KEYWORDS: Tumors, Tumor growth modeling, Multiplexing, Fluorescence resonance energy transfer, Systems modeling, Signal detection, Luminescence, Mode conditioning cables, In vivo imaging, Therapeutic antibodies
Macroscopic fluorescence lifetime FRET imaging (MFLI-FRET) presents a much-needed analytical tool to non-invasively quantify drug-receptor engagement in tumors and other organs in preclinical studies. We demonstrate the specificity and sensitivity of MFLI-FRET for direct and robust measurement of trastuzumab-HER2 target engagement in various types of breast and ovarian cancer tumor xenograft models. Simultaneous metabolic imaging with IRDye 800 CW 2-DG reveals that intracellular delivery of drug is associated with 2-DG lifetime and likely reflects tumors’ microenvironment and perfusion state.
KEYWORDS: In vivo imaging, Fluorescence lifetime imaging, Sensors, In vitro testing, Visible radiation, Tumors, Near infrared, Luminescence, Fluorescence resonance energy transfer, Detector development
Near-Infrared wide-field Fluorescence Lifetime Imaging (FLI) has become an increasingly popular method due to its unique specificity in sensing the cellular micro-environment and/or protein-protein interactions via FRET, but the approach is still challenging due to inefficient detection modules. Here, we report on the characterization of a large gated SPAD array, SwissSPAD2, towards in vivo preclinical imaging of FLI-FRET. Fluorescence decay fitting as well as phasor analysis are used to demonstrate the ability of SwissSPAD2 to accurately quantify short lifetimes and associated lifetime parameters in both in vitro and in vivo experiments, in full agreement with gated ICCD measurements.
KEYWORDS: Tumors, Fluorescence resonance energy transfer, Near infrared, Fluorescence lifetime imaging, Signal detection, Resonance energy transfer, Microscopy, Imaging systems
There is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies in oncology. We have established Macroscopic Fluorescence Lifetime Imaging (MFLI) associated with Forster Resonance Energy Transfer (FRET) to report quantitatively on antibody-target engagement in live intact animals at the organ level. Here, we use FLI FRET imaging to quantify the binding of near infrared labeled antibody drugs to breast cancer cells or tumor xenografts. In combination with antibody engineering, this approach provides a robust analytical tool to optimize the binding and uptake of antibody-target binding during tumor progression and treatment.
Overexpression of Human EGF Receptor 2 (HER2) in cancer is a marker of aggressive metastatic disease and poor prognosis. Anti-HER2 humanized monoclonal antibody trastuzumab (TZM) has been successfully used in the clinic over the last decades. However, a large fraction of eligible patients display resistance to this therapy. This calls for a deeper investigation of HER2 interaction with other members of HER tyrosine kinase receptors and modulation of their endocytic trafficking upon TZM treatment. Forster resonance energy transfer Fluorescence lifetime microscopy (FLIM- FRET) offers a robust approach to monitor HER2 homo and heterodimerization via the reduction of donor fluorophore lifetime. The objective of this study was to assess the dynamics of HER receptor homo and heterodimerization behavior via FLIMFRET by using near-infrared (NIR) FRET pair labeled anti-HER2 and anti-EGFR therapeutic antibodies in HER2- overexpressing breast cancer cells. In addition, we tested our new deep learning platform DL4FLIM adapted for automated analysis of all datasets. Herein, we report a first attempt to quantify NIR FRET pair labeled cetuximab (CTM, as a donor) and TZM (as an acceptor) binding to their receptors EGFR and HER2 respectively in AU565 cells. As a control, we also performed and human isotype IgG FLIM -FRET and found it completely non-specific. Our data demonstrate both the occurrence of FRET between NIR-labeled probes CTM and TZM as well as between CTM-CTM bound to their respective receptors. This proof-of -principal study demonstrated feasibility of monitoring HER2 hetero receptor FRET FLIM to better understand mechanism of TZM resistance.
