A Preliminary Study On The CIK And NK Cells Amplified In Vitro Combined With Cetuximab Enhanced By Reovirus Agaist Colorectal Cancer

Objective: To study the effects of natural killer cells(NK)and cytokine-induced killer cells(CIK)amplified in vitro in combination with cetuximab against EGRF-positive human colorectal cancer cells,and if reovirus can enhance anti-tumor effect of CIK cells and NK cells combined with cetuximab,so as to provide a new therapeutic strategy for clinical treatment of colorectal cancer.Methods: Peripheral blood mononuclear cells(PBMC)were isolated from healthy blood donors to induce CIK cells and NK cells,CIK cells and NK cells were collected at the 14 th day,and flow cytometry was used to analyze the phenotypes of CIK cells and NK cells as well as the expression of EGFR of DLD-1 and Caco-2.Measurement of IFN-? and TGF-? production in supernatant by ELISA;The cytotoxicity of NK cells was measured by RTCA and LDH release assay,respectively.Tissue culture infective dose(TCID50)and methylcellulose plaque assay were adopted to measure the titer of reovirus,and CIK cells and NK cells were infected by reovirus at different titers.Real-time cellular analyzer(RTCA)was used for dynamic monitoring of the effects of CIK cells and NK cells combined with cetuximab on killing colorectal cancer cells(DLD-1 cells and Caco-2 cells),as well as the effects of CIK cells and CIK cells infected by reovirus in combination with cetuximab on killing colorectal cancer cells(DLD-1 cells and Caco-2 cells).Results: The percentage of CD8+T(67.5±16.5%),CD3+CD56+NKT(20.1±13.7%)and CD16+CD56+NK(7.0±6.6%)cells were significant increase after 14 days culture in vitro than before.The level of IFN-? increased from 34.8±35.4 ng/L to 2041.8±697.9ng/L(P<0.01),TGF-? decreased from 1293.8±633.9ng/L to 116.7±26.8ng/L(P<0.01).High levels of NK cell numbers with up to 86.2% purity were obtained after 14 days culture in vitro.EGFR were expressed on tumor cell lines,which were 81.6% and 82.9%,respectively;The titer of reovirus was 2.528×109pfu/L and 1.36×109 pfu/L respectively by the method of TCID50 assay and plaque assay.The cytotocity of NK cells measured by RTCA showed a similar trend with LDH released assay.The cytotoxicity of CIK cells against DLD-1 and Caco-2 cells were 16.04±0.87% and 27.67±3.10% respectively,the cytotoxicity increase to 31.56 ± 3.55% and 35.96 ± 1.73% when CIK cells combined with cetuximab.Cytotoxicity of NK cells were further increased when combined with cetuximab compared with the cytotoxicity of NK cells against DLD-1 and Caco-2 cells.The tumor cell growth inhibition curves of CIK cells and NK cells infected by reovirus of different titers in combination with cetuximab was more obvious.Conclusion: CIK cells and NK cells combined with cetuximab have much higher killing effect on both KRAS cells such as wild type DLD-1 and mutant Caco-2 cells.Reovirus can further enhance antitumor effects of CIK and NK cells combined with cetuximab,so it may lay a foundation for future clinical research.