| The rapid development of cellular immunotherapy has brought new opportunities and challenges for cancer treatment.NK cells have become an important cellular tool in tumor immunotherapy due to their strong anti-tumor effect and the safety of allogeneic NK cell adoptive therapy.Because NK cells cannot be activated effectively in the cytokine deficient tumor microenvironment,clinical trials of NK cell therapy have had poor results.Therefore,to improve the effector function of NK cells and enhance the effect of anti-tumor therapy in vivo is a key scientific problem to be solved urgently.Type Ⅰ Interferons(IFNs)can promote the secretion of cytokines in NK cells,improve the cytotoxicity of NK cells,and play a positive role in immune regulation.Recombinant anti-tumor antiviral protein(novaferon,nova)is a novel recombinant IFN-α-like protein prepared by gene shuffling technology and high throughput screening,which has stronger biological activity than IFN-α.Due to in vivo cytokine depletion,activation of other immune cells,and significant drug side effects,the advantage of exogenous administration of type Ⅰ IFNs to improve NK cell activity is not obvious.In order to overcome the above shortcomings and improve the activity of NK cells more effectively,this study prepared a new type of NK92 cells by lentiviral vector technology to stably express this more active recombinant protein nova,that is,NK92-NOVA cells.In order to explore the anti-tumor efficacy of novel NK cell preparations,the functional changes of NK92-nova cells in effector cytokine secretion,activator receptor expression and tumor killing were first identified,and the possible molecular mechanism of the enhanced anti-tumor activity of NK92-nova cells was initially discussed.The efficacy and safety of NK92-nova cells as a novel NK cell preparation in vivo to inhibit tumor progression were verified.1.Methods(1)Construction of NK92-nova cellHuman NK92 cells were infected with nova lentiviral vector,screened by puromycin and identified by flow cytometry,fluorescence microscopy,q RT-PCR and Western blot.(2)Detection of NK92-nova cell killing activity in vitroNK cells were the effector cells and tumor cells were the target cells.The effector cells and target cells were co-cultured for 4 hours under different effector target ratios.The effects of NK92-nova cells on 6 tumor cell lines(human chronic myeloid leukemia cell line K562 cells,human monocytic leukemia cell line THP-1 cells,human non-small cell lung cancer cell line A549 cells,human colorectal adenocarcinoma cell line SW1116 cells,human liver cancer cell line HepG2 cells and human esophageal cancer cell line TE)were detected by CCK8 The killing effect of-1 cells.HepG2 cells were cultured in the medium containing nova,and after 4 hours of culture(the same time as the killing experiment),nova was fully moistened and washed away,and NK92-vec cells were added to continue the co-culture for 4 hours.CCK8 detected the killing effect of NK92-vec cells combined with nova on HepG2 cells.(3)Effector molecular detection in NK92-nova cellThe secretion levels of IFN-γin wild-type NK92 cells,NK92-vec cells carrying empty lentiviral vector and NK92nova cells were detected by ELISA under the same conditions.Western blot was used to detect the expression levels of perforin,Granzyme B,FASL and TRAIL.NK92 cells were infected with nova lentiviral vector,screened by puromycin and identified by flow cytometry,fluorescence microscopy,q RT-PCR and Western blot.(4)Activation receptor detection in NK92-nova cellFlow cytometry was used to detect the expression levels of NKG2D,NKp44,NKp46 and CD16 on the surface of wild-type NK92 cells,NK92-vec cells carrying empty lentiviral vectors,and NK92-nova cells.NK92 cells were transfected with lentiviral vector,and after puromycin screening,NK92-nova cells with stable expression of nova were constructed.Identification was performed by q RT-PCR,Western blot,fluorescence microscopy and flow cytometry.(5)Type Ⅰ IFN signaling pathway analysisTranscriptome sequencing was performed on NK92-vec and NK92-nova cells,and differential expression analysis was performed by using R software.GO enrichment analysis and KEGG signaling pathway analysis were performed on differentially expressed genes.The changes of the major signaling pathway proteins were detected by Western blot.