Nonlinear unmixing to account for blood absorption in multispectral imaging for improved quantification of intracellular and extracellular EGFR

Head and neck squamous cell carcinomas (HNSCCs) have high levels of chromosomal instability and epidermal growth factor receptor (EGFR) overexpression, both of which drive their tumorigenesis. While drug treatment targeting the extracellular domain of EGFR has shown some success, mutations and alternate intracellular pathways contribute to therapeutic resistance. Therefore, a dynamic in vivo method to monitor binding and downstream cell signaling is warranted. Previous work has demonstrated that paired-agent imaging (PAI) is a powerful tool to quantify extracellular EGFR, and so this work extends the same principles to quantify intracellular protein target engagement. Here, in ovo models were used to grow human HNSCC xenografts - eggs were windowed to reveal the chorioallantoic membrane (CAM) of chicken embryos and tumors were implanted on its surface. A fluorescent cocktail of both intracellular and extracellular, targeted and untargeted agents (four agents total) was intravenously injected and multispectral imaging was performed over two hours. To isolate the relative quantities of each agent, a spectral fitting procedure was employed that accounted for the linear contributions of each fluorescent agent and autofluorescence, and the non-linear absorbing contributions of oxy- and deoxyhemoglobin. This unmixing was performed on a pixel-by-pixel basis to generate distribution maps of each individual dye, and then motion correction was done, followed by a convolution correction to account for delivery differences. Results demonstrated successful unmixing of individual fluorophores such that a ratiometric calculation could be applied to extract both intracellular and extracellular binding potential (BP), which is proportional to EGFR concentration.