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

Kimberley S. Samkoe, Emily Schultz, Allison Solanki, Lei Wang, Jesse Korber, Kenneth M. Tichauer, Summer L. Gibbs

Research output: Chapter in Book/Report/Conference proceedingConference contribution

3 Scopus citations


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.

Original languageEnglish (US)
Title of host publicationVisualizing and Quantifying Drug Distribution in Tissue III
EditorsConor L. Evans, Kin Foong Chan
ISBN (Electronic)9781510623606
StatePublished - 2019
Externally publishedYes
EventVisualizing and Quantifying Drug Distribution in Tissue III 2019 - San Francisco, United States
Duration: Feb 2 2019 → …

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
ISSN (Print)1605-7422


ConferenceVisualizing and Quantifying Drug Distribution in Tissue III 2019
Country/TerritoryUnited States
CitySan Francisco
Period2/2/19 → …


  • Chicken chorioallantoic membrane
  • Epidermal growth factor receptor
  • Intracellular paired agent imaging
  • Multi-spectral imaging
  • Paired-agent imaging

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Biomaterials
  • Radiology Nuclear Medicine and imaging


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