体外扩增的NK细胞运载溶瘤呼肠孤病毒对结直肠癌细胞的杀伤效应
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摘要
目的:评价体外扩增的人NK细胞能否作为呼肠孤病毒的运载细胞并探讨其临床转化应用价值。方法:体外扩增人NK细胞,用NK细胞装载呼肠孤病毒,激光共聚焦显微镜观察呼肠孤病毒与NK细胞结合的部位。NK细胞运载呼肠孤病毒(Reo-NK)到达肿瘤细胞后,CCK-8法检测在中和抗体的存在下病毒发挥的溶瘤作用;qPCR法检测肿瘤细胞内病毒RNA的相对表达量。CCK-8法检测Reo-NK对结直肠癌KRAS基因突变型(DLD-1)和野生型(CaCo-2、HT29)细胞株的杀伤效应,ELISA双抗体夹心法检测NK细胞释放的穿孔素水平。结果:呼肠孤病毒主要结合于NK细胞膜上,在人AB型血清的存在下体外扩增的NK细胞可将呼肠孤病毒传递到肿瘤细胞,且传递后的呼肠孤病毒仍具有显著溶瘤活性(P<0.01);同时检测到肿瘤细胞内病毒RNA的表达量显著增高(P<0.01)。与NK组相比,Reo-NK组对KRAS基因突变型和野生型结直肠癌细胞株的细胞毒作用均显著增强(P<0.05),穿孔素的释放均明显增加(P<0.05)。结论:体外扩增的人NK细胞适合作为呼肠孤病毒的运载细胞,且呼肠孤病毒能够增强NK细胞的细胞毒作用,两者联合后对结直肠癌细胞可发挥更强的杀伤作用,因而具有重要的临床转化应用价值。
Objective: To evaluate whether human NK cells expanded in vitro can be used as carrier cells of reovirus and to investigate its clinical application value. Methods: Expansion of human NK cells in vitro, and flow cytometry was used to analyse the purity of CD3~-CD56~+ cells. Expanded NK cells were loaded with reovirus and observed by confocal microscopy, to determining the location of reovirus on NK cells. CCK-8 assay was used to detect reovirus-induced oncolysis of expanded NK cells carrying reovirus(Reo-NK)to tumor cells in the presence of neutralizing antibodies; Real-time fluorescence quantitative PCR was used to assess the relative expression of viral RNA in tumor cells. Cytotoxicity assay were performed to detect Reo-NK cells against KRAS mutant(DLD-1) and KRAS wild type(CaCo-2, HT29) colorectal cancer cell lines, ELISA matched paired antibodies assay was performed to measure the perforin level released by NK cells. Results: Confocal microscopy demonstrated that NK cells retained reovirus on the surface. Expanded NK cells could delivery reovirus to tumor cells in the presence of neutralizing antibodies, and the reovirus after delivery still had significant oncolytic activity(P<0.01); Corresponding qPCR result displayed that the expression of viral RNA in tumor cells significantly increased over time(P<0.01). Compared with NK group, Reo-NK group evidently enhanced the cytotoxicity on colorectal cancer cell lines with both KRAS gene mutant and wild(all P<0.05), and significantly increased the release of perforin(all P<0.05). Conclusion:In vitro expanded NK cells provide a convincing cell carrier for reovirus, while reovirus enhances the cytotoxicity of NK cells, and the combination of the two show a stronger killing effect on colorectal cancer cells,that has important clinical application value.
