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.
| Original language | English (US) |
|---|---|
| Title of host publication | Visualizing and Quantifying Drug Distribution in Tissue III |
| Editors | Kin Foong Chan, Conor L. Evans |
| Publisher | SPIE |
| ISBN (Electronic) | 9781510623606 |
| DOIs | |
| State | Published - Jan 1 2019 |
| Externally published | Yes |
| Event | Visualizing and Quantifying Drug Distribution in Tissue III 2019 - San Francisco, United States Duration: Feb 2 2019 → … |
Publication series
| Name | Progress in Biomedical Optics and Imaging - Proceedings of SPIE |
|---|---|
| Volume | 10859 |
| ISSN (Print) | 1605-7422 |
Conference
| Conference | Visualizing and Quantifying Drug Distribution in Tissue III 2019 |
|---|---|
| Country | United States |
| City | San Francisco |
| Period | 2/2/19 → … |
Fingerprint
Keywords
- 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
Cite this
Simultaneous extracellular and intracellular quantification of EGFR using paired-agent imaging in an in ovo tumor model. / Samkoe, Kimberley S.; Schultz, Emily; Solanki, Allison; Wang, Lei; Korber, Jesse; Tichauer, Kenneth M.; Gibbs, Summer.
Visualizing and Quantifying Drug Distribution in Tissue III. ed. / Kin Foong Chan; Conor L. Evans. SPIE, 2019. 108590F (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 10859).Research output: Chapter in Book/Report/Conference proceeding › Conference contribution
}
TY - GEN
T1 - Simultaneous extracellular and intracellular quantification of EGFR using paired-agent imaging in an in ovo tumor model
AU - Samkoe, Kimberley S.
AU - Schultz, Emily
AU - Solanki, Allison
AU - Wang, Lei
AU - Korber, Jesse
AU - Tichauer, Kenneth M.
AU - Gibbs, Summer
PY - 2019/1/1
Y1 - 2019/1/1
N2 - 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.
AB - 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.
KW - Chicken chorioallantoic membrane
KW - Epidermal growth factor receptor
KW - Intracellular paired agent imaging
KW - Multi-spectral imaging
KW - Paired-agent imaging
UR - http://www.scopus.com/inward/record.url?scp=85065805129&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85065805129&partnerID=8YFLogxK
U2 - 10.1117/12.2510778
DO - 10.1117/12.2510778
M3 - Conference contribution
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Visualizing and Quantifying Drug Distribution in Tissue III
A2 - Chan, Kin Foong
A2 - Evans, Conor L.
PB - SPIE
ER -