The Effect Of Macrophages On Acquired Resistance To EGFR-TKI In NSCLC Cell Line NCI-H1975

Background:With the high morbidity and mortality,lung carcinoma brings huge harm to patients.Non-small cell lung cancer(NSCLC)occupies 80%～85%of lung carcinoma.Surgical resection and chemotherapy are the main treatment of NSCLC.However,for the patients with multifocality,recurrence or metastases,chemotherapy is the only way.Although traditional chemotherapy is a basis of NSCLC,the clinical application is restricted because of its weak specificity and strong toxicity to patients.During recent years,the molecular targeted drugs have achieved success in treatment of NSCLC represented by epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs).Unfortunately,a problem occurs that more than half of NSCLC patients who were intially sensitive to EGFR-TKIs developed acquired resistance after continuous therapy for 6 months.The main mechanisms of EGFR-TKI acquired resistance are the T790M mutation in exon 20 of EGFR,abnormal activation of proteins in downstream of EGFR pathways,abnormal amplification of c-Met gene and epithelial-mesenchymal transition(EMT)in tumor cells etc.In previous study,we found that EMT emerged in both NCI-H1975/GR carrying T790M mutation and NCI-H1650/GR without T790M mutation during the gefitinib treatment,suggesting that EMT was a common mechanism for NSCLC escaping from the inhibition of EGFR-TKI.Therefore,the factors which affected the process of EMT in tumor cells are worth exploring.EMT in tumor cells depends on the stimulating of cytokines in tumor microenvironment which consists of inflammatory cells,vessels and fibres etc.As a major inflammatory cell,macrophage(MΦ)plays a key role in tumor microenvironment.The function of MΦ is to protect damaged cells and to remove dead cells in somatic organisms.A number of researches show that MΦ could promote the growth,invasion and migration of tumor cells,suggesting that the living of tumor cells need the aids of MΦ in a changed microenvironment.Although tumor cells have gene alternations or abnormal protein expression compared with normal tissue cells,they belong to somatic cells in essence.When damaged by chemotherapy drugs such as EGFR-TKI,a large number of cells in NSCLC produce denaturation and necrosis due to the new microenvironment containing drugs.In this process,whether MΦ can protect the living tumor cells in the new microenvironment is to be explored.Research shows that MΦcould help such as breast cancer cells generate drug resistance with multi-channel.However,there is no report related to whether MΦ can promote NSCLC cells generate EGFR-TKI acquired resistance,which is worth further discussing.Objectives:In this study,we established a separable co-culture system of human NSCLC cell line NCI-H1975(carrying the T790M mutation in exon 20 of EGFR)and MΦin vitro.By comparing the gefitinib sensitivity,cell morphology and EMT occurence between NCI-H1975 cultured alone and NCI-H1975 co-cultured with MΦ,we aim to explore the promoting role of MΦ on acquired resistance to EGFR-TKI of NSCLC and trying to find possible mechanisms.This study could provide new strategy for NSCLC patients to overcome the acquired resistance against EGFR-TKI.Methods:1.The establishment of a separable co-culture system of MΦ and NCI-H1975 cell(1)The culture condition of NCI-H1975 cell、THP-1 and MΦ was optimized,including the concentration of fetal bovine serum(FBS)and antibiotics,as well as the incubation condition etc.(2)The co-culture condition of NCI-H1975 cell and MΦ was explored,including the concentration of FBS and antibiotics,the cells concentration,as well as the incubation condition etc.(3)The objects of study were divide into three groups.1)NCI-H1975 group:NCI-H1975 was cultured alone2)MΦ group:MΦ was cultured alone3)NCI-H1975/MΦ group:MΦ and NCI-H1975 were cultured together2.The choose of gefitinib concentration and working time(1)The concentration of gefitinibThe gefitinib concentration was selected in each group by MTT.(2)The action time of gefitinib3.The protection of MΦ to NCI-H1975 cell in the environment with gefitinibThe effect of MΦ on NCI-H1975 cells about sensitivity to gefitinib,EMT and content of TGF-β1 in culture solution are researched.(1)The susceptibility to gefitinib1)The growth condition of tumor cells2)The density of tumor cells3)The gefitinib inhibition curves of tumor cells4)The IC50 and resistance index of tumor cells(2)EMT of tumor cells in each groupThe expression levels of E-cadherin and Vimentin in tumor cells were detected in NCI-H1975/MΦ group and NCI-H1975 group by both immunocytochemistry and Western-blot.(3)The content of TGF-β1 in culture supernatant of each groupThe content of TGF-β1 in the culture of each group was determined by ELISA.Results:1.A separable co-culture system between MΦ and NSCLC cells was established.(1)The routine culture condition of NCI-H1975、THP-1 and MΦ.These cell lines were cultured in RPMI-1640 complete medium containing 10%FBS,100μg/ml penicillin and 100μg/ml streptomycin in cell incubator with 37℃,5%CO2 and saturated humidity.NCI-H1975 cells showed adherent and long spindle;THP-1 showed a state of suspended growth,circular or elliptic;MΦ showed adherent growth,round or oval,extended pseudopodia.