Effect Of NKTm Cells Plus Cisplatin/cetuximab Treatment On NSCLC Cells In Vitro

【Objective】To investigate inhibitory effect of NKTm cells and combiniton effect of NKTm cells with DDP or cetuximab on NSCLC in vitro. To explore the mechanisms underlying cetuximab's inhibitory effect on NSCLC and find the predictive biomarkers related to the activity of ADCC mediated by cetuximab. To provide evidence for individualized treatment of NSCLC.【Methods】Part one:PLA-801D cells were treated with NKTm cells alone for different time and at effect-to-target cell ratio (E/T) of 50:1,25:1,12:,6:1, and treated with NKTm cells plus DDP administered in different time sequence. Data on treatment efficacy were assayed by MTT. Part two:1. The molecular features of NSCLC cell lines were described using A549, H1975 and PLA-801D as target cells. Protein expression and gene amplification level of EGFR was detected separately using IHC and FISH assay. The Kras and EGFR gene mutations were detected by direct sequencing.2. Inhibition and apoptosis rate of the 3 NSCLC cell lines induced by cetuximab were analyzed by CCK-8 assay and flow cytometry respectively. Gene expression changes in A549 cells were identified by microarray.3. cetuximab mediated ADCC activity and the influencing factors were shown by calculating inhibition rate of A549 cells using CCK-8 assay:1) NKTm cells were blocked by Anti-CD 16 antibody, and ADCC activity was compared between treated groups and control groups; 2) pEGFR-EGFP plasmids were transferred into A549 cells by Nucleofector method to compare the ADCC activity of killing A549 cells with different EGFR expression level; 3) FCGR3A polymorphisms of NKTm cells were detected by direct sequencing, and ADCC activity was compared among NKTm cells with different genotypes.【Results】In our study, NKTm cells showed significant inhibitory effect on NSCLC cells in vitro. The inhibitory effect was positively correlated with E/T ratio. The highest inhibition rate appeared at the 4th h. NKTm cells increased the efficacy of DDP in inhibiting NSCLC, and the best synergistic effect was observed when DDP was administered 4 h after the use of NKTm cells.NSCLC cell lines of A549, H1975 and PLA-801D had low EGFR expression and negative EGFR FISH results. cetuximab had the highest inhibition and apoptosis rate in A549 carrying Kras mutation (GGT>AGT) and the lowest apoptosis rate in H1975 carrying EGFR mutation (L858R), P<0.05. cetuximab had influence on gene expression profiles of A549 cell line, relating to cell cycle, metastasis, resistance to chemotherapy and anti-tumor immunity.The combined use of cetuximab and NKTm cells showed a synergistic inhibitory effect on A549 cells. Application of Anti-CD 16 can block membrane receptor on NKTm cells resulting in lower synergistic inhibition rate (P<0.05), but there were no significant differences between the inhitition rate of Anti-CD 16 group and NKTm cells group. It was indicated that the synergistic effect is realized by ADCC activity. A549 cells transfected with pEGFR-EGFP plasmids expressed higher level of EGFR protein on membrane and were more sensitive to ADCC (P<0.05). The FCGR3A polymorphisms were correlated with ADCC activity of NKTm cells. The NKTm cells with FCGR3A-158V/V genotype had significantly higher ADCC than FCGR3A-158 V/F or F/F genotypes (P<0.05), but ADCC activity between V/F and F/F genotype had no statistical significance.[Conclusion] NKTm cells have inhibitory effect on NSCLC cells in vitro. The synergistic effect was observed when NKTm cells combinited with DDP, and optimal synergistic effect was achieved if DDP is added 4 h after NKTm cells administered. A549 cells with Kras mutation (GGT>AGT) are more sensitive to cetuximab than PLA-801D cells with wild type, while H1975 cells with EGFR mutation (L858R) are resistant to cetuximab mediated apoptosis. Influence on gene expression, relating to cell cycle, metastasis, resistance to chemotherapy and anti-tumor immunity may be the mechanisms underlying cetuximab's inhibitory effect on NSCLC. The combined use of cetuximab and NKTm cells have synergistic inhibitory effect on NSCLC cells and the the synergistic effect is realized by ADCC activity. The ADCC effect may be affected by EGFR protein level of target cells and FCGR3A polymorphisms of NKTm cells.