Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor

Abstract

Rationale: Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes. Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results. Results: First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of Tf-AF700/Tf-QC-1 uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

Keywords: FRET; breast cancer; lifetime imaging; metabolism; target engagement.

Figure 1

Multiplexed Forster resonance energy transfer macroscopy fluorescence lifetime imaging (FRET MFLI) and metabolic in vivo imaging. (A) Schematic representation of IRDye 800CW 2-DG (2-DG) based metabolic imaging and measurement of target engagement via FRET-MFLI in vivo imaging. FRET signal is detected by the reduction of donor fluorophore lifetime upon binding of donor- and acceptor-labeled Tf ligands to the transferrin-receptor (TfR). 2-DG fluorescence intensity indicates the location of 2-DG molecules, either diffused in extracellular space or internalized in tumor cells via glucose transporter 1 (GLUT1). (B-D) Sequential 2-DG and FRET MFLI in vivo imaging. (B) Imaging protocol includes mouse overnight fasting up to 12 h prior to 2-DG injection and imaging at 24 h post-injection (p.i.), followed by transferrin-Alexa Fluor700 (Tf-AF700)/Tf-AF750 FRET pair (acceptor : donor, A:D = 2:1) injection and imaging 1 h p.i. (C) 2-DG fluorescence intensity image (left panel) and respective graph displaying distribution of 2-DG signal per region of interest (ROI) pixel (right panel) of mouse liver (LV), tumor xenograft (TM) and urinary bladder (UB). (D) Left panel displays Tf-AF700 intensity (total Tf, including bound and unbound) and FD% (bound Tf) map of liver (LV), tumor (TM) and urinary bladder (UB) in mouse sequentially injected with 2-DG and Tf-AF700/Tf-AF750 and imaged with MFLI at 24 h and 1 h p.i., respectively. Right panel shows distribution of FRET donor fraction (FD%) signal per ROI pixel. Rectangle box indicates 25%-75% pixel values, horizontal line indicates median value and vertical line indicates range within 1.5 quartile. Statistical analysis of FD% and 2-DG values between tumor and bladder using student t-test is shown in Table S1.

Figure 2

Comparison of AF700/QC1 vs. AF700/AF750 FRET pairs using IgG conjugates in wide-field macroscopy fluorescence lifetime (MFLI) imager. Fluorescence intensity (A) and FRET donor fraction (FD%) (B) maps of AF700/AF750 and AF700/QC1 FRET samples. (C) Donor fluorescence lifetime decay of AF700/AF750 (red line) and AF700/QC1 (black line) samples with acceptor: donor (A:D) ratio 0:1 and 2:1; IRF is instrument response function. (D) Quantification of FD% vs A:D ratio for AF700/AF750 (red) and AF700/QC-1 (black) antibody binding FRET assay. Data presented as a mean ± standard deviation. Statistical analysis of AF700/QC1 vs. AF700/AF750 FRET pairs at increasing A:D ratios (not significant; p > 0.05) using repeated measures Anova is described in Table S2.

Figure 3

(A-B) Transferrin-Alexa Fluor700 (Tf-AF700) and Tf-QC-1 internalization and trafficking in cancer cells. (A) Maximum intensity projections of z-stacks consisting of 10-12 optical slices of T47D human cancer cells loaded with Tf-AF700 or Tf-QC-1 and processed for anti-transferrin receptor (TfR; green) and anti-Tf (red) immunostaining. Scale bar = 20 µm. (B) Quantification of TfR and Tf colocalization analysis using Pearson's correlation coefficient throughout the entire z-stack using Imaris imaging analysis software. Data is presented as a mean of five independent region of interests (ROIs) from n=5 confocal z-stacks. Error bars represent standard deviation. Statistical analysis of colocalization between TfR and Tf-AF700 or Tf-QC-1, as indicated by Pearson's coefficient (not significant; p > 0.05) using two-tailed t-test is shown in Table S3. (C-F) Near infrared (NIR) Forster resonance energy transfer fluorescence lifetime microscopy (FRET FLIM) assay in cancer cells using dark quencher acceptor transferrin-QC-1 (Tf-QC-1) conjugates. (C) NIR FLIM time-correlated single photon counting (TCSPC) data. The representative images of fluorescence intensity and lifetime map (; mean lifetime) in T47D cells treated with transferrin-Alexa Fluor700 (Tf-AF700) (acceptor: donor, A:D=0:1), Tf-AF700 and Tf-AF750 (A:D=2:1) or Tf-AF700 and Tf-QC-1 (A:D=2:1); pseudo-color range= 300-1,500 ps. Both fluorescence intensity and lifetime distributions show punctate endocytic structures containing transferrin receptor (TfR)-Tf complexes. Scale bar= 50 µm. (D) Representative fitting curves and instrument response function (IRF), the fluorescent lifetime decay in the single and double-labeled cells was determined by comparing the fitting of the decay data using both single- and double-exponential decay models. (E) Comparison of fluorescent lifetime distribution in T47D cells treated with Tf-AF700 (A:D=0:1), Tf-AF700 and Tf-AF750 (A:D=2:1) or Tf-AF700 and Tf-QC1 (A:D=2:1). (F) Comparison of FRET donor fraction (FD%) levels in T47D cells treated with Tf-AF700/Tf-AF750 or Tf-AF700/Tf-QC1 FRET pairs at various A:D ratios. Analysis was performed using 10 distinct pixel coordinates from 5 independent region of interests (ROIs). Error bars represent standard deviation. Statistical analysis of Tf-AF700/Tf-QC1 vs. Tf-AF700/Tf-AF750 FRET pairs at increasing A:D ratios (significant; p < 0.05) using repeated measures Anova is described in Table S4.

