Biology subjects

Rudkouskaya, A.

Publications and source records attributed to Rudkouskaya, A..

10 recordsLinked to original sources

bioRxiv2026

Binding to Albumin and Off-Target Toxicity Confound the Use of LRRC8/VRAC Channel Blockers in Cell Physiology Assays

Volume-regulated anion channels (VRACs), formed by leucine-rich repeat-containing 8 (LRRC8) proteins, are ubiquitously expressed chloride channels essential for cell volume regulation and implicated in diverse physiological and pathological processes. Small-molecule VRAC inhibitors have been reported to modulate paracrine signaling, proliferation, differentiation, migration, and apoptosis, and have been patented for potential therapeutic applications in stroke, cardiovascular and metabolic diseases, and cancer. However, growing evidence indicates that many commonly used VRAC blockers exert substantial off-target effects and frequently fail to reproduce phenotypes observed after deletion of the essential VRAC subunit LRRC8A. Here, we systematically compared effects of several widely used pharmacological VRAC inhibitors with outcomes of molecular downregulation of LRRC8A in limiting proliferation of malignant glioblastoma cells derived from surgical specimens. NIH/3T3 fibroblasts served as a non-malignant control. In serum-containing media, structurally diverse VRAC blockers (DCPIB, DIDS, carbenoxolone, phloretin, and bromadiolone) reduced proliferation in a non-uniform manner, with potencies that did not correlate with reported VRAC affinities and varied markedly among cell lines. Radiotracer-based measurements of VRAC activity indicated that these discrepancies were largely attributable to binding of inhibitors to serum albumin. When experiments were repeated under serum-free conditions, all inhibitors except DIDS and phloretin induced extensive death of both malignant and non-malignant cells, confirmed by microscopy and LDH release assays. This cytotoxicity was accompanied by a marked reduction in intracellular ATP levels, consistent with previously reported mitochondrial uncoupling effects. In contrast, LRRC8A knockdown reduced proliferation without substantial cell death. Together, these findings demonstrate that most commercially available VRAC blockers limit proliferation and viability predominantly through VRAC-independent mechanisms. Under standard culture conditions, serum albumin masks much of their intrinsic cytotoxicity. These results underscore the need for rigorous molecular controls in pharmacological studies and provide basis for developing more selective and less toxic VRAC-targeting agents.

pharmacology and toxicology↗

bioRxiv2024

Fluorescence Lifetime Imaging for quantification of targeted drug delivery in varying tumor microenvironments

RationaleTrastuzumab (TZM) is a monoclonal antibody that targets the human epidermal growth factor receptor (HER2) and is clinically used for the treatment of HER2-positive breast tumors. However, the tumor microenvironment can limit the access of TZM to the HER2 targets across the whole tumor and thereby compromise TZMs therapeutic efficacy. An imaging methodology that can non-invasively quantify the binding of TZM-HER2, which is required for therapeutic action, and distribution within tumors with varying tumor microenvironments is much needed. MethodsWe performed near-infrared (NIR) fluorescence lifetime (FLI) Forster Resonance Energy Transfer (FRET) to measure TZM-HER2 binding, using in vitro microscopy and in vivo widefield macroscopy, in HER2 overexpressing breast and ovarian cancer cells and tumor xenografts, respectively. Immunohistochemistry was used to validate in vivo imaging results. ResultsNIR FLI FRET in vitro microscopy data show variations in intracellular distribution of bound TZM in HER2-positive breast AU565 and AU565 tumor-passaged XTM cell lines in comparison to SKOV-3 ovarian cancer cells. Macroscopy FLI (MFLI) FRET in vivo imaging data show that SKOV-3 tumors display reduced TZM binding compared to AU565 and XTM tumors, as validated by ex vivo immunohistochemistry. Moreover, AU565/XTM and SKOV-3 tumor xenografts display different amounts and distributions of TME components, such as collagen and vascularity. Therefore, these results suggest that SKOV-3 tumors are refractory to TZM delivery due to their disrupted vasculature and increased collagen content. ConclusionOur study demonstrates that FLI is a powerful analytical tool to monitor the delivery of antibody drug tumor both in cell cultures and in vivo live systems. Especially, MFLI FRET is a unique imaging modality that can directly quantify target engagement with potential to elucidate the role of the TME in drug delivery efficacy in intact live tumor xenografts.

cancer biology↗

bioRxiv2024

Contrast-enhanced photon-counting micro-CT of tumor xenograft models

Photon-counting micro computed tomography (micro-CT) offers new potential in preclinical imaging, particularly in distinguishing materials. It becomes especially helpful when combined with contrast agents, enabling the differentiation of tumors from surrounding tissues. There are mainly two types of contrast agents in the market for micro-CT: small molecule-based and nanoparticle-based. However, despite their widespread use in liver tumor studies, there is a notable gap in research on the application of these commercially available agents for photon-counting micro-CT in breast and ovarian tumors. Herein, we explored the effectiveness of these agents in differentiating tumor xenografts from various origins (AU565, MDA-MB-231, and SKOV-3) in nude mice, using photon-counting micro-CT. Specifically, ISOVUE-370 (a small molecule-based agent) and Exitrone Nano 12000 (a nanoparticle-based agent) were investigated in this context. To improve tumor visualization, we proposed a novel color visualization method for photon-counting micro-CT, which changes color tones to highlight contrast media distribution, offering a robust alternative to traditional material decomposition methods with less computational demand. Our in vivo experiments confirm its effectiveness, showing distinct enhancement characteristics for each contrast agent. Qualitative and quantitative analyses suggested that Exitrone Nano 12000 provides superior vasculature enhancement and better quantitative consistency across scans, while ISOVUE-370 gives more comprehensive tumor enhancement but with a significant variance between scans due to its short blood half-time. This variability leads to high sensitivity to timing and individual differences among mice. Further, a paired t-test on mean and standard deviation values within tumor volumes showed significant differences between the AU565 and SKOV-3 tumor models with the nanoparticle-based (p-values < 0.02), attributable to their distinct vascularity, as confirmed by immunohistochemistry. These findings underscore the utility of photon-counting micro-CT in non-invasively assessing the morphology and anatomy of different tumor xenografts, which is crucial for tumor characterization and longitudinal monitoring of tumor development and response to treatments.

