| Objective: Cisplatin is one of the effective drugs to treat non-small cell lung cancer,but it found that with the increase of treatment cycle times,clinical cancer cells to cisplatin sensitivity decreases gradually,finally appear to cisplatin resistance.At present it is generally believed that the tumor associated macrophage(tumor associated macrophage,TAM)as an important stromal cell of the tumor microenvironment,not only directly involved in the formation of tumor blood vessels,but also,via the interaction with tumor cells,mediated the EMT of tumor cells and subsequently promoted tumor metastasis.According to the phenotype and function of macrophages,it could be divided into M1 and M2 macrophages.And recent studies have shown that the M1 and M2 macrophages are distribution in the tumor tissue.But whether the M1 and M2 macrophages in lung cancer could affect tumor cell proliferation,migration and sensitivity to cisplatin is rarely reported.This experiment adopts the A549 cells and tumor-burdened mice as models to observe the M1 and M2 macrophages and their secretion of exosomes in the proliferation,migration as well as cisplatin sensitivity of A549 cells in vivo and in vitro.The purpose is to further understand the different types of macrophages on cisplatin therapy and to provide the experimental data for targeting on regulating the macrophages for promoting cisplatin treatment effect.Methods:1 The M1 and M2 macrophages induction Human mononuclear cell leukemia cell line THP 1 was cultured in vitro.PMA(15?g/ml)was used to induce for 36 h and the medium containing PMA was removed,the cells were washed with PBS,then fresh complete medium was added and continued to culture for another 12 h.The adherent cells are M0.1.1 M1 cells differentiation induction.The M0 cells were stimulated with 1?g/ml lipopolysaccharide(LPS)and 20 ng/ml interferon-?(IFN-?)for 24 h,the culture supernatant was removed and the cells were washed once with PBS,and then fresh completely medium was added and continued to culture for another 24 h.The supernatant and cells were harvested respectively,and IL-1?in supernatant was detected by the method of ELISA,the expression of i NOs and Arg-1 in cells were analyzied by Western blot.1.2 M2 cells differentiation induction.The adherent M0 cells were stimulated with IL-4(20ng/ml)in complete medium for 24 h,the culture supernatant was removed and the cells were washed once with PBS,and then fresh completely medium was added and continued to culture for another 24 h.The supernatant and cells were harvested respectively,and IL-1? in supernatant was detected by the method of ELISA,the expression of i NOs and Arg-1 in cells were analyzied by Western blot.2 Preparation of exosomeCollecting the M1 and M2 macrophages culture supernatant,the exosomes of M1 and M2 macrophage were separated by serial centrifugation at low speed centrifugation: 2000 × g,10 min;10000 × g,1 h,then followed by ultracentrifugation at 100,000× g,16 h to pellet the exosomes.The exosome was resuspended with PBS.Samples were observed using the Transmission Electron Microscope with phosphotungstic acid dye.The exosomes were quantified with Nanodrop.3 To observe the role of M1/exo,M2/exo on A549 cell line proliferation,migration as well as the sensibility to Cisplatin3.1Dil labeling of M2/exo:0.5ml(the concentration is 11.5mg/ml)of M2/exo and 5?l fluorescent dye Dil(5?g/ml)were added respectively to a 1.5ml centrifuge tube and put it in the incubator for 30 min,then followed by ultracentrifugation at 100,000 × g,2h to remove the free of dyes.A549 cell line was stimulated with at the concentration of 50?g/ml for 24 h,then M2/exo was uptaked by A549 cells line obserbed by the fluorescence microscope3.2 A549 cell line was cultured in vitro and was stimulated with M1/exo or M2/exo at the concentration of 50?g/ml for 72 h.Then,A549 cells migration ability was detected with the method of transwell and scratch test,respectively.3.3 Additonally,the proliferation of A549 cell line after treating with M1/exo or M2/exo or exosome combined with cisplatin was analyzed by real-time Cellular analysis(RTCA).4 A549 cell line was simulated with M1/exo or M2/exo in vitro for 72 h,then the total protein of A549 was extracted,the level of E-Cadherin,N-Cadherin,PTEN,AKT / p-AKT were analyzed by Western Blot.5 The effect of M1 and M2 macrophages and the exosomes from M1 or M2 on tumor growth and cisplatin sensitivity in vivo5.1 cell culturing and countingCollecting A549 cells in logarithmic phase,and the cell density was adjusted to 7×107/ml.M1 and M2 macrophages were induced according to section 1.1 or 1.2,the cell density were adjusted to for 7×107/ml,respectively.5.2 Establishment of tumor-bearing mice5.2.1Experimental groupsThe aged 5 to 6 weeks,female BALB/c nude mice were divided into 10 groups,there were 5 mice in each group.Group 1: A549 cell line tumor-bearing group.The A549 cells were transplanted into the mice on the right side of