Effects Of Exosomes Released From 4T1 Tumor Cells After X-ray Irradiation On Proliferation And Migration Of Tumor In Vitro And Vivo And Its Mechanism

Objective: Radiation therapy is an effective method of cancer treatment,currently about half of cancer patients rely on radiotherapy.But there are also some problems in radiotherapy.It is difficult to eradicate the tumor,even in some cases,it may cause the spread or metastasis of tumor.Receiving a dose of radiation,some solid tumors are prone to epithelial mesenchymal transition(EMT)to form circulating tumor cells(CTCs),which increases the risk of tumor metastasis.CTCs are equivalent to the seed of the tumor.The appropriate microenvironment is essential for the recruitment and colonization of CTCs.The microenvironment of immune imbalance is beneficial to the colonization and growth of CTCs obviously.Tumor derived exosome(TD-exosome)is an endocytic vesicle with a diameter of 30-150 nm,which is released by tumor cells.TD-exosomes is involved in intercellular communication by carrying informational molecules such as proteins,DNA,RNAs and miRNAs.In this study,we used mouse mammary cancer cell line(4T1)and its tumor-bearing mice as the model to observe the effect of exosomes from 4T1 cells accepted irradiation on 4T1 growth and migration and its effect on the macrophages.To explore the possible mechanism of exosomes secreted by 4T1 cells accepted X-ray irradiation on tumor migration.Methods:1 X-ray irradiation induces apoptosis and epithelial mesenchymal transition(EMT)in 4T1 cells 1.1 4T1 cells were irradiated with different doses of X-ray(0Gy,10 Gy,20Gy,30Gy)in vitro.Apoptosis rate was determined by flow cytometry assay using AnnexinV-PE and 7-AAD staining in 24 hours later.1.2 4T1 cells accepted irradiation between doses from 0Gy(non-IR)to 30Gy(IR).4T1 cells stimulated with X-rays were assessed for Vimentin,N-cadherin and E-cadherin protein expression by western blot.2 X-ray local irradiation promotes lung metastasis in 4T1 tumor-bearing miceSix week old female Balb/c mice were randomly divided into three groups: control group,tumor-bearing mice and X-ray tumor-bearing mice.The tumor-bearing mices were inoculated with 1×106 4T1 cells on the right fourth breast pad.Two weeks later,X-ray irradiation(20Gy)was performed on X-ray tumor-bearing mice.After 10 days,all mice were sacrificed.The effects of X-ray on tumor growth and lung metastasis were observed by weighing the lungs.The upper lobe of the left pulmonary tissue were stained with HE to observe the situation of tumor metastasis.The effect of X-ray on the number of MDSCs cells(CD11b+Gr-1+)and macrophages(CD11b+F4/80+)in mice’s right lobe of lung were determined by flow cytometry assay.3 X-ray irradiation promotes 4T1-GFP cells proliferation4T1 cells in logarithmic growth phase were treated with trypsin containing 0.02% EDTA after 10 Gy,10Gy,20 Gy and 30 Gy X-ray irradiation.Then cells were cultured in 6-well plates.The cell concentration of each irradiation dose group was 3×105 cells/well,each group of 3 holes.1×105 4T1-GFP cells were added to each well.After 48 hours,all cells were collected,the cells of every hole corresponds to a flow tube.The percentage of 4T1-GFP was detected by flow cytometry.4 The effects of exosomes released from 4T1 cells after X-ray on proliferation and migration of 4T1 cells 4.1 Exosomes isolation and electron microscopy observationSupernatant fractions collected from 72 hours cell cultures.The exosomes were purified from the supernatants by differential centrifugation.In brief,cells were removed by centrifugation for 10 min at 2000rpm;30min at 3000rpm;60min at 11000 rpm.Supernatants were collected and centrifuged for 16 hours at 100,000 ×g.Exosomes were pelleted at the final centrifugation step and were resuspended in PBS.Exosome size and particle number was verified by electron microscopy.Exosomes quantity were analysed using the nanoparticle characterization system(Nanodrop).4.2 The effects of exosome on proliferation and migration of 4T1 cells in vitroThe effects of exosomes released from 4T1 cells which accepted irradiation between doses from 0Gy(non-IR)to 30 Gy on proliferation and migration of 4T1 cells by RTCA station and transwell migration assays.4.3 4T1 cells stimulated with exosomes which released from 4T1 cells accepted irradiation between doses from 0Gy(non-IR)to 30 Gy were assessed for Vimentin,N-cadherin and E-cadherin protein expression by western blot.5 The effects of exosomes secreted by 4T1 cells after X-ray irradiation on lung metastasis of breast cancer in tumor-bearing mice and its possible mechanismSix week old female Balb/c mice were randomly divided into four groups: control group,PBS group,4T1/exo group and ir-4T1/exo group.PBS group,4T1/exo group and ir-4T1/exo group mice under ether anesthesia by inhalation of PBS,4T1/exo,ir-4T1/exo respectively,stimulated 3 times per week for 3 weeks,each time 60μl.Three weeks later,half of the mice were sacrificed in each group and the other half of the PBS group,4T1/exo group and ir-4T1/exo group mice were injected into 1 × 105 4T1 cells by tail vein.The effect of exosomes on lung metastasis of breast cancer were observed by weighing the lungs.The upper lobe of the left pulmonary tissue were stained with