HER2-HER3 dimer quantification by FLIM-FRET predicts breast cancer metastatic relapse independently of HER2 IHC status

Weitsman, Gregory; Barber, Paul R.; Nguyen, Lan K.; Lawler, Katherine; Patel, Gargi; Woodman, Natalie; Kelleher, Muireann; Pinder, Sarah; Rowley, Mark; Ellis, Paul; Purushotham, Arnie; Coolen, A. C. C.; Kholodenko, Boris N.; Vojnovic, Borivoj; Gillett, Cheryl; Ng, Tony

doi:10.18632/oncotarget.9963

Cited by 31 publications

(39 citation statements)

References 60 publications

(78 reference statements)

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“…This finding goes against the accepted practice of diagnosis and targeted treatment based on receptor overexpression in tumors. However, in a recent report of Weitsman et al 32 the lack of correlation between the overexpression level of HER2 and degree of HER2-HER3 dimerization as an indicator of metastatic likelihood was also shown. It has also been shown that PD-L1 expression does not accurately determine the therapeutic efficacy of anti-PD-1 drugs 33 .…”

Section: Discussionmentioning

confidence: 90%

Quantitative Imaging of Receptor-Ligand Engagement in Intact Live Animals

Rudkouskaya

Sinsuebphon

Ward

et al. 2017

Preprint

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Maintaining an intact tumor environment is critical for quantitation of receptor-ligand engagement in a targeted drug development pipeline. However, measuring receptor-ligand engagement in vivo and non-invasively in preclinical settings is extremely challenging. We found that quantitation of intracellular receptor-ligand binding can be achieved using wholebody macroscopic lifetime-based Förster Resonance Energy Transfer (FRET) imaging in intact, live animals bearing tumor xenografts. We determined that FRET levels report on ligand binding to transferrin receptors conversely to raw fluorescence intensity. We then established that FRET levels in heterogeneous tumors correlate with intracellular ligand binding but strikingly, not with ubiquitously used ex vivo receptor expression assessment. Hence, MFLI-FRET provides a direct measurement of systemic delivery, target availability and intracellular drug delivery in intact animals. Here, we have used MFLI to measure FRET longitudinally in intact animals for the first time. MFLI-FRET is well-suited for guiding the development of targeted drug therapy in heterogeneous intact, live small animals.The most potent drugs are useless if they are not able to engage their targets in living systems. The majority of failed clinical trials are due to the lack of translation of in vitro data of drug-target engagement into intact living organisms. Therefore a main issue in drug development is to quantify target engagement in a non-invasive manner in longitudinal preclinical studies in live animals. There are two main approaches to estimate drug binding in animal studies: ex vivo invasive methods, e.g. biochemical, radiotracers, flow cytometry, and immunohistochemistry analysis, and measuring binding of the drug to plasma proteins. The latter proved to be a poor indication of drug efficiency (true target engagement) in vivo 1 . Traditional in vivo imaging platforms, including PET, lack the ability to discriminate between unbound and receptor-bound macromolecules 2,3 . Especially in the case of cancer pre-clinical models, where the enhanced permeability and retention (EPR) effect, that results from the aberrant leaky microvasculature and inadequate lymphatic drainage of solid tumors, leads to the accumulation of exogenously added protein ligands and antibodies at the tumor region 4 . Therefore, establishing macroscopic colocalization of labeled ligand or antibodies within the tumor region does not guarantee or even indicate actual ligand-or antibody-target engagement in the tumor cells.Fluorescence lifetime imaging of Forster Resonance Energy Transfer (FLIM FRET) has found numerous powerful applications in the biomedical field 5 . FLIM quantifies FRET occurrence by estimating the reduction of the fluorescence lifetime of the donor fluorophore when in close proximity (2-10nm) to one or more acceptor fluorophores 5,6 . However, to date, FLIM FRET has been confined to microscopic techniques and its translation to the in vivo pre-clinical paradigm is just beginning. Herein, we introduce M...