Iron is highly regulated in the body, since it is an essential element required for life. Fundamental understanding of the key processes that underlie the intracellular transport of iron will have a decisive impact on advancing treatment of diseases that are caused by iron deficiency and iron overload, e.g., anemias and hereditary hemochromatosis. Improved knowledge of iron intracellular transport will also provide insight into many other diseases where iron modulates the pathogenic process, e.g., metabolic syndrome, diabetes, neurodegenerative diseases, and cancer. Measuring the iron-bound form of transferrin in intact biological samples remains a technical challenge that needs to be overcome to understand regulation of endosomal iron release in cells and tissues. Serum transferrin (Tf) is a key regulator of systemic and cellular iron transport. Tf binds ferric iron (Fe3+) for transport throughout the body and delivery into cells via the transferrin receptor (TfR). The iron-bound Tf-TfR complex is endocytosed, and upon acidification of early endosome, the iron is released. Importantly, disruption of iron homeostasis has been linked to cancer progression. Although iron transport has been studied in detail, measurements of iron-bound Tf in tumor tissues are still lacking. Previously, we have developed and validated a Raman hyperspectral imaging technique that identified the iron-bound Tf peak at ~1300 cm-1 Raman shift. Here, we further investigate the variation in peak intensity within frozen tissue sections of T47D and MDA-MB-231 breast cancer tumor xenografts, which represent luminal and basal cancers, respectively. Our results indicate that Raman spectral imaging can be used to evaluate the iron-bound form of Tf in xenograft sections. Measurements of iron-bound Tf in tumor tissues will permit further characterization of iron transport in breast cancer.
KEYWORDS: Luminescence, Fluorescence resonance energy transfer, Near infrared, Visible radiation, Resonance energy transfer, In vitro testing, In vivo imaging, 3D acquisition, 3D image processing, 3D metrology
Quantification of ligand-receptor engagement in human breast cancer cells and tumor xenografts has been performed using fluorescence lifetime Forster resonance energy transfer (FLI-FRET) imaging at multiscale, from in vitro microscopy to in vivo macroscopy and across visible to near-infrared wavelengths. We have developed a 3D convolutional neural network architecture, named FLI-Network (FLI-Net), to process fluorescence lifetime decays acquired by either Time-Correlated Single-Photon Counting (TCSCP)- or gated ICCD- based instruments. FLI-FRET ability to measure target engagement across different imaging platforms as well as post-processing analysis approaches can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.
Iron is an essential element required for human life, and is highly regulated in the body. Iron deficiency leads to many adverse health effects, such as anemias. The exact mechanisms of iron release in cells are not well known. We developed a Raman micro-spectroscopy technique that allows detection of transferrin (Tf) bound iron inside intact human cells. Ferric iron (Fe3+) bound to serum Tf is internalized into cells via the transferrin receptor (TfR). Methods that allow determining when and where Tf releases iron inside a cell lead to a better understanding of disease progression, including cancer. We have previously shown that Raman micro-spectroscopy is able to detect and quantify the Tf-bound iron in human breast cancer T47D cells. In this work, we applied hyperspectral Raman imaging to visualize the spatial distribution of Tf-bound iron in human breast cancer T47D and MDAMB231 cells internalized with iron-loaded Tf. We have also shown that Raman imaging can quantify the amount of iron under different times of Tf internalization prior to fixation. Raman microspectroscopy provides a unique way to determine the amount of iron under different Tf internalization times by employing the Raman metric, which was used to quantify iron content in iron bound Tf (holo-Tf) samples. Importantly, Raman microspectroscopy can be used to follow iron release from Tf in breast cancer cells. Determining the kinetics and location of iron release in cancer cells is key to further our understanding of iron metabolism during cancer progression.