axillary,7×106/mouse.The group was used as control.Group 2: A549+M1 group.Taking the same amount of A549 and M1cells(both cells are 7×106)and mixing them together,then the cells were transplanted into mice on the right side of axillary.Group 3: A549+M2 group.Taking the same amount of A549 and M1cells(both cells are 7×106)and mixing them together,then the cells were transplanted into mice on the right side of axillary.Group 4: A549+cisplatin.The A549 cells were transplanted into the mice on the right side of axillary,7×106/mouse.Then,cisplatin(5mg/kg)was intraperitoneal injected according to Q4d×3 project.Group 5: A549+M1+cisplatin.The mice were transplanted with mixed A549 and M1 macrophage according to Group 2,then cisplatin(5 mg/kg)was intraperitoneal injected according to Q4d×3 project.Group 6:A549+M2+cisplatin.The mice were transplanted with mixed A549 and M2 macrophage according to Group 2,then cisplatin(5 mg/kg)was intraperitoneal injected according to Q4d×3 project.Group 7: A549+M1-exo.The A549 cells were transplanted into the mice on the right side of axillary,7×106/mouse.Then,M1-exo was injected at the right side of axillary on every other day for three weeks.Groups 8: A549+M2-exo.The A549 cells were transplanted into the mice on the right side of axillary,7×106/mouse.Then,M2-exo was injected at the right side of axillary on every other day for three weeks.Group 9: A549 + M1-exo + cisplatin.Same as above.Group 10: A549 + M2-exo + cisplatin.Same as above.The tumor-bearing mice were killed and tumors were weighed on the fourth week after tumor transplanting day.5.3 Observation of the tumor weight.In the four weeks,we anatomize the tumor-burdened mice to separate tumor tissue and weight them.Results:1 ELISA assay showed that IL-1? in M1 macrophages supernatant is significantly lower than that of M2 macrophages(P<0.05).M1 macrophages i NOs protein expression is significantly higher than M2 macrophages(P<0.05);and yet Arg-1 protein expression in M2 macrophages was higher(P<0.05).2 Transmission electron microscopy revealed that exosomes from M1 or M2 culture supernatants were almost 100 nm membrane vesicles.They were round or ellipse in shape.3 Red fluorescence could be seen in the A549 cells under inverted fluorescence microscope,and the result indicated that A549 cells could uptake exosomes.4 Scratch experiment indicated that PBS and M1/exo did not significantly affect the lateral migration of A549(P >0.05);and yet M2/exo can promote the lateral migration of A549 cells(P<0.05).Transwell experiments showed that,comparing with PBS and M1/exo,the M2/exo could significantly promote the longitudinal migration of A549 cells(P<0.05).Cell proliferation experiment showed M2/exo promote A549 cell hyperplasia and drug resistance of cisplatin5 Western blot results demonstrated that the expression of N-Cadherin in M2/exo group was obviously higher than that of M1/exo and the control groups(all P<0.05).The expression levels of E-Cadherin in M2/exo group was obviously lower than that of M1/exo and the control groups(all P<0.05).The data demonstrated that the M2/exo can promote A549 cells migration.Western blot results also demonstrated that the expression level of PTEN in M2/exo group was obviously lower than that of M1/exo group and the control group(all P<0.05).The ratio of p-Akt/Akt in M2/exo stimulated group was significantly higher than that of M1/exo group and the control group(all P<0.05).The data demonstrated that the M2/exo can promote A549 cells proliferation.6 The experiment results of tumor-bearing mice models showed:1)Comparing with A549 and A549+M1 groups,the tumor weight in A549+M2 tumor-bearing mice is significant higher(all P<0.01).Whereas,there were no significant difference about the tumor weight between A549 and A549+M1 groups(P>0.05).The data suggested that M2 macrophages harbored a tumor-promoting role in the A549 model.2)We then examined A549 outgrowth during cisplatin treatment.After treatment with cisplatin,the tumor weight in A549 group was statistically significant lower(P<0.05).After treatment,the tumor weight in A549+M2group was higher than that in A549+M1 group,there was a significant difference(P<0.05).The data further suggested that M2 macrophages have a prmoting drug-resistance role in the A549 model.3)After injecting M2/exo in A549 tumor-bearing mice,A549 growth faster than injecting PBS or injecting M1/exo.There were statistically significant higher tumor weight for mice injected with M2/exo(all P <0.05).4)At the day post A549,A549+M1/exo or A549+M2/exo injection,mice were treated with cisplatin,and evaluated for tumor growth.In striking contrast,M2/exo significantly improved drug-resistance with the higher tumor weight(all P <0.01).Conclusion:1M2 macrophages can promote A549 cell proliferation,migration,and reduce the sensitivity to cisplatin.2M2/exosome can promote A549 cell proliferation,migration,and reduce the sensitivity to cisplatin. |
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