| Section one A radioimmunotherapy study for targeting cancer stem cells with 131I labeled monoclonal antibodies of dual-targets in human colorectal cancer xenograftsObjectiveThe cancer stem cells(CSCs)biomarkers CD133 and CD44 were utilized as therapeutic targets in human colorectal cancer xenografts to evaluate the efficacy of radioimmunotherapy(RIT)with radioiodinated monoclonal antibodies(mAbs)for targeting CSCs and investigate the relative advantage of the utility combined 131I-CD133 mAb and 131I-CD44 mAb(radioiodinated mAbs cocktail).MethodsThe radioiodination of antibodies was performed using the solid-phase iodogen method.HT29 cells(2×106)were inoculated subcutaneously into the right lower limbs of 6-week-old male BALB/c-nu/nu mice to establish tumor xenografts models.Xenografted mice were randomly divided into eight groups.The radioiodinated single-antibody groups were intravenously injected with 14.8 MBq/100 ?l radioiodinated mAbs or IgG.The group receiving the radioiodinated mAbs cocktail was injected with with 7.4 MBq 131I-CD133 mAb and 7.4 MBq 131I-CD44 mAb.The groups receiving unradioiodinated mAbs were injected with 20 ?g/100 ?l of the single mAb(the dose calculated from the radiospecific activity of 131I-CD133 mAb and 131I-CD44 mAb).The group receiving the unradioiodinated mAbs cocktail was injected with 10 ?g of each mAb.The saline group was injected with 100 ?l saline.After administration,tumor size and body weight were measured every 3 days,and survival status was monitored daily.Tumor growth curves and survival curves were generated to assess treatment efficacy.At 30 day after treatment,cell apoptosis in xenografts were measured by TdT-mediated dUTP-biotin nick end labeling(TUNEL)staining,cell proliferation in xenografts were measured by Ki67 staining,the expression levels of biomarkers in xenografts were measured by flow cytometry.Results1.The radiochemical yield of 131I-IgG,131I-CD133 mAb and 131I-CD44 mAb was 67.62±3.86%,74.41±4.65%and 71.27±4.22%,respectively.The specific radioactivity of 131I-IgG,131I-CD133 mAb and 131I-CD44 mAb was 13.57±0.81 ?Ci/?g,15.18±0.54?Ci/?g and 14.36±0.79 ?Ci/?g,respectively.The radiochemical purity after purification of 131I-IgG,131I-CD133 mAb and 131I-CD44 mAb was 88.91±1.66%,93.37±2.79%and 91.08±2.38%,respectively.The labeled antibody had good stability in vitro.2.There is no statistically significant difference in body weight of tumor-bearing nude mice between the groups(P=0.915).There is no statistically significant difference in tumor volume between the groups(P=1.000).The tumor growth curves showed notable tumor growth delay in the CSC-targeting RIT groups compared with the other five groups.The mice in 131I-CD133 mAb,radioiodinated mAbs cocktail and 131I-CD44 mAb groups exhibited significantly smaller mean tumor volume than the other five groups at day 30 after administration.The mean tumor volume in the 131I anti-CD44 mAb group was significantly smaller than that in the 131I anti-CD133 group(P=0.018).The survival curves showed the notably prolonged median survival of tumor-bearing mice in the CSC-targeting RIT groups compared with the other five groups.The median survival of tumor-bearing mice in 131I-CD44 mAb group was significantly longer than that in 131I-CD133 mAb group(P=0.026),while there was no significant difference between radioiodinated mAbs cocktail group and 131I-D133 mAb group(P=0.084)or 131I-CD44 mAb group(P=0.592).3.TUNEL immunofluorescence staining showed increased apoptosis in the CSC-targeting RIT groups compared with the other five groups at 30 d after injection.The quantitative analysis of TUNEL showed statistically significant differences between the CSC-targeting RIT groups and the other groups(P<0.001),while there was no significant difference among the other five groups(P>0.05).The fraction of TUNEL positive cells in 131I-CD44 mAb group was significantly higher than that in radioiodinated mAbs cocktail group(P=0.001),while the fraction of TUNEL positive cells in radioiodinated mAbs cocktail group was significant higher than that in 131I-CD133 mAb group(P<0.001).4.Ki67 immunohistochemical staining showed decreased cell proliferation in the CSC-targeting RIT groups compared with the other five groups