Objective: To evaluate whether human NK cells expanded in vitro can be used as carrier cells of reovirus and to investigate its clinical application value. Methods: Expansion of human NK cells in vitro, and flow cytometry was used to analyse the purity of CD3~-CD56~+ cells. Expanded NK cells were loaded with reovirus and observed by confocal microscopy, to determining the location of reovirus on NK cells. CCK-8 assay was used to detect reovirus-induced oncolysis of expanded NK cells carrying reovirus(Reo-NK)to tumor cells in the presence of neutralizing antibodies; Real-time fluorescence quantitative PCR was used to assess the relative expression of viral RNA in tumor cells. Cytotoxicity assay were performed to detect Reo-NK cells against KRAS mutant(DLD-1) and KRAS wild type(CaCo-2, HT29) colorectal cancer cell lines, ELISA matched paired antibodies assay was performed to measure the perforin level released by NK cells. Results: Confocal microscopy demonstrated that NK cells retained reovirus on the surface. Expanded NK cells could delivery reovirus to tumor cells in the presence of neutralizing antibodies, and the reovirus after delivery still had significant oncolytic activity(P<0.01); Corresponding qPCR result displayed that the expression of viral RNA in tumor cells significantly increased over time(P<0.01). Compared with NK group, Reo-NK group evidently enhanced the cytotoxicity on colorectal cancer cell lines with both KRAS gene mutant and wild(all P<0.05), and significantly increased the release of perforin(all P<0.05). Conclusion:In vitro expanded NK cells provide a convincing cell carrier for reovirus, while reovirus enhances the cytotoxicity of NK cells, and the combination of the two show a stronger killing effect on colorectal cancer cells,that has important clinical application value.
引文
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[24]ZHAO X,CHESTER C,RAJASEKARAN N,et al.Strategic combinations:the future of oncolytic virotherapy with reovirus[J].Mol Cancer Ther,2016,15(5):767-773.DOI:10.1158/1535-7163.MCT-15-0695.
[25]MAITRA R,SEETHARAM R,TESFA L,et al.Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells,and synergizes with irinotecan[J].Oncotarget,2014,5(9):2807-2819.DOI:10.18632/oncotarget.1921.
[26]TWIGGER K,ROULSTONE V,KYULA J,et al.Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway[J/OL].BMC Cancer,2012,12:368[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537694/.DOI:10.1186/1471-2407-12-368.
[2]MARELLI G,HOWELLS A,LEMOINE N R,et al.Oncolytic viral therapy and the immune system:A double-edged sword against cancer[J/OL].Front Immunol,2018,9:866[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932159/.DOI:10.3389/fimmu.2018.00866.
[3]GONG J,SACHDEV E,MITA A C,et al.Clinical development of reovirus for cancer therapy:An oncolytic virus with immune-mediated antitumor activity[J].World J Methodol,2016,6(1):25-42.DOI:10.5662/wjm.v6.i1.25.
[4]BOURHILL T,MORI Y,RANCOURT D E,et al.Going(Reo)Viral:factors promoting successful reoviral oncolytic infection[J/OL].Viruses,2018,10(8):E421[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116061/.DOI:10.3390/v10080421.
[5]SAMSON A,SCOTT K J,TAGGART D,et al.Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade[J/OL].Sci Transl Med,2018,10(422):eaam7577[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276984/.DOI:10.1126/scitranslmed.aam7577.
[6]SAMSON A,BENTHAM M J,SCOTT K,et al.Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer[J].Gut,2018,67(3):562-573.DOI:10.1136/gutjnl-2016-312009.
[7]MAHALINGAM D,FOUNTZILAS C,MOSELEY J,et al.A phase II study of REOLYSIN?(pelareorep)in combination with carboplatin and paclitaxel for patients with advanced malignant melanoma[J].Cancer Chemother Pharmacol,2017,79(4):697-703.DOI:10.1007/s00280-017-3260-6.
[8]何志旭,赵星.溶瘤病毒抗肿瘤临床试验的回顾与展望[J].中国肿瘤生物治疗杂志,2014,21(2):119-124.DOI:10.3872/j.issn.1007-385X.2014.02.001.
[9]WALZER T,VIVIER E.G-protein-coupled receptors in control of natural killer cell migration[J].Trends Immunol,2011,32(10):486-492.DOI:10.1016/j.it.2011.05.002.
[10]SIEGEL R L,MILLER K D,JEMAL A.Cancer statistics,2016[J].CA Cancer J Clin,2016,66(1):7-30.DOI:10.3322/caac.21332.
[11]ADAIR R A,ROULSTONE V,SCOTT K J,et al.Cell carriage,delivery,and selective replication of an oncolytic virus in tumor in patients[J/OL].Sci Transl Med,2012,4(138):138ra77[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893925/.DOI:10.1126/scitranslmed.3003578.
