26 cells was higher in MDSC-CS group than in control group and exo-depleting CS group.Thus,MDSC-CS could promote EMT and migration of CT26 cells,and this promotion is weakened when the MDSC-derived exosomes in MDSC-CS were removed,suggesting that MDSC could induce EMT of CT26 cells by releasing exosomes.（2）The double-layer follicular structure of MDSC-exo was observed by TEM,and the average diameter of MDSC-exo was mainly distributed in the range of 50-150nm by NTA.WB showed that MDSC-exo expressed exosomal protein markers CD63,TSG101 and CD9,but did not express exosomal negative marker Calnexin.It met the typical characteristics of exosomes.（3）Fluorescence microscopy showed that CT26 cells contained MDSC-exo labeled with red fluorescent probe.WB results showed that the expression of E-cadherin protein in MDSC-exo group was lower than that in control group,and the expression of N-cadherin protein was higher than that of control group,q RT-PCR results showed that transcription factor Snail1 mRNA and Snail2 mRNA expression was increased 2.5times（P<0.01）,Zeb1 mRNA expression was increased 2.9 times（P<0.05）,Twist1mRNA expression was increased 3 times（P<0.01）,there was no statistically significant difference in Twist2 mRNA expression（P>0.05）.Transwell migration assay showed that CT26 cells migrated in unit time in MDSC-exo group and invasion than the control group（P<0.001）.In addition,CT26 cell viability increased after MDSC-exo treatment（P<0.01）,the expression of proliferation antigen Ki67 was increased（P<0.001）,apoptosis was inhibited（P<0.001）.These results suggest that MDSC-exo could directly induce EMT in CT26 cells and promote cell migration and invasion.（4）Studies have shown that ROS is a potential factor of EMT.In order to explore the possibility that MDSC-exo induces EMT through ROS,we first confirmed that the intracellular ROS level of MDSCs was higher than that of CT26 cells by FCM,and the intracellular ROS level was significantly increased after CT26 cells were treated with MDSC-exo（P<0.01）.After MDSCs were pretreated with NAC,the intracellular ROS level in MDSCs was decreased（P<0.05）,and the ROS level in MDSC-exo was also significantly decreased（P<0.05）.It was further verified that the changes of ROS levels in CT26 cells were related to ROS carried by MDSC-exo.CT26 cells were treated with MDSC-exo and MDSC^NAC-exo respectively,the intracellular ROS level of CT26 cells in MDSC^NAC-exo group was lower than that in MDSC-exo group（P<0.05）.Finally,we found that MDSC-exo may induce EMT,cell migration and invasion through ROS.We observed that the protein expressions of E-cadherin and Claudin-1 in CT26 cells in MDSC-exo group were lower than those in control group（P<0.001）,and the protein expressions of Vimentin,Snail1 and ZEB1 in CT26 cells were higher than those in control group（P<0.001）.The protein expressions of Claudin-1,Vimentin and Zeb1 in CT26 cells in MDSC^NAC-exo group were lower than those in MDSC-exo group（P<0.05）,while the protein expression of Snail1 was higher than that in MDSC-exo group（P<0.01）.There was no significant difference in E-cadheirn expression（P>0.05）.The number of cell migration and invasion in MDSC-exo group was higher than that in control group（P<0.001）,and the number of cell migration and invasion in MDSC-exo group was lower than that in MDSC-exo group（P<0.01）.These results showed that MDSC-exo induced EMT of CT26 cells and promoted cell migration and invasion through ROS.（5）The results of mouse lung metastasis model showed that the total number of lung tumor nodules in MDSC-exo group was higher than that in the control group,while the total number of lung tumor nodules in MDSC-exo group was lower than that in the MDSC-exo group（P<0.001）,and there was no significant difference between MDSC-exo group and the control group（P>0.05）.The number of tumor nodules with diameter<0.5mm and>2mm in MDSC-exo group was higher than that in the control group（P<0.05）.The number of tumor nodules with diameter 0.5-1mm and 1-2mm was not significantly different from that in the control group（P>0.05）.However,there was no significant difference in the number of tumor nodules with different diameters in the lung between the MDSC^NAC-exo group and the MDSC-exo group（P>0.05）.There was no significant difference in organ index among the three groups（P>0.05）.The results of HE staining showed that the number of CT26 cells in the lung of mice in the MDSC-exo group was significantly higher than that in the control group,while the number of CT26 cells in the lung of mice in the MDSC-exo group was significantly lower than that in the MDSC-exo group.ConclusionMDSC-exo could induce EMT in tumor cells.Specifically,MDSC-exo could enhance the expression of mesenchymal markers of tumor cells,inhibit the expression of epithelial markers of tumor cells,and enhance the the ability of tumor cells to migrate and invade.These effects depended on ROS carried in MDSC-exo.