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Section: Discussionmentioning

confidence: 90%

Quantitative Imaging of Receptor-Ligand Engagement in Intact Live Animals

Rudkouskaya

Sinsuebphon

Ward

et al. 2017

Preprint

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“…This discovery has led to the invention of targeted therapies that specifically disrupt the ability of these proteins to dimerize, and with some success; e.g., a monoclonal antibody for EGFR, cetuximab, is now used alongside chemotherapy for certain colorectal and head & neck tumors, and trastuzumab is used to target HER2 in some breast cancers. We have recently published the use of FLIM/FRET to detect HER family dimerization in archived patient tissue from breast and colorectal cancer studies and clinical trials (30,35) and correlate this with the response to targeted treatment.…”

Section: Use Case I: Segmentationmentioning

confidence: 99%

“…The FRET Efficiency score is calculated for this region using the standard relationship.) B) Weka Trainable Segmentation plugin used to train the segmentation and showing a segmentation result (35). FLIMJ user interface showing a typical transient and fit from the tissue.…”

Section: Use Case I: Segmentationmentioning

confidence: 99%

FLIMJ: an open-source ImageJ toolkit for fluorescence lifetime image data analysis

Gao

Barber

Chacko

et al. 2020

Preprint

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In the field of fluorescence microscopy, there is continued demand for dynamic technologies that can exploit the complete information from every pixel of an image. One imaging technique with proven ability for yielding additional information from fluorescence imaging is Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM allows for the measurement of how long a fluorophore stays in an excited state and is affected by changes in its chemical microenvironment such as proximity to other fluorophores, pH, and hydrophobic regions. This ability to provide information about the microenvironment has made FLIM a powerful tool for cellular imaging studies ranging from metabolic measurement to measuring distances between proteins. The increased use of FLIM has necessitated the development of computational tools for integrating FLIM analysis with image and data processing. To address this need, we have created FLIMJ, an ImageJ plugin and toolkit that allows for easy use and development of extensible image analysis workflows with FLIM data. Built on the FLIMLib decay curve fitting library and the ImageJ Ops framework, FLIMJ offers FLIM fitting routines with seamless integration with other ImageJ components, and the ability to be extended to create complex FLIM analysis workflows. Building on ImageJ Ops also enables FLIMJ’s routines to be used with Jupyter notebooks and integrate naturally with science-friendly programming in e.g. Python and Groovy. We show the extensibility of FLIMJ in two analysis scenarios, lifetime-based segmentation and image colocalization, and validate the fitting routines by comparing against industry FLIM analysis standards.

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“…4B; Conway et al, 2014Conway et al, , 2017Newman et al, 2011). Applications in cell proliferation (Harvey et al, 2008;Mochizuki et al, 2001), survival (Onuki et al, 2002;Tyas et al, 2000), signalling (Shih and Qi, 2017;Weitsman et al, 2016), immune cell activity (Choi and Mitchison, 2013;Li et al, 2016b), metabolism (Fehr et al, 2003;Imamura et al, 2009;Mächler et al, 2016;Okumoto et al, 2005) or migration (Seong et al, 2011;Wang et al, 2005) with a recent move to generate GEMMs of FRET biosensors for high-fidelity intravital imaging (Johnsson et al, 2014;Kamioka et al, 2012;Mukherjee et al, 2016;Nobis et al, 2017).…”

Section: Box 1 Subcellular Imaging Techniquesmentioning

confidence: 99%

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

Nobis

Warren

Lucas

et al. 2018

Journal of Cell Science

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Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo. We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the singlecell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.

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HER2-HER3 dimer quantification by FLIM-FRET predicts breast cancer metastatic relapse independently of HER2 IHC status

Cited by 31 publications

References 60 publications

Quantitative Imaging of Receptor-Ligand Engagement in Intact Live Animals

Quantitative Imaging of Receptor-Ligand Engagement in Intact Live Animals

FLIMJ: an open-source ImageJ toolkit for fluorescence lifetime image data analysis

Molecular mobility and activity in an intravital imaging setting – implications for cancer progression and targeting

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