FLIM‐FRET imaging of ligand‐receptor binding in tumor cells in vitro and in vivo
Authors: Barroso Margarida M , Rudkouskaya Alena , Patel Sonia , Sinsuebphon Nattawut , Intes Xavierr 
Published: November 2023

To improve efficacy of targeted anti‐cancer drugs, we have developed non‐invasive pre‐clinical imaging assays that monitor protein ligand‐receptor or antibody‐receptor binding (i.e. target engagement) in vitro as well as in vivo. Quantitative measurements of receptor‐mediated binding and uptake into tumors of protein ligand or antibody drug conjugates would facilitate the prioritization of the most promising therapeutic agent. A major challenge is the ability to non‐invasively visualize and quantify the binding and internalization of protein ligand or antibody drug conjugates in live subjects, due to the enhanced permeability and retention effect (EPR) that leads to the non‐specific accumulation of drug‐protein conjugates at the tumor region. Currently, only invasive biochemical methods can be used to assess protein ligand or antibody‐receptor binding in tumors. Forster Resonance Energy Transfer (FRET) imaging assays are a key tool to quantify protein ligand or antibody‐receptor binding and internalization as well as receptor dimerization for the development and optimization of targeted drug delivery. Because transferrin receptor (TfnR) is over‐expressed in a wide variety of tumors, it has been used as a carrier for cancer therapeutic agents to enhance their internalization efficiency. FRET occurs when labeled transferrin (Tfn) binds TfnR at the plasma membrane and upon internalization and recycling of Tfn‐TfnR complexes in human breast cancer cells and tumors. Here, we propose to establish time‐resolved fluorescence lifetime FRET (FLIM‐FRET) imaging as the gold standard technology to quantitate target engagement of protein ligands and antibodies across microscopy and macroscopy imaging, visible and near infrared (NIR) wavelength ranges and in vitro and in vivo experiments. To measure target engagement, FLIM‐FRET has been shown to discriminate soluble Tfn from Tfn‐TfnR binding and internalization. Thus, occurrence of FLIM‐FRET leads to reduction of donor lifetime and increase in the fraction of donors involved in FRET (amplitude of short lifetime component –A1‐, also known as FRET donor fraction –FD%‐). FLIM‐FRET and intensity based FRET microscopy imaging assays have shown a positive dependency of FRET efficiency (E%) on the acceptor‐to‐donor ratio. Moreover, we have demonstrated that a linear relationship between FRET levels (E% and FD% parameters) and acceptor‐to‐donor ratio indicates TfnR target engagement in human breast cancer cells and tumors, using in vitro visible microscopy and in vivo NIR macroscopy FLIM‐FRET imaging assays, respectively. Thus, both visible in vitro and NIR in vivo FLIM‐FRET results are quantitatively consistent with the FRET model of receptor dimerization/oligomerization. In summary, FLIM‐FRET was validated across visible and NIR microscopy and macroscopy imaging approaches, demonstrating that FD% is a measure of TfnR engagement by Tfn at the plasma membrane and upon internalization of TfnR‐Tfn complexes into breast cancer cells across a variety of in vitro and in vivo imaging approaches.Support or Funding InformationSupported by NIH grant 1R01EB019443‐01A1