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Updated: Apr 21, 2026
Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells
Published on: May 9, 2025
Quantitative in vivo immunohistochemistry of epidermal growth factor receptor using a receptor concentration imaging
Kimberley S Samkoe1, Kenneth M Tichauer2, Jason R Gunn3
1Department of Surgery, Geisel School of Medicine at Dartmouth College, Lebanon, New Hampshire. Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire. Kimberley.S.Samkoe@Dartmouth.edu.
Abstract:
As receptor-targeted therapeutics become increasingly used in clinical oncology, the ability to quantify protein expression and pharmacokinetics in vivo is imperative to ensure successful individualized treatment plans. Current standards for receptor analysis are performed on extracted tissues. These measurements are static and often physiologically irrelevant; therefore, only a partial picture of available receptors for drug targeting in vivo is provided. Until recently, in vivo measurements were limited by the inability to separate delivery, binding, and retention effects, but this can be circumvented by a dual-tracer approach for referencing the detected signal. We hypothesized that in vivo receptor concentration imaging (RCI) would be superior to ex vivo immunohistochemistry (IHC). Using multiple xenograft tumor models with varying EGFR expression, we determined the EGFR concentration in each model using a novel targeted agent (anti-EGFR affibody-IRDye800CW conjugate) along with a simultaneously delivered reference agent (control affibody-IRDye680RD conjugate). The RCI-calculated in vivo receptor concentration was strongly correlated with ex vivo pathologist-scored IHC and computer-quantified ex vivo immunofluorescence. In contrast, no correlation was observed with ex vivo Western blot analysis or in vitro flow-cytometry assays. Overall, our results argue that in vivo RCI provides a robust measure of receptor expression equivalent to ex vivo immunostaining, with implications for use in noninvasive monitoring of therapy or therapeutic guidance during surgery.
Insights
In vivo receptor concentration imaging (RCI) accurately quantifies protein expression in tumors, outperforming traditional ex vivo methods. This technique offers a superior approach for guiding cancer therapy and monitoring treatment effectiveness.
Area of Science:
- Oncology
- Molecular Imaging
- Biotechnology
Background:
- Receptor-targeted therapeutics are crucial in clinical oncology for personalized treatment.
- Current ex vivo methods for receptor analysis provide static, often physiologically irrelevant data.
- In vivo measurements were previously limited by the inability to separate delivery, binding, and retention effects.
Purpose of the Study:
- To evaluate the efficacy of in vivo receptor concentration imaging (RCI) compared to ex vivo immunohistochemistry (IHC).
- To determine if RCI can accurately quantify epidermal growth factor receptor (EGFR) concentration in xenograft tumor models.
- To assess the correlation of RCI with various ex vivo and in vitro quantification techniques.
Main Methods:
- Utilized a dual-tracer approach with a novel anti-EGFR affibody-IRDye800CW conjugate and a control affibody-IRDye680RD conjugate.
- Administered tracers simultaneously in multiple xenograft tumor models with varying EGFR expression.
- Quantified in vivo EGFR concentration using RCI and compared results with ex vivo IHC, immunofluorescence, Western blot, and in vitro flow cytometry.
Main Results:
- In vivo RCI demonstrated a strong correlation with pathologist-scored ex vivo IHC and computer-quantified ex vivo immunofluorescence.
- No significant correlation was found between RCI and ex vivo Western blot analysis or in vitro flow cytometry.
- RCI provided a robust measure of in vivo receptor expression equivalent to ex vivo immunostaining.
Conclusions:
- In vivo RCI is a reliable method for quantifying receptor expression, offering an advantage over static ex vivo analyses.
- This technique has significant implications for noninvasive monitoring of cancer therapy response.
- RCI can potentially guide therapeutic interventions, including during surgical procedures.