at 30 d after injection.The quantitative analysis of Ki67 staining showed statistically significant differences between the CSC-targeting RIT groups and the other groups(P<0.001),while there was no significant difference among the other five groups(P>0.05).The fraction of Ki67 positive cells in 131I-CD44 mAb group was significantly lower than that in radioiodinated mAbs cocktail group(P=0.011),while the fraction of Ki67 positive cells in radioiodinated mAbs cocktail group was significant lower than that in 131I-CD133 mAb group(P<0.001).5.The expression levels of CD133 and CD44 detected by flow cytometry showed a varying expression of CD133 and CD44 in various groups at day 30 after injection.The percentage of CD133+ cells,CD44+cells and CD133+/CD44+cells in the saline group was significantly higher than that in the CSC-targeting RIT groups(P<0.001),while there was no significant difference with the other groups(P>0.05).The percentage of CD133+ cells in 131I-CD133 mAb group was significantly lower than those in other groups(P<0.001).The percentage of CD44+cells in 131I-CD44 mAb group was significantly lower than those in other groups(P<0.001).The percentage of CD133+/CD44+cells in radioiodinated mAbs cocktail group was significantly lower than those in other groups(P<0.01).ConclusionOur results suggest that the CSC-targeting RIT with two radioiodinated mAbs alone and in combination can effectively eradicate CSCs and consequently inhibit tumor development.Although the therapeutic effect of the utility combined 131I-CD133 mAb and 131I-CD44 mAb at the same dose is not better than the single-target therapy with 131I-CD44 mAb,but the utility combined 131I-CD133 mAb and 131I-CD44 mAb could specifically eradicate more CD133+/CD44+cells than single-target therapy,which may result in enhanced CSC-targeting eradication.Section two Monitoring therapy response by radioimmunoimaging for targeting cancer stem cells with 131I labeled monoclonal antibodies of dual-targets in human colorectal cancer xenograftsObjectiveThe radioiodinated monoclonal antibodies(mAbs)were utilized as radiotheranostic probes for specific targeting cancer stem cells(CSCs)surface biomarkers(CD133 and CD44).The feasibility of dynamic monitoring CSC population changes in radioimmunotherapy(RIT)was investigated by several procedures including in vivo single photon emission computed tomography(SPECT),ex vivo biodistribution analysis,immunohistochemistry and western blot.The relative advantage of the utility combined 131I-CD133 mAb and 131I-CD44 mAb in radioimmunoimaging(RII)were evaluated.MethodsThe radioiodination labeling of antibodies was performed using the solid-phase iodogen method.HT29 cells(2×106)were inoculated subcutaneously into the right lower limbs of 6-week-old male BALB/c-nu/nu mice to establish tumor xenografts models.Xenografted mice were randomly divided into four groups.The radioiodinated single-antibody groups were intravenously injected with 14.8 MBq/100 ?l mAbs or IgG.The radioiodinated mAbs cocktail group was injected with with 7.4 MBq 131I-CD133 mAb and 7.4 MBq 131I-CD44 mAb.In vivo SPECT imaging was used to dynamically monitor CSC population changes at 1,5,10,15,20 and 25 d after injection.At day 5,10,15 and 20 after injection,4 mice in each group were sacrificed.The radioactivity uptake of tumors was quantified by ex vivo biodistribution analysis.The expression levels of CD 133 and CD44 after treatment were assessed by immunohistochemistry.The expression levels of biomarkers in xenografts were measured by western blot.Results1.At day 1 after injection,the whole bodies of mice showed uptakes of the radiolabeled agents in 131I-IgG,131I-CD133 mAb,131I-CD44 mAb and radioiodinated mAbs cocktail groups.SPECT images at day 5 and 10 after injection showed that 131I-CD44 mAb group had more intense accumulation of radiolabeled agents in the tumor area than that of 131I-CD133 mAb and radioiodinated mAbs cocktail groups SPECT images at day 15 and 20 after injection showed that 131I-CD133 mAb group had more intense accumulation of radiolabeled agents in the tumor area than 131I-CD44 mAb and radioiodinated mAbs cocktail groups.SPECT images at day 25 after injection showed that all CSC-targeting RIT groups had significant