Experimental Study Of Noninvasive Monitoring Of The Gene Therapy With Somatostatin Receptor Imaging

Objective: A bicistronic adenoviral vector encoding the Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) and somatostatin receptor 2a gene (hSSTr2a), which were used as the supervised therapeutic gene and the reporter gene respectively, was constructed and infected the tumor cell and tumor model. A series of in vitro and in vivo studies were carried out to investigate the feasibility of SSTR imaging in monitoring the expression level of HSV1-tk gene and forecasting the antitumor effect of HSV1-tk/GCV treatment,Methods:1. Biodistribution and SSTR imaging of tumor model with ^99mTc-HYNIC-TOCA and ^99mTc-P829Tyr³-Octreoteate (TOCA) was labeled with ^99mTc using HYNIC as bifunctional chelator, EDDA and tricine as coligand. The nude mice bearing human small cell lung cancer underwent static whole body planar imaging after the injection of ^99mTc-HYNIC-TOCA or ^99mTc-P829. ROIs were drawn and the radioactivity ratios of T/NT were calculated in every planar imaging. The dynamic biodistribution study was operated in Kunming mice and the percent uptake ratios (%ID/g) of two imaging agents in different tissue at different time were measured.2. Expression of the recombined bicistronic adenoviral vector Ad5-TIS in the human lung adenocarcinoma cellBy inserting HSV1-tk and hSSTr2a gene into the upstream and downstream of the internal ribosome entry site (IRES) respectively, a recombined bicistronic adenoviral vector (Ad5-TIS) was constructed. The vector was used to infect the human lung adenocarcinoma cell line A549, which was negative for hSSTr2a, withmultiplicity of infection (MOI) 10.0. The expression of the transfected genes was tested by the reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunocytochemistry (ICC).3. Noninvasive monitoring of the expression of Ad5-TIS in tumor with the somatostatin receptor imagingWhen A549 cell was infected with Ad5-TIS at multiplicity of infection (MOI) 0.0, 0.4, 2.0, 10.0, 20.0 or 40.0, the semi-quantitative RT-PCR and the cell uptake experiment with the SSTR specific ligand, ^99mTc-HYNIC-TOCA, were performed to evaluate the expression level of the transfected genes. In the subcutaneous A549 tumor xenografts, 0.0, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸ or 1×10⁹I.U. of Ad5-TIS was injected into the tumor. 48h later, the expression level of the transfected genes was estimated by semi-quantitative RT-PCR and the SSTR imaging.4. The anti-tumor effect of HSV1-tk/GCV in tumor cellThe human lung adenocarcinoma cell line A549 was infected with Ad5-TIS atMOI 0.0, 0.4, 2.0, 10.0, 20.0 or 40.0 for 48h. The infected cell was treated with GCV in different dose (0, 1, 10 and 100μg /ml) for 24h, and the proliferation inhibit ratio (IR) was obtained with MTT test. The Annexin V-FITC/PI flow cytometry (FCM) was used to calculate the ratio of the apoptosis cell and the necrotic cell (KR) in the infected cell after treated with 10μg /ml GCV.5. The anti-tumor effect of HSV1-tk/GCV in tumor modelIn the A549 tumor bearing model, 1×10⁸I.U. of Ad5-TIS was injected into the tumor, and the other group of tumor bearing model was treated with 0.9% sodium chloride in same volume. 48h later, a 14day intraperitoneal GCV treatment (50mg/kg/d) was performed in the tumor model. The volume and the weight of tumors were measured at the end of the treatment. The apoptosis of the tumor tissue was tested by the terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling (TUNEL) method.Results:The radiochemical purity of ^99mTc-HYNIC-TOCA could reach 94.2±1.9% and keep to 93.5+2.2% after 6h at room temperature. In the images of tumor bearing mice, two imaging agent were both mainly concentrated in kidney and tumor. In early images, the ratios of tumor-to-contralateral limbs (T/C) in ^99mTc-HYNIC-TOCA group were higher than which in ^99mTc-P829 group. The greatest ratios of T/C in two groups (7.45 ± 0.40 and 5.71 ±0.69) were gained until 4h post-injection. In kunming mice, the %ID/g of kidney in both groups were the highest and the half-clearance time in blood were less than 60min.The uptake ratio of ^99mTc-P829 in liver was significantly greater than the ratio of ^99mTc-HYNIC-TOCA (t=2.17, P<0.05).When A549 cell was infected with Ad5-TIS at MOI 10.0, the specific electrophoresis bands of HSV1-tk and hSSTr2a were obtain by RT-PCR. In the result of western blotting of hSSTr2a, the specific band only appeared in the infected group. The specific fluorescence could be observed in the cytoplasm and on the cell membrane by the ICC of hSSTR2. But in the A549 without infection, all results were negative.The results of the semi- quantitative RT-PCR showed that, in the infected tumor cell and tissue, the expression level of the transfected genes was promoted with the increasing titer of the Ad5-TIS and the expression of two genes kept good coherence (cell: r²_S/T=0.981 , P<0.01; tissue: r²_S/T=0.797, P<0.05). With the augment of Ad5-TIS, the cell uptake ratio of ^99mTc-HYNIC-TOCA was increased from 4.2 ± 0.46% to 13.8 ± 1.14%, and the radioactivity ratio of tumor to Contralateral (T/C) was also increased from 1.72±0.22 to 5.65±0.30 in the SSTR imaging of the tumor model. Both of these two ratios have favorable correlation with the expression level of HSV1-tk and hSSTr2a in cell or tissue (cell: r²_T/U=0.965, r²_S/U=0.959, P<0.01; tissue: r²_T/I=0.942, r²_S/I=0.889, P<0.01).When A549 cell was infected with Ad5-TIS at MOI 0.4-40.0, the specific electrophoresis bands of HSV1-tk and hSSTr2a were obtain by RT-PCR. But in the MOI 0.0 group, both results were negative. The expression level of the transfected gene, the uptake ratio of ^99mTc-HYNIC-TOCA, the IR in MTT and the KR in FCM were all promoted with the increasing titer of the Ad5-TIS. The KR has positivecorrelation not only with the expression of two genes respectively (r²_T/K=0.871, r²_S/K=0.854, P<0.01) but also with the cell uptake ratio (r²_U/K=0.933, P<0.01).In the infected tumor model, the expression of two genes was proved by RT-PCR, and the radioactivity ratio of tumor to Contralateral (T/C) in SSTR imaging was significantly higher than that of uninfected group (T/C₍₊₎=3.26±0.24, T/C_(-)=1.72±0.22, P<0.01). Consisted with the results of SSTR imaging and RT-PCR, the antitumor effect in the infected group was distinct with the GCV treatment. The growth of the tumor was slower (V₍₊₎=498.50± 42.68mm³, V_(-)=1470.00± 80.02 mm³, P<0.01; W(+)=624.33 ± 63.51mg, W(-)=1803.00 ± 223.70mg, P<0.01), and the apoptosis in TUNEL test was remarkable.Conclusion: The bicistronic recombined adenoviral vector encoding hSSTr2a and HSV1-tk gene could be co-expressed effectively in tumor cell and tumor model. Cooperating with GCV, the HSV1-tk gene could exert its therapeutic effect. The SSTR reporter gene system could be applied to monitor the expression of the transfected suicide gene and to forecast the antitumor effect of the HSV1-tk/GCV.