Experimental Study On Human NK Cells Carrying Oncolytic Reovirus Combined With Cetuximab Against Colorectal Cancer Cells In Vitro And In Vivo

Objective:To solve problems existing in clinical application of oncolytic reovirus and molecular targeted drug cetuximab,the purpose of this study was to verify the enhanced killing effect of expanded human NK cells carrying reovirus(Reo-NK)combined with cetuximab on colorectal cancer cells in vitro and in vivo.We expect to provide a new strategy for the combined treatment of colorectal cancer in clinic.Methods:1.Peripheral blood mononuclear cells(PBMC)from healthy volunteers were isolated by Ficoll density gradient centrifugation,subsequently NK cells were induced and activated by irradiation-inactivated K562 engineering cells and cytokine interleukin-2(IL-2)in vitro.Cell viability was calculated by trypan blue staining,and the expression of CD3 and CD56 on the surface of NK cells was analyzed by flow cytometry.Real-time cellular analyzer(RTCA)was used to verify the oncolytic activity of reovirus live virus and UV-inactivated virus(UVReo).Expanded NK cells were loaded with reovirus and observed by confocal microscopy,to determining the location of reovirus on NK cells.2.Cell viability assay explored the optimal AB serum concentration that can protect virus-susceptible cell mouse fibroblast L929 cells from reovirus.The oncolytic effect of reovirus was dynamically monitored by CCK-8 when NK cells loaded with reovirus and delivered virus to DLD-1 cells under the blocking of neutralizing antibody AB serum.Real-time fluorescence quantitative PCR(qPCR)was used to assess the relative expression of viral RNA in tumor cells.Cytotoxicity assay were performed to detect the synergetic killing effects of Reo-NK combined with cetuximab on EGFR positive KRAS mutant colorectal cancer cell line DLD-1 and KRAS wild type colorectal cancer cell lines CaCo-2 and HT29.Measurement of the perforin level in supernatant after co-incubation of NK cells and colorectal cancer cells treated with different conditions by ELISA matched paired antibodies assay.3.Establishment of nude mice xenograft model with DLD-1 cells,mice were randomly assigned to four groups.Reo-NK in combination of cetuximab,either Reo-NK alone or cetuximab alone were administered,and control group treated with PBS,each treatment repeated every3 days,for 3 times in total.Anti-tumor efficacy of combination therapy was evaluated by observing and measuring changes in tumor volume of nude mice after treatment in comparison to tumor volume of untreated nude mice,qPCR was used to assess the relative expression of viral RNA in tumor tissues and the liver and kidney of nude mice were taken for pathological analysis,to identifying whether this therapeutic regimen with organ toxicity by HE staining.Results:1.The number of NK cells obtained from PBMCs reached more than 10~9 after 14 days expansion process in vitro,with the cell survival rate of(96.47±2.66)%and purity of(89.03±3.90)%.RTCA verified that the oncolytic activity of reovirus against DLD-1 cells was positively correlated with virus titer,and the non-oncolytic activity of UVReo.Confocal microscopy demonstrated that NK cells retained reovirus on the surface.2.The optimal concentration of AB serum for neutralizing reovirus was 15%.Expanded NK cells could delivery reovirus to DLD-1 cells in the presence of neutralizing antibodies,and the reovirus after delivery still had significant oncolytic activity(P<0.001).Corresponding qPCR result displayed that the expression of viral RNA in DLD-1 cells evidently increased over time(P<0.001).Compared with NK group,pretreatment with reovirus enhanced the killing effect of expanded NK cells,after combined with cetuximab,a stronger antibody-dependent cell-mediated cytotoxicity(ADCC)against DLD-1 cells can be achieved(P<0.001).This combined group of Reo-NK and cetuximab significantly enhanced the cytotoxicity on either KRAS gene wild-type or mutant colorectal cancer cells(all P<0.01),and obviously promoted the perforin release from NK cells(all P<0.01).3.After 3 times of treatment in tumor-bearing nude mice,a evidently tumor growth restriction was observed in Reo-NK combined with cetuximab group,and the tumor weight of combined group was lower than that of Reo-NK alone?cetuximab alone or PBS group(P<0.05).Meanwhile,a great level of viral RNA expression was detected in the tumor tissues of nude mice in the combined treatment group.The results of HE staining showed that the therapeutic regimen did not cause distinct pathological changes in liver and kidney organs of nude mice.Conclusion:Expanded human NK cells carrying reovirus combined with cetuximab exerts a stronger cytotoxicity on either KRAS gene wild or mutant colorectal cancer cells,and this combined treatment regimen significantly delayed the tumor growth in DLD-1 xenografts.