Human EGF receptor 2 (HER2) is an important oncogene and marker of aggressive metastatic cancer, found in up to 20% of oncologic patients. Anti-HER2 humanized monoclonal antibody trastuzumab (TZM) has been successfully used over the last two decades. However, both primary and acquired TZM resistance calls for the deeper investigation on TZMHER2 binding, internalization and trafficking/degradation in cancer cells in vitro and in vivo. Fluorescence lifetime FRET imaging (FLIM FRET) offers a unique approach to monitor TZM-HER2 binding followed by their uptake into target cells via the reduction of donor fluorophore lifetime. In this study, we characterized for the first time TZM-AF700 uptake and its relation to HER2 expression in AU565 human breast cancer cell line using confocal microscopy. Further, we have quantified the dimerization of HER2 via NIR FLIM FRET in vitro microscopy. Extensive analysis confirmed high specificity and efficiency of TZM FRET signal. Interestingly, we observed a significant heterogeneity of FRET within the cells: the highest TZM FRET levels occurred at the plasma membrane, whereas less if any donor lifetime reduction was registered in the perinuclear endosomes. These results suggest that HER2 dimers undergo dissociation or degradation upon TZM binding and trafficking. Overall, this study provides a good foundation for in vivo TZM FRET imaging of target engagement in preclinical studies.
Fluorescence lifetime imaging (FLI) has become an invaluable tool in the biomedical field by providing unique, quantitative information about biochemical events and interactions taking place within specimens of interest. Applications of FLI range from superresolution microscopy to whole body imaging using visible and near-infrared fluorophores. However, quantifying lifetime can still be a challenging task especially in the case of bi-exponential applications. In such cases, model based iterative fitting is typically employed but necessitate setting up multiple parameters ad hoc and can be computationally expensive. These facts have limited the universal appeal of the technique and methodologies can be specific to certain applications/technology or laboratory bound. Herein, we propose a novel approach based on Deep Learning (DL) to quantify bi-lifetime Forster Resonance Energy Transfer (FRET). Our deep neural network outputs three images consisting of both lifetimes and fractional amplitude. The network is trained using synthetic data and then validated on experimental FLI microscopic (FLIM) and macroscopic data sets (MFLI). Our results demonstrate that DL is well suited to quantify wide-field bi-exponential fluorescence lifetime accurately and in real time, even when it is difficult to obtain large scale experimental training data.
KEYWORDS: Fluorescence resonance energy transfer, Luminescence, Near infrared, In vivo imaging, Multiplexing, Confocal microscopy, Fluorescence lifetime imaging, Resonance energy transfer, Organisms, Absorbance
Fluorescence lifetime imaging (FLI) is widely regarded as the most robust means to utilize Förster resonance energy transfer (FRET) to study protein-protein interactions. Upon donor excitation, FLI estimates FRET occurrence by determining the reduction of the fluorescence lifetime of the donor when in close proximity (2-10nm) of an acceptor. Recently, macroscopic FLI-FRET (MFLI-FRET) in living mice has been attained by using a near-infrared (NIR)-labeled transferrin (Tf) FRET pair. To harness the potential of multiplexing FLI-FRET in live organisms, it is necessary to employ NIR dark acceptor fluorophores to avoid spectral cross-contamination. IRDye QC-1 (QC-1, LI-COR) is a dark quencher that has a broad absorbance spectrum encompassing the NIR range. Herein, we demonstrate that QC-1 is an effective acceptor for quenching of Alexa Fluor 700 (AF700) via FRET in IgG antibody interactions. Additionally, we characterized the cellular uptake of Tf conjugated to QC-1 using confocal microscopy, NIR FLI microscopy, and wide-field MFLI imaging. The AF700/QC-1 FRET pair exhibits a linear trend in FRET with increasing A:D ratio. In vivo MFLI-FRET imaging was performed under reflectance geometry to compare Tf AF700/AF750 and Tf AF700/QC1 at A:D ratio 2:1 2, 6, and 24h post-injection. FRET was detected in the liver, an important organ for pharmacokinetic studies that shows elevated expression of transferrin receptor (TfR), but not in the bladder, an important organ for drug clearance. Although we observed slightly less FRET using AF700/QC-1 compared to AF700/AF750, both in vitro and in vivo, we found that QC-1 is suitable for FRET imaging and multiplexing approaches.
