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Simultaneous extracellular and intracellular quantification of EGFR using paired-agent imaging in an in ovo tumor model
Author(s): Kimberley S. Samkoe; Emily Schultz; Allison Solanki; Lei Wang; Jesse Korber; Kenneth M. Tichauer; Summer L. Gibbs
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Paper Abstract

Quantification of protein concentrations is often a static and tissue destructive technique. Paired-agent imaging (PAI) using matched targeted and untargeted agents has been established as a dynamic method for quantifying the extracellular domain of epidermal growth factor receptor (EGFR) in vivo in a variety of tumor lines. Here we extend the PAI model to simultaneously quantify the extracellular and intracellular regions of EGFR using novel cell membrane permeable fluorescent 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 on the chorioallantoic membrane (CAM) of the embryonated chicken egg. In total six fluorescent molecules were administered and monitored over 1 h using multi-spectral imaging. EGFR concentrations were determined using both extracellular 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 to IRDye 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 647 carboxylate. Results from simultaneous imaging of both the extracellular and intracellular binding domains of EGFR indicate that concentrations of intracellular EGFR are higher than extracellular. This is anticipated as EGFR exists in two distinct populations in cells, cell membrane bound and internalized, activated protein. iPAI is a promising new method for quantifying intracellular proteins in a rapid tumor model on the chicken CAM.

Paper Details

Date Published: 28 February 2019
PDF: 8 pages
Proc. SPIE 10859, Visualizing and Quantifying Drug Distribution in Tissue III, 108590F (28 February 2019); doi: 10.1117/12.2510778
Show Author Affiliations
Kimberley S. Samkoe, Dartmouth College (United States)
Dartmouth-Hitchcock Medical Ctr. (United States)
Emily Schultz, Illinois Institute of Technology (United States)
Allison Solanki, Illinois Institute of Technology (United States)
Lei Wang, Illinois Institute of Technology (United States)
Jesse Korber, Illinois Institute of Technology (United States)
Kenneth M. Tichauer, Oregon Health and Science Univ. (United States)
Summer L. Gibbs, Illinois Institute of Technology (United States)


Published in SPIE Proceedings Vol. 10859:
Visualizing and Quantifying Drug Distribution in Tissue III
Kin Foong Chan; Conor L. Evans, Editor(s)

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