| ObjectiveIn this study,the expression level of both markers(CD133 and CD44)in different colon cancer celllines was detected.The difference of cells uptaking131I-AC133mAbin different cells was perfomed in vitro studies.The ability of labeled antibodies targeting CD1 33-positive tumor cells in tumor xenografts was studied.And we would explore the feasibility of radioimmunoimaging tracing cancer stem cells in vivo.It would provide experimental evidence for radioimmunotherapy of colorectal cancer.Methods1.The expression levels of CD133 and CD44 in colon cancer cells(HT29,HCT116 and DLD-1 cell)were detected by Flow cytometry.And we performed the Western-blot analysis on the colon cancer cells.2.Transwell experimental study would be conducted to analyze the correlation between cell invasiveness and CD133 and CD44 expression level.3.1311-labeled monoclonal antibody was specific for CD133 molecule which is one of the surface markers colon tumor stem cells.131I-labeled monoclonal antibody was purified on the application of PD-10 desalting column.The labeling efficiency,radiochemical purity and stability of 131I-labeled AC 133 mAb were carried out.4.Cell uptake experiments were performed in vitro.And the relationship between the CD133 molecule expression levels and cellular uptake in the three cellswould be analyzed.5.The experimental group and blocking group were established in tumor xenografts model,respectively.131I-labeled monoclonal antibody was injected into the nude mice models on the tail vein.SPECT/CT imaging and biodistribution experiments were performed at different points in time.And the characteristics of the labeled antibody in vivowereanalyzed.6.Autoradiography studies and immunofluorescence analysiswere carried out in vivo.And the relationship between the distribution of labeled antibodies and CD133 expression in vivo would be analyzed.Results1.The expression levels of CD133 molecules in the colon cancer cell line HT29 and HCT116 cells were 91.33±1.66%,90.83±2.47%,respectively,which were significantly higher than the expression level of the CD 133 molecule in DLD-1 cells(3.87±0.57%,P<0.01).However,the expression levels of CD44 in the colon cancer cells(HT29,HCT116 and DLD-1 cells)were 24.53±8.35%,6.0±0.82%and 8.87±3.9%,respectively.2.Western-blot analysis showed that the both cells(HT29 and HCT116 cells)highly expressed CD 133.And it was low in DLD-I cells.3.The cell Transwell experiments showed higher amounts HT29 cells and HCT116 cells were observed through the microscopic.But there was only a small amount of DLD-1 cells.4.The radiochemical yield of 131I-labeled AC133 mAb was 68.37 ± 2.45%.The radiochemical purity of the labeled antibody was 91.18 ± 3.41%after purification,and the labeled antibody had good stability in vitro.Statistical analysisrevealed thatthe peak of uptaking 131I-AC133 mAb in HT29 and HCT116 cells was 29.63± 3.92%and 33.99± 6.65%at the time of4h,respectively.The blocking group uptake ratios were 3.22 ± 0.18%and 3.67 ± 0.56%,respectively.There was a significant statistical difference between the experimental group and the blocking group(P<0.01).The uptake ratio in DLD-1 cells with 131I-AC133 mAb was 2.89 ±0.90%at the time of 4h,whichwas significantly lower than that of the HT29 and HCT116 cells(P<0.01).5.The results on SPECT/CT imaging showed that HT29 and HCT116 tumor bearing mice injected 131I-AC133 mAb at 7 days had the highest uptake.Meanwhile,the results on biodistribution studies showed that the radioactivity uptake in the tumor was 5.49 ± 0.42%ID/g and 5.04± 0.05%ID/g,respectively.They were significantly higher than that in the DLD-1 tumor bearing mice(1.20±0.11%ID/g,P<0.05).The ratios of tumor-to-muscle(T/M)at 7 day were 11.34±0.09(HT29),11.31 ± 1.05(HCT116)and 3.43±0.53(DLD-1),respectively.The good consistency between the radioactivity distribution and immunofluorescence analysis in different tumor tissues was maintained.6.The tumor tissue uptake was no obvious at each time point in blocking experiments.The radioactivity biodistribution in the blocked tumor tissue was significantly lower than that in the test group.7.HT29 and HCT116 tumor image could be blocked at each time point examined.The biodistribution study revealed that radioactivity uptake in tumor tissue was also low in blocking groups.At 7 day post-injection of labeled antibody,the radioactivity uptake of blocking group in HT29 and HCT116 tumor xenograftswere 2.18 ± 0.38%ID/g and 2.06 ± 0.35%ID/g,respectively.8.The immunofluorescence analysis and autoradiography studies revealed that the radioactivity distribution of 131-AC133mAb was consistent with the expression of the CD133 in vivo.ConclusionThe expressionlevels of CD133 in colon cancer cell lines(HT29 and HCT116 cells)were relatively higherthanthat in DLD-1 cells.The expression levelof CD44 moleculein HT29 cells washigherthan that in HCT116 and DLD-1 cells.Meanwhile cells uptake study,SPECT/CT tomography,biodistribution and autoradiography were consistent with the difference.And the targeting antibody 131I-AC133 mAb was specific for the CD 133 antigen.It wasfeasiblefor radioimmunoimaging on the evaluation of CD 133-positive tumor cells with molecular probes,which provided an experimental basis for radioimmunoimaging and radioimmunotherapy incancer stem cells. |
PDF Full Text Request