There is a great unmet need to non-invasively quantify the active versus passive delivery of drugs in preclinical studies. Quantifying probe-receptor interactions, or target engagement in living systems, is critical as it directly correlates with drug efficacy. We selected the transferrin receptor (TfR) as a target, since TfR is overexpressed in breast cancer cells. MFLI-FRET enables the quantification of transferrin (Tf) internalization into the cells by measuring FRET between receptor-bound Tf donor and acceptor near infrared (NIR) fluorophore pairs, based on the reduction of donor fluorophore lifetime in live intact mice. In this study we compared FRET levels in aggressively growing triple negative MDA-MB-231 breast cancer tumors to estrogen receptor positive T47D tumors, which are less dense and slowly growing. Unlike in T47D xenografts, in MDA-MB-231 tumors FRET donor fraction (FD%) was very low or undetectable in first few hours post injection. Only by 24-48 h p.i. FD% reached comparable to T47D FD% levels. Immunohistochemical analysis of excised tumors showed that TfR density levels were similar in both types of tumors. This suggests that ligand penetration inside the MDA-MB-231 tumors is impaired due to microenvironment features, such as the perfusion defects, elevated stroma stiffness and interstitial fluid pressure. We plan to measure functional blood flow using contrast-enhanced Doppler Ultrasound imaging in tumors to further validate MFLI-FRET data. Overall, MFLI-FRET is well suited for guiding the development of targeted drug therapy in preclinical studies as analytical tool to monitor and quantify drug penetration in heterogeneous breast cancer xenografts.
Receptor-targeting has been considered a method of choice for drug delivery in oncology because many types of cancer have elevated expression of a specific receptor. Recently, our group has demonstrated that quantification of interacting receptors, particularly homo-dimers, can be achieved in vivo using lifetime-based Förster Resonance Energy Transfer (FRET). However, quantification of FRET is typically performed in microscopy by either intensity- or lifetime-based measurement. Herein, we report on cross-validation between intensity and lifetime-based FRET for in vivo applications. In particular, we demonstrated dynamic in vivo FRET quantification using both intensity and lifetime measurements for assessing transferrin receptor engagement in live intact animals. Using hybridized oligonucleotides as FRET standard, we obtained the same FRET quantification via both intensity and lifetime-based measurements. Overall, both measurement methods provided the same FRET quantification trends of receptor engagement over 120 minutes of imaging. However, intensity FRET approach required 18 measurements (17 of which were used for calibration), whereas lifetime FRET required only one measurement. Hence, macroscopic fluorescence lifetime FRET presents superior, more rapid and simple method for the assessment of targeted drug delivery in longitudinal preclinical studies.
KEYWORDS: Near infrared, Luminescence, Fluorescence resonance energy transfer, Receptors, Microscopy, In vivo imaging, Cancer, In vitro testing, Proteins, Therapeutic antibodies
Monitoring the binding of protein ligands and therapeutic antibodies to their respective receptors (target engagement) is crucial to compound prioritization in anti-cancer targeted drug screening. However, current in vivo optical imaging techniques cannot distinguish between co-localization and actual receptor-ligand binding at the tumor region. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The homodimeric nature of TfR allows for measuring fluorescence lifetime FRET (FLI-FRET) to quantitate the TfR-Tf binding and internalization into cancer cells, based on the reduction of donor fluorophore lifetime. Near infrared (NIR) FLI-FRET has been used to directly visualize and quantitate TfR-Tf binding and internalization by providing the fraction of donor-labeled entity that is interacting with its respective receptor. NIR FLI-FRET has been validated at multiscale, using both in vitro microscopy as well as in vivo macroscopy whole-body deep imaging assays using different NIR FRET pairs. Accuracy of NIR FLI-FRET quantitation has been compared between fluorescence intensity and lifetime measurements using both microscopy and macroscopy fluorescence imaging. NIR FLI-FRET employs well-characterized quantitative lifetime-based metrics, standard in FRET microscopy, but with the additional benefit of a seamless multiscale technological platform. In summary, we have successfully demonstrated quantitative imaging of receptor-mediated binding and uptake of Tf using NIR FLI-FRET microscopy and macroscopy imaging in vitro and in vivo, respectively. This novel approach can be extended to other receptors, currently targeted in oncology. Hence, NIR FLI-FRET can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.