[12]JENNINGS V A,ILETT E J,SCOTT K J,et al.Lymphokine-activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites[J].Int J Cancer,2014,134(5):1091-1101.DOI:10.1002/ijc.28450.
[13]ILETT E J,BARCENA M,ERRINGTON-MAIS F,et al.Internalization of oncolytic reovirus by human dendritic cell carriers protects the virus from neutralization[J].Clin Cancer Res,2011,17(9):2767-2776.DOI:10.1158/1078-0432.CCR-10-3266.
[14]BERKELEY R A,STEELE L P,MULDER A A,et al.Antibodyneutralized reovirus is effective in oncolytic virotherapy[J].Cancer Immunol Res,2018,6(10):1161-1173.DOI:10.1158/2326-6066.CIR-18-0309.
[15]ZHAO X,OUYANG W W,CHESTER C,et al.Cytokine-induced killer cell delivery enhances the antitumor activity of oncolytic reovirus[J/OL].PLoS One,2017,12(9):e0184816[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602626/.DOI:10.1371/journal.pone.0184816.
[16]JUNG I H,KIM D H,YOO D K,et al.In vivo study of natural killer(NK)cell cytotoxicity against cholangiocarcinoma in a nude mouse model[J].In Vivo,2018,32(4):771-781.DOI:10.21873/invivo.11307.
[17]CHESTER C,FRITSCH K,KOHRT H E.Natural killer cell immunomodulation:targeting activating,inhibitory,and co-stimulatory receptor signaling for cancer immunotherapy[J/OL].Front Immunol,2015,6:601[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667030/.DOI:10.3389/fimmu.2015.00601.
[18]PAUL S,LAL G.The molecular mechanism of natural killer cells function and its importance in cancer immunotherapy[J/OL].Front Immunol,2017,8:1124[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601256/.DOI:10.3389/fimmu.2017.01124.
[19]DAHLBERG C I,SARHAN D,CHROBOK M,et al.Natural killer cell-based therapies targeting cancer:possible strategies to gain and sustain anti-tumor activity[J/OL].Front Immunol,2015,6:605[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663254/.DOI:10.3389/fimmu.2015.00605.
[20]HALL K,SCOTT K J,ROSE A,et al.Reovirus-mediated cytotoxicity and enhancement of innate immune responses against acute myeloid leukemia[J].Biores Open Access,2012,1(1):3-15.DOI:10.1089/biores.2012.0205.
[21]EL-SHERBINY Y M,HOLMES T D,WETHERILL L F,et al.Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo[J].Clin Exp Immunol,2015,180(1):98-107.DOI:10.1111/cei.12562.
[22]ZHAO X,RAJASEKARAN N,CHESTER C,et al.Natural killer cells activated by oncolytic reovirus enhance cetuximab mediated antibody dependent cellular cytotoxicity in an in vitro and in vivo model of colorectal cancer[J/OL].Blood,2015,126(23):3439[2019-02-23].http://www.bloodjournal.org/content/126/23/3439.
[23]MERKT W,STURM P,LASITSCHKA F,et al.Peripheral blood natural killer cell percentages in granulomatosis with polyangiitis correlate with disease inactivity and stage[J].Arthritis Res Ther,2015,17(1):337-347.DOI:10.1186/s13075-015-0851-7.
[24]ZHAO X,CHESTER C,RAJASEKARAN N,et al.Strategic combinations:the future of oncolytic virotherapy with reovirus[J].Mol Cancer Ther,2016,15(5):767-773.DOI:10.1158/1535-7163.MCT-15-0695.
[25]MAITRA R,SEETHARAM R,TESFA L,et al.Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells,and synergizes with irinotecan[J].Oncotarget,2014,5(9):2807-2819.DOI:10.18632/oncotarget.1921.
[26]TWIGGER K,ROULSTONE V,KYULA J,et al.Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway[J/OL].BMC Cancer,2012,12:368[2019-02-23].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537694/.DOI:10.1186/1471-2407-12-368.