3T3-MDR1 cells were utilized as a positive control. in small animal PET imaging of Pgp in a mouse xenograft model of NCI/ADR-Res cells, which are chemoresistant through overexpression of Pgp. Quantification analysis of the PET images demonstrated that the tumor uptake of the radioactive probe was 9.9 1.4, 12.1 1.2, and 10.5 1.0%ID/g at 4, 24, and 48h post injection. The tumor-to-muscle ratio was 20.9 at 48 h post injection based on biodistribution studies. Fluorescence imaging was performed following PET experiments, and it demonstrated excellent tumor accumulation of this dual-modality probe in the NCI/ADR-Res tumors. Further, an image-guided surgery was performed successfully using the fluorescence modality of the probe, demonstrating potential utility of this probe in image-guided surgical removal of Pgp-positive drug resistant tumors in the patients. In conclusion, this study clearly demonstrated that the Pgp-targeted antibody probe, 64Cu-DOTA-Pab-IR800, could provide a promising diagnosis tool Rabbit Polyclonal to SYTL4 for detection of Pgp-expressing tumors immunostaining assay, and then examined its application in detection of Pgp expression with PET/fluorescence imaging in a mouse xenograft model of chemoresistant OvCa tumors. Further, we tested the feasibility of using the fluorescent moiety of this probe for an image-guided surgery. Methods Materials The chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was purchased from Macrocyclics Inc. (Dallas, TX, USA). The fluorescence dye IRDye800-NHS (IR800-NHS) was purchased from LI-COR Inc. (Lincoln, NE, USA). 64Cu was produced at University of Wisconsin in the form of 64CuCl2 in 0.1 N HCl. Mouse IgG1 isotype control (IgG) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Anti-Pgp Antibody Production Anti-Pgp Cinobufagin monoclonal antibody (Pab) Cinobufagin was produced in house using the hybridoma cell line 15D319 from ATCC (Rockville, MD, USA). Briefly, 15D3 hybridoma cells were initially cultured in DMEM media (Corning Inc., Corning, NY, USA) containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, USA). The FBS content was reduced by serial dilution until culturing in serum-free hybridoma medium (Thermo Fisher Scientific, Rockford, IL, USA). The antibody-containing Cinobufagin media was harvested every 24 h and the antibody was purified with HiTrap Protein G HP columns (GE Healthcare Life Sciences, Piscataway, NJ, USA). The identity and purity of the antibody was assessed by SDS-PAGE. Chemistry and radiochemistry Pab and control IgG were modified with DOTA using a method published previously.20 Briefly, Cinobufagin DOTA was first reacted with EDC and Sulfo-NHS. Then 50-folded excess of activated DOTA was added to Pab or IgG and the pH was adjusted to 8.5 using borate buffer. Additional 3-folded Cinobufagin excess of IR800-NHS was added to the Pab as well. Reaction mixture was incubated at 4 C overnight followed by purification with a PD-10 desalting columns (GE). DOTA-Pab-IR800 and DOTA-IgG were then labeled with 64Cu as described previously.20 Briefly, 37 MBq of 64Cu was added to 60 g of the DOTA-Pab-IR800 or IgG-DOTA and the pH was adjusted to 5.5 with 0.25 M NH4OAc. The mixture was incubated at 40 C for 1 h with constant shaking and purified using a PD-10 column. The radioactivity of the eluted fractions was measured by a -counter (PerkinElmer, Waltham, MA, USA), and the radioactive fraction containing 64Cu-DOTA-Pab-IR800 or 64Cu-DOTA-IgG was collected for further experiments. The purity of the radioactive probes was examined by SDS-PAGE followed by autoradiography. SDS-PAGE was performed with a polyacrylamide gel (Bio-Rad, Hercules, CA, USA). After electrophoresis, the gel was stained with Coomassie Blue Staining buffer (Bio-Rad), and digitally scanned. The 64Cu radioactivity in the gel was detected using autoradiography. Cell lines Cell line 3T3-MDR1 is a mouse fibroblast cell line stably transfected with a cDNA coding for human Pgp (also called Multi-Drug Resistance Gene 1, MDR1) and was obtained from Dr. Gottesmans lab at National Cancer Institute (NCI, Bethesda, MD, USA). This cell line was maintained in DMEM medium supplemented with 10% FBS, 400 IU/mL penicillin plus 100 g/mL streptomycin (Corning Inc.), and 60 ng/ml colchicine. Adriamycin-resistant OvCa cell line NCI/ADR-RES was also obtained from Dr. Gottesmans lab at NCI and was maintained in the same condition as 3T3-MDR1 cell line. Cell immunostaining and flow cytometry Immunostaining followed by flow cytometry was performed to.