Tumor was clearly delineated against healthy pancreas, and additional lesions could be seen around the posterior surface of the pancreas (Physique 4D). Open in a separate window Figure 4. Fluorescence-Guided Surgery in orthotopic pancreatic cancer. Ester. AR9.6-IRDye800 and IgG-IRDye800 antibody conjugates were synthesized by reacting NHS ester dye with free amines around the antibody to form stable amide bonds (Figure 2A). IgG was used as a non-specific isotype control throughout this study. Conjugation reactions resulted in an average of 3 dyes per protein as determined by absorbance spectroscopy (Physique 2B). Fluorescence spectra of the antibody conjugates exhibited that fluorescence was not quenched upon conjugation to the protein. Open in a separate window Physique 2. Synthesis and characterization of antibody conjugates. (A) Schematic of IRDye800 NHS Ester conjugation to AR9.6 and IgG. (B) Representative absorbance and emission spectra from both antibody conjugates. (C) Western blot of MUC16 expression in human pancreatic cancer cell lines. (D) Fluorescent western blot confirming binding of AR9.6-IRDye800, and lack of binding in IgG control. (E) Immunofluorescence of antibody conjugate binding. Images acquired at 400X magnification. Scale bar = 20 m. The expression of MUC16 was assessed by western blot in 5 pancreatic cancer cell lines: T3M4, Capan1, Colo357, CFPAC, and HPAC (Physique 2C). An immortalized normal pancreas cell line (HPNE) served as a negative control, and the ovarian cancer cell line, OVCAR3, which has well-documented MUC16 expression, served as a positive control (25,26). A range of moderate to high expression of MUC16 was seen across all pancreatic cancer cell lines. To confirm that conjugation of AR9.6 to IRDye800 did not drastically impact antigen recognition and cell binding, a fluorescent western blot and fluorescence microscopy were conducted, as shown in Determine 2D and ?and2E.2E. Fluorescent western blotting in the 800 nm channel exhibited that AR9.6-IRDye800 could still recognize MUC16 after dye conjugation. Secondary antibody binding (700 nm channel) confirmed that this fluorescence seen in the 800 nm channel was due to the presence of AR9.6-IRDye800 binding, as shown by colocalization between the 700 and 800 nm channels in Figure 2D. As expected, the non-specific IgG-IRDye800 conjugate did not bind to MUC16, and secondary antibody staining confirmed that AR9.6 was not present. Fluorescence microscopy showed strong fluorescence signal from AR9.6-IRDye800 in Cav3.1 MUC16 expressing pancreatic cancer cell lines, which was consistent with OVCAR3 cells (positive control). AR9.6-IRDye800 did not bind to MUC16 negative HPNE cells, and the IgG-IRDye800 control did not bind to cells, regardless of MUC16 expression levels (Figure 2E). Determination of AR9.6-IRDye800 optimal imaging time from tumor signal dynamics To monitor the tumor accumulation of the antibody conjugates over time, AR9.6-IRDye800, IgG-IRDye800, Omtriptolide and unconjugated IRDye800 were assessed for 6 days in a subcutaneous Omtriptolide T3M4 xenograft model of pancreatic cancer. Physique 3A depicts representative images of tumor accumulation from the three groups over 144 h based on images acquired daily around the Pearl? Trilogy. Strong fluorescence signal was observed throughout the Omtriptolide mouse at 4 h, while robust enhancement of the tumor was observed within 24 h after injection of AR9.6-IRDye800, and signal was retained in the tumor at 144 h. Diffuse signal was observed with IgG-IRDye800 at 24 h, while unconjugated IRDye800 was cleared within 24 h as expected (27). Tumor to background ratios (TBRs) were highest for AR9.6-IRDye800 at 144 h after injection (4.47 1.43), as compared to 2.12 0.12 for the IgG-IRDye800 control and 0.89 0.11.