Preclinical imaging using near-infrared (NIR) fluorescent markers is a non-invasive approach to molecular imaging with many applications ranging from drug delivery monitoring to immune cell tracking and image-guided surgery. It is however limited by signal absorption and scattering within tissues, which make it difficult to localize and quantify signal sources. Moreover, broad absorption and emission spectra make it difficult to use multiple NIR dyes for multiplexed imaging. Fluorescence lifetime imaging (FLI) helps solve some of these problems by providing a contrast mechanism that is sensitive to the intracellular environment but not to signal intensity and can separate dyes with similar spectra but different lifetimes. So far, however, in vivo FLI has found few applications due to the complexity of standard FLI data acquisition and analysis. Here we show that in vivo wide-field time-gated macroscopic FLI (MFLI) imaging, combined with phasor analysis of NIR fluorescence lifetime data, addresses most of these issues. We illustrate these capabilities with a study of in vivo transferrin ligand-receptor engagement dynamic in anesthetized mice using Förster resonant energy transfer (FRET), and validate our results by comparing them with standard lifetime fitting. Our approach speeds up data analysis by several orders of magnitude and requires less data, while providing easy-to-use and interpret data visualization, opening up the possibility of real-time in vivo MFLI of fast dynamic processes.
KEYWORDS: Fluorescence resonance energy transfer, In vivo imaging, Receptors, Cancer, Optical imaging, Tumors, Imaging systems, Deep tissue imaging, Luminescence, Oncology
Targeted drug delivery in cancer treatment has been a major area of development in the past decades. However, there is a great need in preclinical studies to not only assess the drug distribution but also monitor and quantify target engagement in vivo to ensure maximal drug delivery efficacy. Macroscopic Fluorescence Lifetime Imaging of Förster Resonance Energy Transfer (MFLI-FRET) is a unique non-invasive imaging methodology to monitor in vivo receptor-target interactions and directly discriminate between unbound and internalized ligands in pre-clinical studies. In this study, we capitalized on the homodimeric nature of the transferrin receptor (TfR) to quantify transferrin (Tf) internalization into cancer cells by measuring FRET between receptor-bound Tf-labeled donor and acceptor near-infrared (NIR) fluorophore pairs. We found a strong correlation between FRET levels and Tf internalization into tumor cells despite the significant heterogeneity of tumors regarding their size and cellular density. In contrast, no correlation between MFLI-FRET and TfR levels was observed, underscoring the insufficient link between receptor density and intracellular drug delivery. Additionally, we compared results of in vivo MFLI-FRET imaging with intensity-based FRET (using the IVIS pre-clinical optical imaging system). Intensity-based imaging failed to provide reliable and consistent results, showing significantly higher FRET quantification in negative control samples. Overall, we demonstrated that MFLI-FRET enables real time in vivo information on receptor ligand engagement in deep tissues, conversely to current commercial systems. Hence, MFLI-FRET is well positioned to play a critical role in accelerating the optimization of targeted drug delivery efficacy in pre-clinical studies.
