Macroscopy fluorescence lifetime (MFLI) FRET imaging
By integrating our knowledge on the endocytic trafficking of receptor-drug complexes with our expertise on Forster Resonance Energy Transfer (FRET) and fluorescence lifetime (FLIM) imaging, we work to establish novel in vitro and in vivo deep-tissue imaging assays to measure drug-target engagement in tumors, a crucial parameter to increase delivery and targeting of anti-cancer therapy.
Xavier and Margarida during their presentation on Advances in Target Therapy for Breast Cancer" at MCP translational forum.
Fluorescence intensity vs. lifetime near-infrared (NIR) FRET using macroscopy deep-tissue imaging in living, intact animals
Smith JT, Sinsuebphon N, Rudkouskaya A, Michalet X, Intes X, Barroso M. In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET. Biophys Rep (N Y). 2023 May 9;3(2):100110.
In vivo comparison of intensity- and MFLI-FRET dynamic imaging. (A) Maps at selected time points of intensity-based fDE in the liver and the urinary bladder of a mouse injected with Tf A:D 80:40 ug (total time points: 210). (B) Corresponding intensity-based fDE time trace. (C) Maps at selected time points of lifetime-based fDE in the liver and the urinary bladder of a mouse injected with Tf A:D 80:40 ug (total time points: 127). (D) Corresponding lifetime-based fDE time trace. For (B) and (D), solid lines mark the average value, and the shaded areas indicate the standard deviation across all pixels within each organ ROI.
Measuring drug-target engagement in living, intact tumor xenografts using MFLI FRET imaging
In vivo Trastuzumab-HER2 receptor engagement. Mice were injected with 20 µg of AF700-TZM and 40 µg of AF750-TZM and imaged by MFLI at 24 h post-injection (p.i.). Mouse 1 was imaged with ICCD and mouse 2 with SS2. A and B, fluorescence intensity images (Smith et al., 2022)
Dynamic MFLI FRET imaging monitors receptor-ligand binding and internalization in living and intact animals
In vivo imaging of single retroorbital i.v. injection of Transferrin (Tf)-AF700 (donor)
In vivo imaging of A:D ~ 2:1 double-labeled injection of Tf-AF700 (donor) and Tf-AF750 (acceptor).
Fluorescence signal accumulates in the liver (top) and urinary bladder (excretion) (below), but an increase in FRET signal is detected only in the liver where the majority of the receptor-mediated Tf uptake occurs (Smith et al., 2019).
In vivo Trastuzumab-HER2 drug-receptor engagement using MFLI FRET in vivo imaging of live intact HER2+ tumor xenograts
Rudkouskaya A, Smith JT, Intes X, Barroso M. Quantification of Trastuzumab-HER2 Engagement In Vitro and In Vivo. Molecules. 2020, 25(24):5976.
Live small animal NIR MFLI-FRET for preclinical studies. (A) AU565 tumor xenograft; (B) Injection of AF700–trastuzumab (TZM) and/or AF750–TZM; (C) Live small animal imaging using wide-field time-resolved MFLI FRET; (D) Ex vivo validation. Inset top left: Trastuzumab (TZM)-AF700 and TZM–AF750 ligands. Inset bottom right: TZM–HER2 MFLI-FRET events upon binding of donor- and acceptor-labeled antibodies to HER2 dimer at the surface of cancer cells.
Quantification of trastuzumab (TZM)–HER2 engagement via MFLI-FRET in vivo imaging. Mice were injected with TZM–AF700 (M1) or TZM–AF700 and TZM–AF750 (M2-M3) and subjected to MFLI-FRET at 48 h p.i. Panels show TZM donor maximum intensity ROIs (both soluble and bound probe) and FRET donor fraction (FD%) map (bound and internalized probe) in the tumors (T), livers (LV), and urinary bladders (UB).
Watch this webinar to learn how Macroscopy FLI FRET Imaging Monitors Cancer Drug-Target Engagement in Vivo
Transferrin-receptor (TfR)-transferrin (Tf) binding using FRET FLIM in vitro imaging of cancer cells and MFLI FRET in vivo imaging of tumor xenografts in intact live animals
Non-invasive multiplexed imaging of glucose metabolism and TfR-Tf receptor-ligand engagement in breast tumor xenografts using NIR MFLI FRET in intact live animals
Working together with Xavier Intes's group at Rensselaer Polytechnic Institute, Biomedical Engineering, we have validated Macroscopy Fluorescence Lifetime (MFLI) FRET imaging to measure the target engagement of near infrared (NIR)-labeled probes upon binding to their respective receptors in tumors in intact live animals.
