Simultaneous extracellular and intracellular quantification of EGFR using paired-agent imaging in an in ovo tumor model

摘要

Quantification of protein concentrations is often a static and tissue destructive technique. Paired-agent imaging (PAI) usingmatched targeted and untargeted agents has been established as a dynamic method for quantifying the extracellular domainof epidermal growth factor receptor (EGFR) in vivo in a variety of tumor lines. Here we extend the PAI model tosimultaneously quantify the extracellular and intracellular regions of EGFR using novel cell membrane permeablefluorescent small molecules, TRITC-erlotinib (targeted) and BODIPY-N-erlotinib (non-binding control isoform)synthesized in house. An EGFR overexpressing squamous cell carcinoma cell xenograft tumor, A431, was implanted onthe chorioallantoic membrane (CAM) of the embryonated chicken egg. In total six fluorescent molecules wereadministered and monitored over 1 h using multi-spectral imaging. EGFR concentrations were determined using bothextracellular and intracellular PAI methods. The fluorescent molecules used for extracellular PAI were ABY-029, an anti-EGFR Affibody molecule conjugated to IRDye 800CW, and a Control Imaging Agent Affibody molecule conjugated toIRDye 680RD. The intracellular PAI (iPAI) fluorescent molecules were cell membrane penetrating TRITC-erlotinib,BODIPY-N-erlotinb, and BODIPY TR carboxylate, as well as cell membrane impermeant control agent, Alexa Fluor 647carboxylate. Results from simultaneous imaging of both the extracellular and intracellular binding domains of EGFRindicate that concentrations of intracellular EGFR are higher than extracellular. This is anticipated as EGFR exists in twodistinct populations in cells, cell membrane bound and internalized, activated protein. iPAI is a promising new method forquantifying intracellular proteins in a rapid tumor model on the chicken CAM.