Immuno-PET and activatable near-infrared fluorescence dual-modality imaging of L1-CAM expression in cholangiocarcinoma model.

Objectives L1 cell adhesion molecule (L1-CAM) is implicated in the control of proliferation, migration, and invasion of several tumors including cholangiocarcinoma. We prepared a chimeric anti-L1-CAM antibody (cA10-A3) tagged with both ⁶⁴Cu and indocyanine green (ICG) and evaluated for the feasibility of dual-modal PET/NIRF imaging of L1-CAM expression and image-guided surgery using dual labeled antibody in cholangiocarcinoma xenograft model.

Methods We labeled cA10-A3 with both ⁶⁴Cu via p-SCN-Bn-NOTA and a NIRF dye, ICG as an activatable fluorescent probe. Immunoconjugates were treated with detergent and reducing agent to re-activate the fluorescence of bound ICG. Serum stability, immunoreactivity test and flow cytometry analysis were performed. Cell binding assay of ⁶⁴Cu-NOTA-cA10-A3-ICG was performed in ACHN, SK-OV-3-Luc and SCK-L1-Luc cells. Biodistribution and PET/NIRF imaging of ⁶⁴Cu-NOTA-cA10-A3-ICG were obtained at 24, 48 and 72 h in SCK-L1-Luc xenografted mice. Blocking experiment with cold cA10-A3 was also performed.

Results The conjugates had minimal fluorescence signal in PBS, but became fluorescent by re-activation. ⁶⁴Cu-labeled antibody showed high radiolabeling yield, high stability (> 99%) and favorable immunoreactivity (0.98). Cell bound radioactivity and fluorescence intensity of ⁶⁴Cu-NOTA-cA10-A3-ICG in cell lines were well correlated with the levels of L1-CAM expression by flow cytometry analysis. In biodistribution study, tumor uptakes of ⁶⁴Cu-NOTA-cA10-A3-ICG were 39.3 ± 7.5, 41.2 ± 5.9 and 45.0 ± 6.2 %ID/g at 24, 48 and 72 h, respectively. In ex vivo NIRF imaging, the fluorescent activity of SCK-L1-luc tumor was higher than those of most major organs, except for liver. Considering the absolute tumor uptake, tumor-to-background ratio and half-life of ⁶⁴Cu, the optimal time point for PET imaging was 48 h. Excellent contrast for NIRF imaging was achieved at 72 h when tumor-to-backgrounds ratio peaked. In blocking study, tumor uptakes were markedly reduced (P < 0.05) by cold cA10-A3 treatment.

Conclusions We showed the feasibility for immune-PET and intraoperative optical imaging of L1-CAM expressing cholangiocarcinoma using dual-labeled cA10-A3. Dual-labeled L1-CAM targeting agent may provide useful information to improve delineation of tumors and image-guided surgery for cholangiocarcinoma.