The red filter sets were used to image Alexa680 fluorescence and the NIR filter sets were utilized for ICG fluorescence. and 50-fold dequenching capacity under detergent treatment, respectively.In vitromicroscopy and flow cytometry using MDA-MB-468 (EGFR+/HER2-) and 3T3/HER2 cells (EGFR-/HER2+), demonstrated specific fluorescence signal for each cell type based on binding to Pan-Alexa680(SQ) or Tra-ICG. Anin vivoimaging study employing a cocktail of Pan-Alexa680(SQ) and Tra-ICG (each 50 g) was injected into mice with orthotopic MDA-MB-468 and 3T3/HER2 tumors SEL120-34A in the breast. Each probe visualized only the target-specific breast tumor. == Conclusions == Multi-color target-specific fluorescence breast cancer imaging can be achievedin vivoby employing two activatable SEL120-34A fluorescent probes administered as a cocktail. The images allowed us to see a specific receptor expression in each breast tumor without post-image processing. == Introduction == Antibodies are macromolecules with high binding specificity for target antigens. This makes them excellent scaffolds for molecular imaging probes. However, a central limitation of using antibodies for imaging is usually that they are slow to obvious from the body resulting in non-specific background contamination, reducing the target-to-background ratio. In order to overcome the high background transmission found with labeled intact antibodies, labeled antibody fragments, which show more rapid clearance, have been developed. However, faster clearance from your blood pool prospects to lower concentrations and, therefore, lower tumor uptake [1]. Optical imaging has many theoretical advantages for tumor identification and characterization. A central advantage is usually that simultaneous multi-color imaging can be employed to characterize expression profiles SEL120-34A [2,3]. By using two or more different antibodies labeled with distinct colors, unbound antibody in the normal tissue can be mathematically subtracted [4,5], since the bio-distribution of antibodies is similar in normal organs and only differs markedly at the tissue expressing the target antigen. However, this approach is somewhat inefficient as it subtracts the target transmission as well as the background transmission. Therefore, another approach to reducing background transmission from two-antibody-based optical is usually to take advantage of the unique feature of fluorescence that allows it to be quenched or activated depending on its chemical state. Such activatable probes (for example, labeled antibodies) are designed to generate transmission only after being bound and processed by the target cancer cells; therefore, high accumulation of antibodies in the target tissue can be clearly visualized while the background transmission remains quenched [6,7]. This approach permits many high tumor-to-background ratios to be achieved [8]. In this study, two different antibodies against human epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor type 2 (HER2) were conjugated with activatable near-infrared (NIR) fluorophores, namely Rabbit Polyclonal to SLC6A6 Indocyanine Green (ICG) and AlexaFluor680 (Alexa680), which emit light at different wavelengths. We then employed these two activatable antibodies as a cocktail in mice with EGFR and HER2 positive tumors in order to demonstrate the feasibility of this approach. == Materials and methods == == Reagents == Panitumumab (Pan), a fully human IgG2monoclonal antibody (mAb) directed against the extracellular domain name of the human EGFR (HER1), was purchased from Amgen (Thousand Oaks, CA, USA). Trastuzumab (Tra), a recombinant humanized mAb directed against the human HER2, was purchased from Genentech Inc. (South San Francisco, CA, USA). ICG-Sulfo-OSu was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Alexa680-NHS ester was purchased from Invitrogen Co. (Carlsbad, CA, USA). All other chemicals used were of reagent grade. == Synthesis of Alexa680 or ICG conjugated antibodies == Panitumumab (0.5 mg, 3.4 nmol) was incubated with Alexa680-NHS ester (39.3 g, 34 nmol) in 0.1 M Na2HPO4(pH 8.6) at room temperature for one hour, followed by the purification with a size exclusion column (PD-10; GE Healthcare, Piscataway, NJ, USA). ICG labeling of trastuzumab was also performed by reacting mAb with ICG at a ratio of 1 1:6 in the same manner as Pan-Alexa680. The concentrations of Alexa680 and ICG were calculated by measuring the absorption with the UV-Vis system (8453 Value UV-Vis system; Agilent Technologies, Santa Clara, CA, USA) to confirm the number of fluorophore molecules conjugated with each antibody molecule. The protein concentration was also determined by measuring the absorption at 280 nm with a UV-Vis.