Quantitative In Vivo Immunohistochemistry of Epidermal Growth Factor Receptor Using a Receptor Concentration Imaging Approach

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. Using multiple xenograft tumor models with varying epidermal growth factor receptor (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 immunohistochemistry and computer-quantified ex vivo immunofluorescence. In contrast, no correlation was observed with ex vivo Western blot 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 immuno-staining, with implications for use in non-invasive monitoring of therapy or therapeutic guidance during surgery.

Review of fluorescence guided surgery systems: identification of key performance capabilities beyond indocyanine green imaging

“…Using molecular signals in the surgical field is a natural progression that follows the development of molecular pathology to identify lesion phenotype in conjunction with image guidance from MRI or CT, as a decision tool for patient management; there is a compelling evidence that these phenotypes can be imaged in vivo to allow better real-time definition of the surgical margin. [10][11][12][13][14][15][16] In addition to ICG, there is extensive ongoing research using fluorescein and 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) imaging for neurosurgery, [17][18][19][20][21] a procedure that has gained clinical approval in a handful of countries (Approved by the European Medicines Agency in September 2007 and is approved for use in all European Union, European Economic Area, and European Free Trade Association states). Additionally, PpIX fluorescence imaging using blue light illumination has local approvals in some countries for subspecialty use in bladder cancer detection [22][23][24] and gynecologic oncology [25][26][27] with clinical trials underway.…”

Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

“…However, it should be noted that the receptor concentrations estimated using both pairs of imaging agents for the two cell lines in all conditions, matched well with expected levels of EGFR. These agents have also been used extensively and validated in other work, where EGFR concentrations matched well with gold standard and expected levels (Leigh et al , 2013; Wang et al , 2016; Wang et al , 2015; Wang et al , 2014b; Tichauer et al , 2012a; Tichauer et al , 2012b; Samkoe et al , 2014). This body of work suggests that both non-specific binding and internalization are relatively negligible, at least within 1 h of initial administration of the imaging agents.…”

Quantification of cell surface receptor expression in live tissue culture media using a dual-tracer stain and rinse approach

“…This possibility was considered based on the fact that while untargeted tracer signal began to flatten at the last few washes, 40% of untargeted tracer remains in the gel. [9][10][11][12][13] The and rinse e, several…”

“…This approach employs a targeted fluorescent tracer, the signal from which is normalized mathematically to the retention of a simultaneously administered "untargeted" fluorescent tracer signal used to account for nonspecific retention of the targeted tracer. [9][10][11][12][13] To test the dual-tracer approach, 0, 5×10 4 , 5×10 5 , and 5×10 6 live human glioma (U251) cells were mixed in 0.6% agarose and cell media and placed into a six-well plate to create a 3D microenvironment. Equal concentrations of both imaging agents were then added to the tops of the gels and rinsed off after 1 hour.…”