(6)Analysis of NK92-nova cell tumor inhibition in vivoBy establishing subcutaneous transplanted tumor model of NOD/SCID immunodeficient mice,the inhibitory effect of NK92-nova cell therapy on HepG2tumor bearing mice in vivo was detected by direct measurement.H&E staining was used to detect the effect of NK92-nova cell therapy on tumor morphology and important organs.The effects of nova on the expression and degranulation of NK92 cell surface activator receptors were detected by flow cytometry.2.Results(1)NK92-nova cells were successfully prepared.Flow cytometry,fluorescence microscopy,q RT-PCR and Western blot results showed that NK92-nova cells expressing green fluorescent protein and nova protein were successfully prepared.(2)NK92-nova cells have enhanced anti-tumor activity.In vitro killing experiments,NK92-nova cells showed significantly increased antitumor activity against K562,THP-1,A549,SW1116,HepG2 and TE-1 tumor cell lines.The antitumor activity of NK92-vec cells combined with nova protein was similar to that of NK92-vec cells alone against HepG2 cells,and the results showed that nova could directly induce the activation of NK92 cells,rather than enhance the killing activity of NK cells by inhibiting HepG2 cells.(3)NK92-nova cells showed enhanced expression of effector molecules.ELISA results showed that the secretion level of IFN-γin NK92-nova cells was significantly increased,and the secretion capacity of IFN-γwas significantly enhanced in the presence of inducible activator or tumor cell HepG2.Western blot results showed that the expression levels of perforin,Granzyme B,FASL and TRAIL were significantly increased in NK92-nova cells.(4)The expression of NK92-nova cell activator receptor was significantly up-regulated.Flow cytometry showed that the expressions of NKG2D,NKp44 and NKp46 on the surface of NK92-nova cells were significantly up-regulated.CD16 expression was detected on the surface of NK92-nova cells,and transcriptome sequencing showed that CD16 gene was differentially expressed and significantly upregulated in NK92-nova cells.NK92-nova cells,in coordination with trastuzumab,showed approximately 40%substantial cytotoxicity against HER2~+breast cancer cell line BT474.(5)The enhanced activity of NK92-nova cells is related to the activation of type Ⅰ IFN signaling pathway.A difference analysis of the transcriptome data of NK92-nova and NK92-vec cells showed that there were 243 significantly up-regulated and 206 significantly down-regulated genes in NK92-nova cells compared with NK92-vec cells.GO functional enrichment analysis showed that the main biological function of differentially expressed genes was type Ⅰ IFN signaling.In addition,KEGG pathway enrichment analysis showed that upregulated genes in NK92-nova cells were involved in NK cell-mediated cytotoxicity.The expression of IFN receptor IFNAR2 on the surface of NK92-nova cells was significantly increased,and the phosphorylation levels of type Ⅰ IFN signaling pathway proteins JAK1,STAT1 and STAT2 were significantly increased,which significantly reduced the killing effect of NK92-nova cells under the action of the JAK1 inhibitor Ruxilitinib.(6)NK92-nova cell therapy inhibited tumor progression in tumor-bearing miceIn NOD/SCID mouse xenotransplantation model,tumor volume and tumor weight in NK92-nova cell therapy group were significantly lower than those in NK92-vec cell therapy group and control group.H&E staining showed that the necrotic areas of tumor increased in NK92-nova treatment group,and there was no obvious toxic and side effects on the heart,liver,spleen,lung and kidney.3.ConclusionIn summary,the novel NK cell preparation NK92-nova has enhanced anti-tumor activity,and the increase of its anti-tumor activity is related to the secretion of cytokine IFN-γ,the expression levels of cytolytic effervescent molecules perforin,Granzyme B,FASL and TRAIL,and the increased expression of activator receptors.Activation of type Ⅰ IFN signaling pathway in NK92-nova cells promotes the killing activity of NK cells.It was further verified in tumor-bearing mice that adoptive treatment with NK92-nova cells significantly inhibited HCC progression in xenografted tumor models without systemic toxicity. |
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