The Radioimmunoimaging Of Colon Cancer With^99mTC Labeled AC133.1Monoclonal Antibody

Contents:1. Flow cytometry was used to assess the CD133expression level of three clone cancer cell lines;2. AC133.1was conjugated with SHNH and labeled with99mTc. The characteristic of99mTc-SHNH-AC133.1was evaluated;3. Tumor models were established. SPECT imaging and biodistribution studies were performed;4. Flow cytometry analysis and immunohistochemistry of tumor tissues were performed to analyze the CD133expression. And the immunoactivity of labeled antibody was evaluated by immunofluorescence staining.Methods:1. Flow cytometry analysis of CD133expressionCD133expression level on tumor cells was evaluated by flow cytometry. HCT116、 Lovo and DLD1cells were digested. After washing three times with PBS,1×106of each cell lines were suspended in100μl MACS running buffer.1μl blocking reagent were added to block the nonspecific binding to non-target cells. And then cells were incubated with1μl phycoerythrin (PE)-conjugated CD133/2(293C) antibody or mouse IgG2b-PE (isotype control) at4℃for10min. Each sample was independently analyzed on FACS Canto Ⅱ.2. The radiolabelling, purification, stability study and Cell uptake study in vitroAC133.1monoclonal antibody is prepared by inoculating the bybridoma cells (HB-12346) into abdominal cavity of female Balb/c mice to induce antibody containing ascites. Ascites were harvested and purified. Mouse IgG were used as the control antibody. AC133.1antibody and control IgG were conjugated with succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH), and labeling with99mTc. The mixture was purified by PD-10column. The radiochemical purity was determined by instant thin-layer chromatography (ITLC). The stability of99mTc labeled AC133.1and control IgG was measured in human serum at37℃for several time points by ITLC. The cell uptake study was performed by incubating cells with purified99mTc labeled antibody. The blocking study was performed to incubate cells with500-fold (5μg) of unlabeled AC133.11h before addition of99mTc-SHNH-AC133.1.3. SPECT imaging and biodistribution studyXenograftes of human HCT116, Lovo and DLD1cells were established. Each animal was intravenously injected with37MBq (1mCi)"mTc-SHNH-AC133.1or control99mTc-SHNH-IgG SPECT imaging were performed at4,12,24and36h postinjection. For the blocking, each tumor model in a group of four animals were predosed with approximately60-fold (600μg) unlabeled AC133.1antibody. After SPECT imaging, organs of interest were harvested, weighted and counted in a y-counter. The percentage of injected dose per gram of tissue (%ID/g) was calculated and the physical decay was corrected.4. Flow cytometry, immunohistochemistry and confocal immunofluorescence staining of tumor tissuesFlow cytometry and immunohistochemistry staining of tumor tissues were performed to evaluate the CD133expression level and to further validate the in vivo results. And immunofluorescence staining was performed to evaluate the immunoactivity of labeled antibody.Results:1. Flow cytometryThe surface CD133expression level of tumor cells was analyzed by flow cytometry. Among the three cell lines, CD133expressed at a high level on HCT116cells (84.44±0.40%, n=3), followed by Lovo cells (68.77±0.20%, n=3), and DLD1cells presented the lowest level of expression (5.1±0.12%, n=3).2. Radiolabelling and cell uptake studyAC133.1and control IgG could be labeled efficiently with high radiochemistry purity (97.7±2.4%and98.7±1.3%n=3, respectively) and specific activity (4.07MBq/μg and3.33MBq/μg). The99mTc labeled mAb was stable in human serum.The percentage of99mTc-SHNH-AC133.1bound to HCT116and Lovo cells after incubation4h were15.38±4.94%and13.85±0.90%, which were significant higher than DLD1cells (3.37±0.31%)(P<0.05). The binding specificity was also confirmed by the marked decrease binding on HCT116cells and Lovo cells by blocking with excess unlabeled AC133.1. In addition, the cell uptake of the control99mTc-SHNH-IgG were very low.3. SPECT imaging and biodistributionHCT116and Lovo tumor lesions were clearly visualized after12h. Tumor uptake increased gradually in the two tumor models. In contrast, DLD1tumors, with low level of CD133expression, were barely detectable at all time points. The radioactivity signal of tumor area exhibited background level in control IgG group. Biodistribution studies showed high tumor uptake of99mTc-SHNH-AC133.1in HCT116and Lovo xenograftes (8.82±0.73%ID/g and7.37±0.26%ID/g, respectively), while DLD1tumors with low level of CD133uptook only3.40±0.80%ID/g. Moreover, the tumor uptake of99mTc-SHNH-AC133.1in the two positive tumor models was significantly reduced by pre-saturation of the receptor via pre-injection of excess unlabeled antibody. The uptake in HCT116and Lovo tumors were3.74±0.84%ID/g and2.84±0.60%ID/g, respectively.4. Flow cytometry, immunohistochemistry and confocal immunofluorescence stainingCompared to the cell lines, the expression level of HCT116、Lovo and DLD1tumor cells isolated from the exnografts showed lower CD133expression (46.31±0.23%,26.57±0.62%and4.93±0.05%, respectively). Immunohistochemistry staining reveled that HCT116and Lovo tumors had high level of CD133expression, and DLD1tumors had no obvious expression. Immunofluorescence staining showed AC133.1mAb can specifically target CD133receptor in vivo without losing its immunoreactivity by conjugating with SHNH.Conclusion:Our study showed99mTc labeled AC133.1had high radiochemistry and specific activity. It exhibited high tumor uptake in CD133positive tumors. The high specificity and excellent tumor targeting properties of99mTc-SHNH-AC133.1may provide a new perspective for tracking or locating cancer stem cells. Introduction:EphA2is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe,99mTc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated.Methods:The SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by99mTc labeling. The radiochemistry purity and specific activity were measured. Immunofluorescence staining was carried out to detect the expression of EphA2in A549lung cancer cells and OCM-1melanoma cells. Saturation binding experiments were performed by incubating A549cells with increasing concentrations of radiolabelled peptide in vitro. The Scatchard plot was drawn and the KD value were calculated. To test the probe in vivo, nude mice bearing either A549or OCM-1derived tumors were established, injected with99mTc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo y-counting of dissected tissues from the mice was also performed to evaluate biodistribution.Results:99mTc-HYNIC-SWL was prepared with a high radiochemistry purity (98.23±0.39%, n=3) and specific activity (37MBq/μg).Immunofluorescence staining showed that A549cells intensively expressed EphA2, while OCM-1cells had little expression.99mTc-HYNIC-SWL displayed high binding affinity with A549cells (KD=2.6±0.7nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549tumors (1.44±0.12%ID/g) than in OCM-1tumors (0.43±0.20%D3/g) at1h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58±0.20%ID/g) was seen. These data suggest that99mTc-HYNIC-SWL specifically targets EphA2in tumors.Conclusions:The expression of EphA2can be noninvasively investigated using99mTc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of99mTc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging.