The Effects Of¹⁸⁸Re Labelled Aptamers Of U87-EGFRvⅢ Cells In Glioma Animal Model

Systematic evolution of ligands by exponential enrichment(SELEX), which was established by Gold and Ellington from the U.S. in1990, is an in vitro screening technology for target molecule screening to obtain nucleic acid ligands with high affinity and specificity. Cell-SELEX technology, which is developed on the basis of SELEX technology, uses intact cell as target, can screen specific target molecules from cells with unknown molecular organizations thus improves the efficiency of screening and expands the scope of target molecules greatly. Aptamers that bind specifically to tumor cells can be screened out by Cell-SELEX technology, so Cell-SELEX technology has a wide range of applications in basic research, clinical diagnosis and treatment of tumor.Glioma is a common malignant intracranial tumor. As glioma presents infiltrative growth pattern, the effect of traditional treatments including surgery, radiotherapy and chemotherapy is extremely limited. It is reported that the incidence of glioma account for70%of all brain tumors, among them Glioblastoma multiforme (GBM) is the most common type. It has a poor prognosis(5years survival ratio is less than3%) and is difficult to cure, often tends to relapse. Due to a variety of factors such as chemotherapy resistance and blood brain barrier impermeable to drugs, so for the efficacy of traditional treatment methods is limited. Thus research on targeted therapy against glioma is of great social and clinical significance. EGFRvIII is a mutant results from deletion of2-7exons in the extracellular region of epidamal growth factor receptor(EGFR), and is relate to tumor invasiveness and tumorigenicity. EGFRvIII is only expressed in malignant tumor cells and not expressed in normal cells and tissues, thus it is a good therapeutic target of tumor. Using Cell-SELEX to select aptamers against U87cells that overexpress EGFRvIII (U87-EGFRvIII cells) is important for the study of targeted therapy against glioma, and the Cell-SELEX technology is simple, fast and economic.Aptamers own some features such as small molecule weight, rapid plasma clearance rate, high tissue penetration, low immunogenicity, good stability and so on. It is reported aptamers can be used effectively on targeted therapy, detection and diagnosis of diseases. The first aptamer drug "Mucagen" has been approved by the FDA in2005, furthermore aptamer AS1411against nucleolar proteins is under clinical trials, all these indicate the important role of aptamers in treatment of diseases. With the progress of SELEX technology, aptamer has become an increasingly important research tool for bioscience and medical research. So the use of Cell-SELEX technology on target screening of glioma cells and studies about glioma clinical diagnosis, treatment and tumor basic research has a great application value and prospect, the obtained specific aptamers will also become effective molecular recognition tools in basic research and applied research.This experiment research contains three aspects:Firstly, we screened aptamers for U87-EGFRⅧ cells and identified the binding specificity and affinity of aptamers at cellular level; Secondly, we preliminarily observed the inhibition effect on tumor growth of selected aptamer U2in animal models of tumor-bearing nude mice; Thirdly, we studied the metabolism and target effect of188Re labelled aptamer in vivo.(1) We screened aptamers for U87-EGFRvIII cells by Cell-SELEX technology. In the fourth round we began to apply the subtractive SELEX technology, using U87MG as control cells to screen aptamers. The amount of DNA library was inputed according to the certain proportion strictly during the process of screening, and every inputed sub-library must be identified by denaturing polyacrylamide gel electrophoresis. As number of screening rounds was constantly increasing, time for washing and number of washing of cells were increasing gradually, while incubating time of the cells with library was decreasing gradually. We applied the PCR technology to obtain double-stranded DNA, used the mothed of streptavidin-biotin magnetic bead separation to separate single-stranded DNA and obtain FITC-labelled ssDNA as the next screening library. The reaction amounts of various reagents and the reaction system were fumbled gradient by gradient in the PCR process, finally a band with a single strip, high purity and molecular weight of76bp was identified as the purpose strip. A total of11cycles were performed, the obtained ssDNA of last round was then cloned and sequenced. Online software was used to analyze sequence homology, and four sequences with identical homology were found out as the selected aptamers, which were named aptamers U2, U8, U19and U31, respectively.Secondly, we synthetized the5’ end FITC-labelled aptamers U2, U8, U19and U31from the company and identified their binding specificity and affinity, FITC-labelled original library GN was used as control. We detected the enrichment of bound oligonucleotide as the number of screening rounds was increased by flow cytometry; and observed the binding intensity of aptamers with target cells U87-EGFRvIII by proteinase K digesting U87-EGFRvIII cells for3min and10min. We found that the oligonucleotides enriched as the number of screening rounds increased. Flow cytometry showed the binding peak positions of aptamers with U87-EGFRⅧ were shifted to the right compared with original library GN group and blank control group, indicates