Non-Invasive In Vivo Imaging of Near Infrared-labeled Transferrin in Breast Cancer Cells and Tumors Using Fluorescence Lifetime FRET

Abstract

The conjugation of anti-cancer drugs to endogenous ligands has proven to be an effective strategy to enhance their pharmacological selectivity and delivery towards neoplasic tissues. Since cell proliferation has a strong requirement for iron, cancer cells express high levels of transferrin receptors (TfnR), making its ligand, transferrin (Tfn), of great interest as a delivery agent for therapeutics. However, a critical gap exists in the ability to non-invasively determine whether drugs conjugated to Tfn are internalized into target cells in vivo. Due to the enhanced permeability and retention (EPR) effect, it remains unknown whether these Tfn-conjugated drugs are specifically internalized into cancer cells or are localized non-specifically as a result of a generalized accumulation of macromolecules near tumors. By exploiting the dimeric nature of the TfnR that binds two molecules of Tfn in close proximity, we utilized a Förster Resonance Energy Transfer (FRET) based technique that can discriminate bound and internalized Tfn from free, soluble Tfn. In order to non-invasively visualize intracellular amounts of Tfn in tumors through live animal tissues, we developed a novel near infrared (NIR) fluorescence lifetime FRET imaging technique that uses an active wide-field time gated illumination platform. In summary, we report that the NIR fluorescence lifetime FRET technique is capable of non-invasively detecting bound and internalized forms of Tfn in cancer cells and tumors within a live small animal model, and that our results are quantitatively consistent when compared to well-established intensity-based FRET microscopy methods used in in vitro experiments.

Reduced temporal sampling effect on accuracy of time-domain fluorescence lifetime Förster resonance energy transfer

“…[31][32][33] This system has been designed to quantitatively image FRET donor fractions in vitro, ex vivo, and in vivo. 29,31,34 Briefly, the system uses a Maitai laser as a source and is coupled to a digital micromirror unit (DLP Discovery 4100 Kit and D2D module, Texas Instruments Inc., Dallas, Texas) to obtain a spatially controllable wide-field excitation [or active wide-field illumination (AWFI)]. The fluorescence signals are collected in transmittance geometry via an ultrafast-gated, intensified CCD (ICCD) camera (Picostar HR, LaVision GmbH, Goettingen, Germany) which has a 12-bit CCD and a resolution of 1376 × 1040 pixels.…”

“…If the short-lifetime component is represented by τ 1 (FRET positive signals), then the parameter of particular interest is A 1 , as it represents the quenched fraction of the FRET donor, or in our application, the internalized amount of Tfn. 29,31,33,34 Here, human Tfn molecules labeled with a NIR FRET pair (Alexa Fluor 700-Alexa Fluor 750) were added to MadinDarby canine kidney (MDCK) cells and T47D cells (human ductal breast epithelial tumor cell line), both lines expressing human TfnR. 34,48,49 This specific FRET pair was chosen due to the significant spectral overlap and Forster distance (≈7.76 nm).…”

Wide-field fluorescence molecular tomography with compressive sensing based preconditioning

“…Similar compressive imaging work in FMT was reported later on by Ducros et al who used a wavelet base [5]. Beside validations in well-controlled settings, the method has been applied in vivo to quantify FRET signals [6], with applications in drug delivery monitoring [7].…”

Spatial light modulator based active wide-field illumination for ex vivo and in vivo quantitative NIR FRET imaging

“…In our previous work [16], we have demonstrated that this designed FRET NIR pair is able to discriminate the bound and internalized Tfn from free, soluble Tfn. We also have shown that the FD% indicates the fraction of intracellular receptor-bound Tfn, which includes receptor bound-Tfn at the plasma membrane as well as during clathrin-mediated internalization and subsequent endocytic recycling pathway back to the plasma membrane.…”

“…In our previous work [16], we have demonstrated that AF700-Tfn behaves as a FRET donor that can effectively transfer energy to AF750-Tfn acceptor molecules upon internalization of Tfn-transferrin receptor (Tfn-TfnR) complexes into cancer cells using FLIM. We also have shown that FD% levels, which are directly correlated to FRET efficiency, are positively dependent on A:D ratios and independent of acceptor levels upon Tfn-TfnR binding and internalization into cancer cells.…”