HE to observe the situation of lung metastasis.Take the different groups of mice lung tissue.The pulmonary homogenate supernatant were prepared from the lower lobe of the left pulmonary tissue(about 0.037g).The levels of cytokines CXCL1,IL-1α,G-CSF and IL-10 in the pulmonary homogenate supernatant were detected by ELISA.6 The cytokines contained in exosomes released from 4T1 cells before and after X-rayProtein chip technique was used to detect the cytokines contained in exosomes(4T1/exo,ir-4T1/exo)released from 4T1 cells before and after X-ray.The cytokines contained in the exosomes released from 4T1 cells before and after X-ray were examined by ELISA.7 The effects of culture supernatant of mouse bone marrow-derived macrophages(BMDM)stimulated with 4T1/exo and ir-4T1/exo on proliferation and migration of 4T1 cells 7.1 BMDM cells cultureCells in the femur bone marrow cavity of Balb/c mice were taken and cultured in RPMI 1640 medium.The cytokine GM-CSF were added to cell culture medium for 7 days,and the final concentration was 50ng/ml.7.2 The culture supernatant of BMDM cells stimulated with exosome released from 4T1 cells before and after X-ray was collected.The effects of culture supernatant of BMDM cells on proliferation and migration of 4T1 cells were detected by RTCA station and transwell migration assays.The levels of cytokines CXCL1,IL-1α and G-CSF in the culture supernatant of BMDM cells were detected by ELISA.7.3 The effects of exosomes on the expression of M1 and M2 related proteins in BMDM cellsBMDM cells were stimulated by exosomes for 48 hours in vitro to extract total protein.BMDM cells stimulated with exosome released from 4T1 cells before and after X-ray were assessed for NF-κB IKappaB,IKKa protein expression by western blot.7.4 The effect of exosomes on BMDM cells p38 MAPK signaling moleculeBMDM cells were stimulated by exosomes for 48 hours in vitro to extract total protein.BMDM cells stimulated with exosome released from 4T1 cells before and after X-ray were assessed for p38 MAPK,p-p38 protein expression by western blot.Results:1 X-ray increased the apoptosis rate of 4T1 cells was significantly higher than that of non irradiated(P<0.01),and the rate of 20 Gy group was higher than that of 10 Gy group and 30 Gy group(P<0.01).Western blot showed that E-Cadherin expression was decreased by X-ray(20Gy,30Gy)(P<0.05).2 X-rays can reduce the weight of the tumor-bearing mice’s tumors significantly(P<0.05),but it can promote pulmonary metastasis.The results of flow cytometry showed that the number of MDSCs cells and macrophages in the lungs was increased by X-rays(P<0.01).3 The 4T1-GFP cells were co-cultured with X-ray irradiated 4T1 cells.And 4T1 cells irradiated by 20 Gy or 30 Gy were significantly promoted the proliferation of 4T1-GFP cells compared with 0Gy or 10 Gy.(P<0.05).4 Transmission electron microscopy revealed that exosomes from tumor cell culture supernatants were 30 to 100 nm membrane vesicles.They were round or ellipse in shape.5 RTCA station and transwell migration assays results showed that exosome secreted by 4T1 cells irradiated by 20 Gy dose of X-ray compared with non irradiated group(control group)could promote the proliferation and migration of 4T1 cells(P<0.05).The results of Western blot showed that exosome secreted by 4T1 cells irradiated by 30 Gy dose of X-ray compared with other three groups of irradiation dose group(0Gy,10 Gy,20Gy)can significantly lower the expression of E-Cadherin(P<0.05).6 The results of mice’s lung weight showed that compared with the non-inhalation exosomes group,inhalation of exosomes(4T1/exo and ir-4T1/exo)in mice increased lung weight significantly(P<0.01).And ir-4T1/exo group compared with 4T1/exo group promote lung weight increased significantly(P<0.01).The pulmonary tissue showed that ir-4T1/exo promotes pulmonary metastasis.ELISA results showed that the levels of CXCL1 and G-CSF in ir-4T1/exo group were higher than those in 4T1/exo group(P<0.01).7 Compared with ir-4T1/exo,the levels of cytokines RANTES,MCP1 and CXCL1 were significantly increased in 4T1/exo(P<0.05).The ELISA results showed that the content of CXCL1 in ir-4T1/exo was higher than that in 4T1/exo(P<0.01).8 RTCA station and transwell migration assays results showed that supernatant of BMDM cells stimulated by ir-4T1/exo compared with ir-4T1/exo could promote the proliferation and migration of 4T1 cells(P<0.01).The results of ELISA showed that the content of CXCL1,G-CSF and IL-1 were increased in the culture supernatants of BMDM cells stimulated by 4T1/exo and ir-4T1/exo(P<0.01).9 Western blot showed that the expression of NF-κB IKappaB and NF-κB IKKa in BMDM cells did not change after stimulated with exosomes.The expression of MAPK p-p38 was up-regulated by exosomes.Conclusion:1 X-ray local irradiation promotes lung metastasis in 4T1 tumor-bearing mice.2 The exosomes released from 4T1 cells after X-ray irradiation have a positive effect on the proliferation or migration of 4T1 in vitro and in vivo.3 Supernatant of BMDM cells stimulated by exosomes which released from 4T1 cells after X-ray could promote the proliferation and migration of 4T1 cells.4 The exosomes released from 4T1 cells after X-ray irradiation can activate the inflammatory factor related signaling pathway.