intense accumulation of radiolabeled agents in the tumor area.Almost no radiolabeled agent could be seen in the tumor area in 131I-IgG group until day 20 after injection.At day 25 after injection,the images of tumors in 131I-IgG group were slightly clearer than before.2.The biodistribution results at 5,10,15 and 20 d after injection showed the radioactivity uptakes(%ID/g)of tumors and the tumor-to-background dose(T/B)ratios in the CSC-targeting RIT groups were significantly higher than those in 131I-IgG group(P<0.001).At day 5 after injection,the%ID/g of tumor and T/B ratios in 131I-CD44 mAb group were significantly higher than those in 131I-CD133 mAb group(P<0.05).At day 10 after injection,the%ID/g of tumor and T/B ratios in 131I-CD44 mAb group were significantly higher than those in 131I-CD133 mAb group(P<0.05),meanwhile the T/B ratios in radioiodinated mAbs cocktail group were significantly higher than those in 131I-CD133 mAb groups(P<0.001).At day 15 after injection,the%ID/g of tumor and T/B ratios in 131I-CD133 mAb group and radioiodinated mAbs cocktail group were significantly higher than those in 131I-CD44 mAb group(P<0.01),meanwhile the T/B ratios in 131I-CD133 mAb group were significantly higher than those in radioiodinated mAbs cocktail group(P<0.001).At day 20 after injection,the%ID/g of tumor and T/B ratios in 131I-CD133 mAb group were significantly higher than those in 131I-CD44 mAb group(P<0.01),meanwhile the T/B ratios in 131I-CD133 mAb group were significantly higher than those in radioiodinated mAbs cocktail group(P<0.05).3.At 5,10,15 and 20 d after injection,the immunohistochemical staining for CD133 and CD44 showed that the tumor section had less brown staining in the CSC-targeting RIT groups than 131I-IgG group.The quantitative analysis of CD133 and CD44 expression showed that the mean IOD in the CSC-targeting RIT groups were significantly lower than those in 131I-IgG group(P<0.01).The expression of CD133 at each timepoint in 131I-CD133 mAb group was significantly lower than that in radioiodinated mAbs cocktail group and 131I-CD44 mAb group(P<0.01),meanwhile the expression of CD 133 at day 15 and 20 after administration in radioiodinated mAbs cocktail group was significant lower than that in 131I-CD44 mAb group(P<0.05).The expression of CD44 at each timepoint in 131I-CD44 mAb group was significantly lower than that in radioiodinated mAbs cocktail group and 131I-CD133 mAb group(P<0.01),meanwhile the expression of CD44 at day 10 and 20 after administration in radioiodinated mAbs cocktail group was significant lower than that in 131I-CD133 mAb group(P<0.05).4.The western blot images showed that the levels of CD 133 and CD44 in the CSC-targeting RIT groups were notably lower than those in 131I-IgG group.The densitometric analysis of CD133 and CD44 expression showed that the relative signal intensity in the CSC-targeting RIT groups were significantly lower than those in 131I-IgG group(P<0.01).At 5,10,15 and 20 d after injection,the expression of CD133 in 131I-CD133 mAb group was significantly lower than that in radioiodinated mAbs cocktail group and 131I-CD44 mAb group(P<0.001),meanwhile the expression of CD133 in radioiodinated mAbs cocktail group was significant lower than that in 131I-CD44 mAb group(P<0.001).The expression of CD44 at each timepoint in 131I-CD44 mAb group was significantly lower than that in radioiodinated mAbs cocktail group and 131I-CD133 mAb group(P<0.001),meanwhile the expression of CD44 at day 5,10 and 20 after administration in radioiodinated mAbs cocktail group was significant lower than that in 131I-CD133 mAb group(P<0.05).ConclusionThe results from in vivo SPECT imaging and ex vivo biodistribution analysis were consistent with the expression levels of CD133 and CD44 assessed by immunohistochemistry and western blot,which demonstrated the feasibility of utilizing the radioiodinated CSC-targeting mAbs as radiotheranostic probes for dynamic RII monitoring of CSCs in RIT.The radioiodinated mAbs cocktail can specifically monitor the dynamic changes of CSCs as an evaluation indicator of RIT efficacy,it may not necessarily produce more clear RII images than 131I-CD133 mAb or 131I-CD44 mAb. |
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