(2)After experiments of more than twenty times,a stable co-culture system of separation between M Φ and NCI-H1 975 cells was established.1)Cells culture medium:RPMI-1640 complete medium containing 10%FBS,100μg/ml penicillin and 100μg/ml streptomycin2)Cells were placedNCI-H1975 was placed in six orifice,the concentration was 4×105/ml;THP-1 was placed in Transwell chamber,the concentration was 2.5×105/ml.The chamber was placed above the six orifice.Cells culture medium was exchanged every 48h.3)Co-cultured condition:37℃,5%CO2 and saturated humidity.4)Co-cultured cells growth state:Two kinds of cells were growed in good state,and there was no significant difference of conventional culture.2.Gefitinib concentration and action time(1)Gefitinib concentrationThe growth inhibition rate of tumor cells was compared by MTT.The inhibition rate of NCI-H1975 cell to gefitinib was increased with the increase of drug concentration gradually.The IC50 value was 14.61±0.03μM。Choose three concentrations of gefitinib(10μM,20μM,30μM)respectively,at the same time add gefitinib to the NCI-H1975 group and NCI-H1975/MΦ group.(2)Gefitinib action timeGefitinib action time in each experimental group was selected 48 h.3.The protection of MΦ to NCI-H1975 cells during gefitinib environment(1)The sensitivity to gefitinib of tumor cells in each group1)The morphological differences of tumor cells in each group during gefitinib environmentThe conclusion showed that the status of tumor cells in NCI-H1975/MΦ group was better than that in NCI-H1975 group,and the difference was more significant with the increase of gefitinib concentration.2)The concentration of living tumor cells in each groupIn the presence of 10μM gefitinib,the number of living tumor cells in NCI-H1975 group and NCI-H1975/MΦ group was(2.53±0.25)×105/ml and(7.47±1.30)×105/ml,respectively.(P<0.05);In the presence of 20μM gefitinib,they were(1.63±0.107)×105/ml and(5.28±0.13)×105/ml,respectively(P<0.05);In the presence of 30μM gefitinib,they were(1.169±0.011)×104/m and(4.81±0.045)×105/ml,respectively(P<0.05)Generally,the living number of tumor cells in NCI-H1975/MΦ group was larger than that in NCI-H1975 group,and the difference was more significant with the increase of gefitinib concentration.3)The growth inhibition curves of tumor cells in each group.With gefitinib of the same concentration,the growth of tumor cells in both NCI-H1975/MΦ group and NCI-H1975 group was inhibited with the prolongation of drug treatment.However,the degree of growth inhibition of gefitinib in NCI-H1975/MΦ group was lower than that in NCI-H1975 group,and the difference was more significant with the increase of gefitinib concentration.4)The gefitinib inhibition rate、IC50 and resistance index of tumor cells in each groupa.The gefitinib inhibition rateThe growth inhibition rate of tumor cells in NCI-H1975/MΦ group was lower than that in NCI-H1975 group at the same concentration of gefitinib.b.The IC5Φ valueThe IC5Φ value of tumor cells in NCI-H1975/10 and NCI-H1975/MΦ/10 were 15.547±0.46μM and 22.897±0.5μM,respectively(P<0.05);the IC50 value of tumor cells in NCI-H1975/20 and NCI-H1975/MΦ/20 were 16.137± 0.12μM and 23.01±0.2μM,respectively(P<0.05);the IC50 value of tumor cells in NCI-H1975/30 and NCI-H1975/MΦ/30 were 16.71±0.8μM and 23.19±0.019μM,respectively(P<0.05).Thus the IC50 value of tumor cells in NCI-H1975/MΦ group was higher that in NCI-H1975 group.c.The resistance index of tumor cellsThe resistance index of tumor cells in NCI-H1975/MΦ/10、NCI-H1975/MΦ/20、NCI-H1975/MΦ/30 were 1.567、1.575 and 1.587.The resistance index of tumor cells was stable with gefitinib of the same concentration.(2)The effect of macrophages on EMT in tumor cells with gefitinibThe results of Immunocytochemistry and Western-blot showed that the expression level of E-cadherin in tumor cells of NCI-H1975/MΦ group was lower than that of NCI-H1975 group,while the expression level of Vimentin in tumor cells in the former group was stronger than that of the latter group(P<0.05).Thus EMT was more likely to occur in NCI-H1975 cells at the presence of MΦ with gefitinib.(3)The TGF-β1 content in tumor cellsWith gefitinib of 10μM,the concentration of TGF-β1 in NCI-H1975 group,MΦgroup and NCI-H1975/MΦ group were 0、0 and(0.121 ±0.012)ng/ml,respectively(P<0.05);With gefitinib of 20μM,they were 0、0 and(0.122±0.008)ng/ml,respectively(P<0.05);With gefitinib of 30μM,they were 0、0 and(0.133±0,005)ng/ml,respectively.(P<0.05)The result showed that TGF-β1 content in NCI-H1975/MΦ co-cultured supernatant increased significantly at the presence of MΦ.Conclusions:1.A stable co-culture system of MΦ and NSCLC cells was established.2.MΦ could play a promoting role on the acquired resistance of NSCLC cells(harboring T790M mutation)to EGFR-TKI in vitro.3.EMT could be one of the mechanisms of acquired resistance of NSCLC cells(harboring T790M mutation)to EGFR-TKI.4.The presence of MΦ might promote TGF-β1 content increase significantly in co-cultured supernatant with NSCLC cells.