that the aptamers had enriched. As proteinase K can destroy cell surface, therefore, destruction of the cell surface through proteinase K digesting by different levels of3min and10min would affect the bond strength of aptamers with U87-EGFRⅧ in certain extent. The impact was getting more and more greater as the digestion time increased, that is, the greater the peak positions were shifted to left.Finally, we used pull-down experiment and western blot analysis to identify the target protein through which aptamers combined with U87-EGFRvIII cells. Result showed that aptamers U2and U8could specifically bind to the target protein of U87-EGFRvIII, compared with original library GN group and U87MG cells group. In the binding experiment of3’end biotin-labelled aptamer U2, cell lysate was analysed by Western blot, and EGFR antibody was used as control. Result showed that aptamers U2could bind specifically to the target protein of U87-EGFRⅧ.(2) We conducted experiments about inhibition effect of aptamer U2on tumor growth in vivo and tumor inhibitory effect, target effect and metabolism of Re labelled aptamer U2in animal models of tumor-bearing nude mice.Firstly,4to6weeks old male mice were randomly divided into four groups, with7mice for each group. They were blank group, in vivo jet-PEITM reagent treated group, original library GN treated group and U2aptamer treated group, respectively. We injected U87-EGFRvIII cell suspensions subcutaneously to establish the animal models of glioma-bearing nude mice, then we injected U2and GN intratumorally with in vivo jet-PEITM transfection reagent, and we preliminarily observed inhibition effect of aptamer U2on glioma growth. We found that tumors grew very slowly and there were no significant changes on tumors size in the aptamer U2treated group; tumors grew rapidly and there was necrotic tendency from the first administration to the time of peeling off tumors (two weeks) in blank group; there were no significant differences on tumor size and volume compared in vivo jet-PEITM reagent treated group to original library GN treated group(the Volume V=n/6xaxb2,a is length,b is width).There was no statistically significant difference among groups(n=7,P>0.05) on tumor size when tumors formed(2weeks after U87-EGFRvIII cell suspensions injection).There was significant difference between U2treated group and blank group,in vivo jet-PEITM treated group(n=7,P=0.000)on tumor weight and volume1week after treatment by One-way ANOVA analysis,and there was difference between U2treated group and GN treated group(n=7,P<0.05).These results indicated that aptamer U2had certain inhibitoty effect on glioma growth.Next we injected188Re Iabelled U2and GN intratumorally’ with each mice200pmol (approximately2μg).188Re radioactivity of the labelled aptamer U2,GN and free188Re was0.075mCi and1abelled rate was72%.There was significant difference between188Re-U2group and188Re-GN group,free188Re group,blank group(n=9,P <0.01)on tumor weight and volume by One-way ANOVA analysis,which indicated188Re labelled aptamer had inhibitory effect on glioma growth in tumor-bearing nude mice animal model.This experiment also showed that aptamer U2had the capability to carry188Re for the treatment of glioma.It is likely that aptamer u2could affect expression of EGFRvIII protein on tumor cell surface to suppress cell proliferation, thus inhibits tumor growth.Because aptamer U2was screened against U87-EGFRⅧ cells,the mechanism of how aptamer affects glioma cells growth needs to be further studied.We injected intratumorally188Re labelled aptamer U2,GN and free188Re nuclide and detected the metabolism of aptamer and free188Re within3hours. SPECT showed that188Re.GN and free188Re could be detected at the tumor site immediately after injection,and radionuclide imaging was gradually weaken after0.5hour,and was undetectable after1hour；imaging in other organs of nude mice such as liver,bladder can be detected and were gradually weaken after3hours,finally it can only be detected in bladder. SPECT showed similar results of188Re labeled U2, GN and free188Re injected into mice through caudal vein. These results suggested that188Re labelled U2, GN and free188Re excreted mainly through the urinary system. We injected a certain amount of188Re labelled U2, GN and free188Re through caudal vein and perform SPECT detection to study target effect. Results showed through blood circulation188Re-U2could be detected within the tumor1hour later, but188Re-GN and free188Re could not be detected within tumor. Isolated Organs SPECT also showed similar results, and imaging of Re-GN and free188Re could be detected in other organs weakly, especially in liver. It is possible due to the reason that liver is an organ rich in blood supply. The above results preliminarily indicate that the selelcted aptamer can specifically target glioma formed by planting U87-EGFRvⅢ cell suspensions.In summary, we have used the streptavidin-biotin bead to isolate ssDNA and used Cell-SELEX technology to screen aptamers for the U87-EGFRvⅢ cells. We identified the binding specificity and affinity of obtained aptamers. These aptamers have certain inhibitory effect on tumor growth in animal model of tumor-bearing nude mice;188Re labelled aptamers can specifically target glioma and inhibit tumor growth in tumor-bearing nude mice. These results provide experimental evidence that aptamers can be used for targeted therapy of glioma, and may also provide an experimental basis for targeted therapy and metabolic safety studies of glioma.