| Background: Oncolytic virus is a type of naturally occurring or artificially transformedvirus which can selectively infect and lyse tumor cells but has little effect on normalcells. Because cell cycle related transcription factor E2F-1is highly expressed in certaintumors, oncolytic virus targeting this gene was tested in animal models such as bladdercancer, gastric cancer, liver cancer etc. This is also a good choice for gene therapy ofcolorectal cancer. However, experimental results of ONYX-015, the first oncolytic virusused in clinical trials, showed that inhibitory effect as monotherapy is not obvious, andit need to improve the anti-tumor effect in combination with other therapies. In recentyears, as adoptive immunotherapy has made considerable progress, it is regarded as asafe and effective method can be combined with oncolytic viruses. On the other hand,researchers inserted immune regulatory gene into the viral genome so that the viruscould release immunomodulatory factors while directly killing tumor cells. This"virus-gene" therapy optimized anti-tumor immune response and became new strategicdirection for tumor treatment. Interleukin15, as a promising candidate for tumorimmunotherapy, has not been widely used in the construction of the oncolytic virus.Oncolytic virus expressing IL-15combined adoptive immunotherapy simultaneously orsequentially may further enhance the anti-tumor effect.Objective: To research E2F-1expression in colorectal cancer, construct oncolyticadenovirus targeting E2F-1gene with IL-15expressing simultaneously and explore thefeasibility and effectiveness of combined therapy with novel oncolytic virus andadoptive immune cells (CIK, CTL) in treatment of colorectal cancer.Methods:1）Western blot was used to determine the expression of E2F-1in colorectalcancer cell lines SW620, SW480and LS174T. Immunohistochemistry for E2F-1wasperformed in28cases of rectal cancer,32cases of colon cancer,24cases of gastriccancer and normal tissue.2）Three adenoviral vectors were constructed through plasmids recombinantion and viral packaging, including E2F-1promoter oncolytic virusAd-E2F, Ad-E2F/IL15(expressing IL-15simultaneously) and CMV promoterreplication-defective adenovirus Ad-EGFP(as control virus).3）CIK and CTL cells wereisolated and amplified from healthy volunteer’s PBMC and cell phenotype was testedby flow cytometry.4）Fluorescence microscope was used to observe Ad-EGFP infectedcells. The effect on cell proliferation after Ad-E2F infection was detected by MTT assay.The effect on cell cycle of tumor cells was detected by flow cytometry. The cytotoxicityof treatment combined CIK cells with Ad-E2F was detected by LDH release assay invitro. A nude mouse model of orthotopic rectal cancer was established. In vivocytotoxicity assays using a combination of Ad-E2F and CIK cells was observed in thismodel.5）The effect on cell proliferation after virus(Ad-EGFP, Ad-E2F or Ad-E2F/IL15)infection was compared by MTT assay. ELISA was used to detect IL-15expressionlevels in cell supernatant. Different anti-tumor effect of adenovirus was compared insubcutaneous tumor model. The virus’ anti-tumor effect was observed in mice by PETscan. Cytotoxicity of combined treatment with CTL cells and Ad-E2F/IL15wasdetected by LDH release assay in vitro, and was observed in subcutaneous tumor model.Results:1）Western blot analysis revealed the expression of the E2F-1gene in theSW620, SW480, and LS174T human colorectal adenocarcinoma cell lines. Accordingto immunohistochemistry analysis, E2F-1positive rate in rectal cancer, colon cancerand gastric cancer was67.9%(19/28),53.1%(17/32) and83.3%(20/24) respectively,and that was higher than in normal mucosa (all p＜0.01). After endonuclease and PCRidentify, recombinant adenovirus Ad-EGFP, Ad-E2F and Ad-E2F/IL15was constructedsuccessfully and achieved ideal titer.3）Cell phenotype of CIK and CTL meet therequirements for the following experiment.4）Type5adenovirus Ad-EGFP couldefficiently infect tumor cells but had little impact on the CIK cells. Ad-E2F could inhibitthe proliferation of high-E2F-1expression tumor cell line SW620, and there was anobvious dose-response relationship, but it had little effect on normal cell line Wi38andCIK cells. Cell cycle changed in SW620cells after the cells were infected with Ad-E2F:the proportion of cells reduced in G1phase and significantly increased in S phase.Compared with the application of either method alone, combined therapy with CIK cellsand Ad-E2F produced stronger cytotoxic effects on SW620cells(p＜0.01). Orthotopic animal model of colorectal cancer was constructed successfully and the tumor initiationrate was80%. In vivo experiments showed that combined therapeutic approachinvolving an intratumoral injection of Ad-E2F and an intravenous injection of CIK cellsproduced a significantly greater inhibition of cancer growth. Also, t test showed that thedifference between combined treatment group with PBS group (p<0.0001), or CIKgroup (p=0.0002) or Ad-E2F group (p<0.0001) was statistically significant.5）Ad-E2F/IL15could lyse tumor cells while expressing IL-15effectively. However, therewas no significant effect on the proliferation of normal cells and CTL cells. In vivoexperiment showed that Ad-E2F/IL15group had greater anti-tumor effect than eithergroups (PBS&Ad-E2F/IL15, p=0.0002; Ad-EGFP&Ad-E2F/IL15, p=0.0008; Ad-E2F&Ad-E2F/IL15, p=0.0054). Combined therapy with Ad-E2F/IL15and CTL couldinduce enhanced antitumor activity both in vitro and in vivo.Conclusions:1) E2F-1gene is highly expressed in colorectal cancer and it can be usedas targets for cancer gene therapy.2）E2F-1promoter oncolytic viruses can lyse tumorcells, but had little effect on normal cells, while the expression of IL-15can improve theanti-tumor effect.3）Type5adenovirus has weak ability to infect or suppress adoptiveimmune cells, so these two methods can be used together.4）Combined therapy withoncolytic virus (Ad-E2F or Ad-E2F/IL15) and adoptive immune cells (CIK or CTL) hassynergistic anti-tumor effect. |
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