Figure 4

Near infrared (NIR) Forster resonance energy transfer macroscopy fluorescence lifetime imaging (FRET MFLI) of transferrin-QC-1 (Tf-QC-1) as an acceptor in live intact animals. (A) Intensity and FRET donor fraction (FD%) maps in Matrigel plugs containing T47D cancer cells preloaded with Tf-AF700/Tf-AF750 or Tf-AF700/Tf-QC-1 ligands with indicated acceptor: donor (A:D) ratio. Anesthetized animals were imaged using wide-field MFLI imager. Because of the different size of injections and the localization of the plug on the animal body, significant variation in fluorescence intensity was detected across the different A:D ratios and FRET pairs in Matrigel plugs. (B) FRET quantification in cancer cells containing Matrigel plugs in vivo: box plots showing pixel distribution of FRET donor fraction at various A:D ratios.

Figure 5

Whole-body Forster resonance energy transfer macroscopy fluorescence lifetime imaging (FRET MFLI) using dark quencher acceptor QC-1 in live intact animals. Nude mice were tail-vein injected with 40 µg/mL transferrin-Alexa Fluor 700 (Tf-AF700) and 80 µg/mL Tf-AF750 or Tf-QC-1 (acceptor: donor, A:D = 2:1) and imaged using MFLI imager at 2, 6 and 24 h post-injection (p.i.). (A) Representative liver (LV) and urinary bladder (UB) region of interest (ROI) images of donor intensity (total Tf, including bound and unbound) and FRET donor fraction (FD%) (bound Tf) levels at 24 h p.i. (B) Graph displaying distribution of FD% signal per ROI pixel in livers and bladders at 24 h p.i. (C) Time course of Tf uptake in livers of mice injected with Tf-AF700 (n=1), Tf-AF700/Tf-AF750 (n=4) and Tf-AF700/Tf-QC-1 (n=3) from two independent experiments. Error bars represent standard deviation. All mice images are shown in Figure S6.

Figure 6

IRDye 800CW 2-DG (2-DG) and Forster resonance energy transfer macroscopy fluorescence lifetime imaging (FRET MFLI) using dark quencher acceptor QC-1 in live intact animals. (A) Imaging protocol including fasting, followed by 2-DG and near-infrared transferrin (NIR-Tf) pair injections and MFLI imaging using consecutive imaging at 750 nm and 695 nm excitation steps. (B) Region of interest (ROI) images of 2-DG, Tf-AF700 intensity and FRET donor fraction (FD%) levels of liver (LV), tumor (TM) and urinary bladder (UB) in mice injected with 2-DG and Tf-AF700/Tf-QC-1 and imaged with FRET MFLI at 6 h post injection (p.i.). (C) Distribution of 2-DG fluorescence intensity per ROI pixel in tumors and bladders. (D) Distribution of FD% signal per ROI pixel in tumors and bladders. Asterisks indicate p<0.05 (significant). Statistical analysis is presented in Table S5.

Figure 7

Simultaneous IRDye 800CW 2-DG (2-DG) and Forster resonance energy transfer macroscopy fluorescence lifetime imaging (FRET MFLI) using dark quencher acceptor QC-1 in live intact animals: (A) Imaging protocol including fasting, followed by 2-DG and near-infrared transferrin (NIR-Tf) pair injections and MFLI imaging step at 695 nm excitation. (B) Images of 2-DG, Tf-AF700 intensity and FRET donor fraction (FD%) levels of liver (LV), tumor (TM) and urinary bladder (UB) in mice injected with 2-DG and Tf-AF700/Tf-QC-1 and simultaneously imaged using FRET MFLI at 24 h post injection (p.i.); single excitation at 695 nm. 2-DG mask was used to determine organ region of interest (ROIs) with the same number of pixels for extraction of FRET and 2-DG data. (C) Magnified pixels of 2-DG intensity and FD% in tumors. (D) Distribution of 2-DG fluorescence intensity per ROI pixels in tumors and bladders. (E) Distribution of FD% signal per ROI pixels in tumors and bladders. Asterisks indicate p<0.05 (significant). Statistical analysis is presented in Table S6. (F) Graph of normalized to liver values 2-DG intensity and (G) FD%. Error bars represent 95% confidence interval, asterisk indicate p<0.05 (significant). Statistical analysis is presented in Table S7.

Figure 8

Immunohistochemical staining of consecutive tumor sections from M1 (A-D), M2 (E-H) and M3 (I-L) tumor xenografts using hematoxylin and eosin (A, E & I), anti-human transferrin (Tf) (B, F & J), anti-human transferrin receptor (TfR) (C, G & K) and anti-glucose transporter 1 (GLUT1) (D, H & I). NovaRED was used as peroxidase substrate (brown color), sections were counterstained with methyl green. White dashed lines delineate necrotic core (NC). Scale bar = 100 µm.