Theranostic evaluation of radiolabeled “cocktail” antibodies targeting cancer stem cells in human colorectal cancer xenografts

Objectives: Mounting evidence indicates that a combination of cell surface biomarkers (CD133 and CD44) is more reliable for the identification of cancer stem cells (CSCs) which have been confirmed the main reason for conventional anticancer treatment failure. In our previous work, ¹³¹I labeled CD133 monoclonal antibody (mAb) was prepared to trace CD133+ CSCs in vivo by radioimmunoimaging (RII) and treat CD133+ CSCs by radioimmunotherapy (RIT) in colorectal cancer. The aim of this study was to perform the theranostics value with radioiodinated CSC-targeting mAbs in human colorectal cancer xenografts, and to evaluate the relative advantage of a “cocktail” treatment of ¹³¹I-CD133 mAb and ¹³¹I-CD44 mAb.

Methods: CD133 mAb and CD44 mAb were labeled with ¹³¹I by the iodogen coating method, and IgG isotype was also labeled used as a control. Tumor-bearing mice were randomly divided into 8 groups (n=10) received intravenous injection of: ¹³¹I-CD133 mAb group (14.8 MBq/100 μl), ¹³¹I-CD44 mAb group (14.8 MBq/100 μl), ¹³¹I-IgG isotype control group (14.8 MBq/100 μl), radioiodinated mAbs cocktail (7.4 MBq ¹³¹I-CD133 mAb and 7.4 MBq ¹³¹I-CD44 mAb) group, CD133 mAb (20 μg/100 μl) group, CD44 mAb (20 μg/100 μl) group, unradioiodinated mAbs cocktail (10 μg CD133 mAb and 10 μg CD44 mAb) group and saline (100 μl) group. In vivo SPECT/CT imaging was used to noninvasively monitor CSC population changes after treatment at day 1, 5, 10, 15, 20 and 25. The radioactivity uptakes of tumors at day 5, 10, 15 and 20 after injection were assessed by the ex vivo biodistribution. The protein expressions of CD133 and CD44 in tumor tissues at day 5, 10, 15 and 20 after treatment were assessed by immunohistochemical and western blot analysis. The tumor growth curves and survival curves were constructed to assess the treatment efficacy. The proliferation and apoptosis in xenografts at day 30 after treatment were assessed by Ki-67 and TUNEL staining. The expression levels of CD133 and CD44 in xenografts at day 30 after treatment were detected by flow cytometry. Results: The results of RII showed that the CSC-targeting RIT groups (¹³¹I-CD133 mAb, ¹³¹I-CD44 mAb and radioiodinated mAbs cocktail groups) had intense accumulations of radiolabeled agents in the tumor areas, while almost no signal could be seen in the tumor area in ¹³¹I-IgG group. The ex vivo biodistribution at day 5, 10, 15 and 20 after injection showed that the radioactivity uptakes of tumors in the CSC-targeting RIT groups were significantly higher than that in ¹³¹I-IgG group. In the CSC-targeting RIT groups, immunohistochemical and western blot analysis at day 5, 10, 15 and 20 after injection indicated significantly reduced specific target expressions in subcutaneous xenografts. Consequently, the tumor growth curves and survival curves showed that the CSC-targeting RIT significantly inhibited tumor growth and prolonged mean animal survival, while the best therapeutic outcome was observed in ¹³¹I-CD44 mAb group (Supporting Figure). Significantly decreased proliferation and increased apoptosis in xenografts assessed by Ki-67 and TUNEL staining further confirmed the therapeutic efficacy of CSC-targeting RIT. The flow cytometry showed that the lowest percentage of CD133+ CSCs, CD44+ CSCs and CD133+/CD44+ CSCs post treatment was found in ¹³¹I-CD133 mAb group, ¹³¹I-CD44 mAb group and radioiodinated mAbs cocktail group, respectively. Conclusions: Our results suggest that the CSC-targeting RII and RIT with ¹³¹I labeled CD133 and CD44 can effectively trace CSCs and eradicate CSCs and consequently inhibit tumor development, meanwhile the radioiodinated mAbs cocktail may generate enhanced CSC-targeting specificity. Acknowledgment: This work was supported by the National Natural Science Foundation of China (No. 81371599).