KEYWORDS: Fluorescence resonance energy transfer, In vivo imaging, Fluorescence lifetime imaging, Breast cancer, Receptors, Cancer, Luminescence, Near infrared, Mammography, Tumors
Fluorescence Lifetime Förster Resonance Energy Transfer (FLIM-FRET) is a unique non-invasive imaging platform to monitor and quantify in vivo target engagement in pre-clinical studies. FLIM FRET is a valuable tool in targeted drug delivery due to its nanoscale-range molecular resolution that detects near-infrared labeled ligand binding to dimerized receptors followed by their uptake into cancer cells in vivo. Various imaging platforms, including PET, lack the ability to directly discriminate between unbound and internalized ligands. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The dimeric nature of TfR allows for the quantification of Tf internalization into cancer cells by measuring FLIM FRET between receptor-bound Tf donor and acceptor NIR fluorophore pairs, based on the reduction of donor fluorophore lifetime in live mice. We analyzed tumor morphology, the level of expression of TfR, estrogen receptor (ER) and Tf accumulation in human breast cancer tumor xenografts. We found a remarkable heterogeneity of breast cancer tumors regarding their size, cell density, TfR and ER expression and Tf uptake. The results of this study confirm a strong correlation between in vivo NIR FLIM FRET and ex vivo evaluation of Tf uptake into tumor tissues, thus validating FD% as a robust measure of the target engagement of TfR-Tf in tumor cells in vivo.
KEYWORDS: Fluorescence resonance energy transfer, In vivo imaging, Fluorescence lifetime imaging, Therapeutics, Receptors, Optical imaging, Resonance energy transfer, Oncology, Near infrared, Microscopy, Tumors, Cancer
To guide the development of targeted therapies with improved efficacy and accelerated clinical acceptance, novel imaging methodologies need to be established. Toward this goal, fluorescence lifetime Förster resonance energy transfer (FLIM-FRET) imaging assays capitalize on the ability of antibodies or protein ligands to bind dimerized membrane bound receptors to measure their target engagement levels in cancer cells. Conventional FLIM FRET microscopy has been widely applied at visible wavelengths to detect protein-protein interactions in vitro. However, operation at these wavelengths restricts imaging quality and ability to quantitate lifetime changes in in vivo small animal optical imaging due to high auto-fluorescence and light scattering. Here, we have analyzed the uptake of iron-bound transferrin (Tf) probes into human breast cancer cells using FLIM-FRET microscopy in the visible and near-infrared (NIR) range. The development of NIR FLIM FRET microscopy allows for the use of quantitative lifetime-based molecular assays to measure drug-target engagement levels at multiple scales: from in vitro microscopy to in vivo small animal optical imaging (macroscopy). This novel approach can be extended to other receptors, currently targeted in oncology. Hence, lifetime-based molecular imaging can find numerous applications in drug delivery and targeted therapy assessment and optimization.
Targeted drug delivery is a critical aspect of successful cancer therapy. Assessment of dynamic distribution of the drug provides relative concentration and bioavailability at the target tissue. The most common approach of the assessment is intensity-based imaging, which only provides information about anatomical distribution. Observation of biomolecular interactions can be performed using Förster resonance energy transfer (FRET). Thus, FRET-based imaging can assess functional distribution and provide potential therapeutic outcomes. In this study, we used wide-field lifetime-based FRET imaging for the study of early functional distribution of transferrin delivery in breast cancer tumor models in small animals. Transferrin is a carrier for cancer drug delivery. Its interaction with its receptor is within a few nanometers, which is suitable for FRET. Alexa Fluor® 700 and Alexa Fluor® 750 were conjugated to holo-transferrin which were then administered via tail vein injection to the mice implanted with T47D breast cancer xenografts. Images were continuously acquired for 60 minutes post-injection. The results showed that transferrin was primarily distributed to the liver, the urinary bladder, and the tumor. The cellular uptake of transferrin, which was indicated by the level of FRET, was high in the liver but very low in the urinary bladder. The results also suggested that the fluorescence intensity and FRET signals were independent. The liver showed increasing intensity and increasing FRET during the observation period, while the urinary bladder showed increasing intensity but minimal FRET. Tumors gave varied results corresponding to their FRET progression. These results were relevant to the biomolecular events that occurred in the animals.
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