Blocking Transforming Growth Factor-β Signaling Pathway Augments Antitumor Effect Of Adoptive NK-92 Cell Therapy

Objective Transforming growth factor (TGF)-βis a potent suppressor in tumor microenvironment, which may lead to tumor evasion from the host immune surveillance and tumor progression. The present study is to synthesize the gene of human dominant-negative TGF beta receptorⅡ(DNTβRⅡ) and construct its eukaryotic expression vector pIRES2-AcGFP-DNTβRⅡ; block TGF-βsignaling pathway in NK-92 cells by transfection with pIRES2-AcGFP-DNTβRβⅡplasmid using Nucleofector technology; investigate the in vitro and in vivo antitumor effect of this TGF-βinsensive NK-92 cells against human Calu-6 lung cancer cells which secrete high levels of TGF-β1.Methods (1) According to the Genebank Database, the sequence of target gene DNTβRβⅡwas determined. The single chain oligo was synthesized chemically and then ligated into full length gene by PCR. The synthesized gene was cloned into plasmid PMD18-T and transferred into competent cells DH5α, then it was inserted into the corresponding restriction site on eukaryotic expression vector pIRES2-AcGFP, the DNTβRⅡgene was confirmed by double enzyme digesting and DNA sequencing analysis. After transfer into COS-7 cells, expression of DNTβRⅡwas identified by inverted fluorescent microscope. (2) The expression of TGF-βtypeⅠ(TβRⅠ) and typeⅡreceptors (TβRⅡ) in parental NK-92 cells were detected by fluorescence activated cell sorter (FACS). Mean levels of TGF-β1 secreted by 4 human tumor cell lines(Calu-6,A549,MDA-MB231 and HT-29) were determined by ELISA. NK-92 cells were transfected with recombinant plasmid pIRES2-AcGFP-DNTβRⅡand control plasmid pIRES2-AcGFP using Amaxa Nucleofector technology. RT-PCR and inverted fluorescent microscope were used to identify the expression of DNTβRⅡ, FACS was used to analyse the transfection efficiency, then western blot was used to detect the phosphorylated satus of Smad2. TGF-β1 was added at the final concentration of lOng/ml with Calu-6 cells for 24h. The cytotoxicity of two types transfected NK-92 cells against Calu-6 cells was detected and analyzed by CCK-8 kit. (3) According to the model of tumor formation,24 BALB/C-nu mice received a single subcutaneouly injection of 1x106 Calu-6 cells into the back; at the same time, 1x107 NK-92 cells transfected with DNTβRⅡpositive vector (group C) or GFP control plasmid (group B) were injected via tail vein while there was no intervention added in group A.10 days afer transplantion, tumor formation rate of mice bearing Calu-6 cells in 3 groups was compared. According to the model of tumor suppression, 1x106 Calu-6 cells were injected subcutaneouly into the back of BALB/C mice, then saline (group a),1x107 NK-92 cells transfected with plasmid pIRES2-AcGFP (group b) or pIRES2-AcGFP-DNTβRⅡ(group c) were respectively injected via tail vein on the day 10,17 following transplantion. Serum IFN-y levels of tumor-bearing mice in different groups were detected by ELISA on the day next to the second adoptive transfer.56 days after transplantion, all animals were sacrificed and histologic examination was done.Results (1) The synthesized fragment was consistent with the target gene DNTβRⅡby DNA sequence analysis. After the recombinant expression plasmid pIRES2-AcGFP-DNTβRⅡtransferring into COS-7 cells, DNTβRⅡwas instantaneously transfected and expressed successfully. (2) The highest level of TGF-β1 was secreted by Calu-6 cell-line while the lowest level of TGF-β1 was secreted by HT-29 cell-line. High expression of TβRⅠand TβRⅡwas identified by FACS. Using nucleofector technology, the transfection efficiency was 18.80% for the plasmid pIRES2-AcGFP-DNTβRⅡand 28.53% for the control vector pIRES2-AcGFP to NK-92 cells. The expression of DNTβRⅡin NK-92 cells was confirmed by inverted fluorescent microscope and RT-PCR. By the irritation of TGF-β1, western blot detected inactive Smad2 status in NK-92 cells transfected with pIRES2-AcGFP-DNTβRⅡ. Parental NK-92 cells displayed lower cytotoxity against Calu-6 cells incubated with TGF-β1 than that without TGF-β1 (E:T ratio 10:1,32.93%±0.80% vs.17.90%±0.75%, p< 0.001; E:T ratio 20:1, 46.33%±1.40% vs.26.46%±1.10%, p<0.001). The cytotoxity of NK-92 cells transfeced with DNTβRⅡvector was higher than that with control GFP vector against Calu-6 cells cultured with TGF-β1(E:T ratio 29.73%±0.96% vs. 15.43%±0.97%, p<0.001; E:T ratio 20:1,45.0%±1.20% vs.24.83%±1.21%, p< 0.001). (3) The tumor formation rate of mice bearing Calu-6 cells in group C was lower than group A(p<0.05). After adoptive transfer, decreased tumor volume, increased survival, lower lung metastasis rate and higher levels of serum IFN-y were shown in group c which received adoptive transfer of NK-92 cells transfected with DNTβRⅡpositive vector.Conclusion (1) The eukaryotic expression vector pIRES2-AcGFP-DNTβRⅡwas constructed successfully, providing the basis for blocking TGF-βsignaling pathway in NK-92 cells. (2) The pIRES2-AcGFP-DNTβRⅡplasmid was successfully transferred and expressed in NK-92 cells by nucleofector technology. (3) pIRES2-AcGFP-DNTβRⅡplasmid transfection can block TGF-βsignaling pathway in NK-92 cells at the receptor level. (4) Blocking TGF-P signaling pathway in NK-92 cells can resist immunosupression effect of TGF-P in vitro. (5) Blocking TGF-βsignaling pathway can augment antitumor efficacy of adoptive NK-92 cell therapy in vivo. (6) Relieving immuosupression in tumor microenvironmet may enhance the efficacy of adoptive therapy in clinical practice.