bioengineering↗

bioRxiv2023

in vivo quantitative FRET small animal imaging: intensity versus lifetime-based FRET

Forster Resonance Energy Transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2-10 nm range. FRET is currently being extended to in vivo optical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the 3-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated ICCD, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fD E of the FRET efficiency E and the fraction of donor molecules involved in FRET, fD, are described in detail for both methodologies. Dynamic in vivo FRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of near infrared-labeled transferrin FRET pair and benchmarked against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required 9 measurements (6 of which are used for calibration) acquired from three mice, MFLI FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice. WHY IT MATTERSFRET measurements in live animals open a unique window into drug-target interaction monitoring, by sensing the close proximity between a donor and acceptor-labeled molecular probes. To perform these measurements, a 3-cube fluorescent intensity measurement strategy can be adopted, as is common for in vitro FRET microscopy studies. However, it is challenging to translate this already cumbersome approach to in vivo small animal imaging. Here, we compare this standard approach, for which we provide a revised analytical framework, to a conceptually much simpler and more powerful one based on fluorescence lifetime measurements. Our results demonstrate that the technical challenge of in vivo fluorescence lifetime macroscopic imaging is well worth surmounting to obtain quantitative, whole-animal information regarding molecular drug-target engagement.

biophysics↗

bioRxiv2021

In vitro and in vivo NIR Fluorescence Lifetime Imaging with a time-gated SPAD camera

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche photodiode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Forster Resonant Energy Transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated-ICCD camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

bioengineering↗

bioRxiv2020

Deep Learning Enhanced Hyperspectral Fluorescence Lifetime Imaging

Acquiring dense high-dimensional optical data in biological applications remains a challenge due to the very low levels of light typically encountered. Single pixel imaging methodologies enable improved detection efficiency in such conditions but are still limited by relatively slow acquisition times. Here, we propose a Deep Learning framework, NetFLICS-CR, which enables fast hyperspectral lifetime imaging for in vivo applications at enhanced resolution, acquisition and processing speeds, without the need of experimental training datasets. NetFLICS-CR reconstructs intensity and lifetime images at 128x128 pixels over 16 spectral channels while reducing the current acquisition times from [~]2.5 hours at 50% compression to [~]3 minutes at 99% compression when using a single-pixel Hyperspectral Macroscopic Fluorescence Lifetime Imaging (HMFLI) system. The potential of the technique is demonstrated in silico, in vitro and in vivo through the monitoring of receptor-ligand interactions in mice liver and bladder and further imaging of intracellular drug delivery of the clinical drug Trastuzumab in live animals bearing HER2-positive breast tumor xenografts.

biophysics↗

bioRxiv2019

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

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. To date, non-invasive quantitative imaging modalities that can comprehensively assess simultaneous cellular drug delivery efficacy and therapeutic response are lacking. In this regard, Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Forster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in vivo and subsequent intracellular internalization, which is critical to assess the delivery efficacy of targeted therapeutics. However, implementation of multiplexing optical imaging with FRET in vivo is challenging to achieve due to spectral crowding and cross-contamination. Herein, we report on a strategy that relies on a dark quencher that enables simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. 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) using in vitro NIR FLI microscopy and in vivo wide-field MFLI imaging. Second, we report on simultaneous in vivo imaging of the metabolic probe IRDye 800CW 2-deoxyglucose (2-DG) and MFLI-FRET imaging of NIR-labeled transferrin FRET pair (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. Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

bioengineering↗

bioRxiv2019

Ultra-fast fit-free analysis of complex fluorescence lifetime imaging via deep learning

Fluorescence lifetime imaging (FLI) provides unique quantitative information in biomedical and molecular biology studies, but relies on complex data fitting techniques to derive the quantities of interest. Herein, we propose a novel fit-free approach in FLI image formation that is based on Deep Learning (DL) to quantify complex fluorescence decays simultaneously over a whole image and at ultra-fast speeds. Our deep neural network (DNN), named FLI-Net, is designed and model-based trained to provide all lifetime-based parameters that are typically employed in the field. We demonstrate the accuracy and generalizability of FLI-Net by performing quantitative microscopic and preclinical experimental lifetime-based studies across the visible and NIR spectra, as well as across the two main data acquisition technologies. Our results demonstrate that FLI-Net is well suited to quantify complex fluorescence lifetimes, accurately, in real time in cells and intact animals without any parameter settings. Hence, it paves the way to reproducible and quantitative lifetime studies at unprecedented speeds, for improved dissemination and impact of FLI in many important biomedical applications, especially in clinical settings.

bioengineering↗

bioRxiv2018

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using near-infrared dyes

1We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We first show that fast and precise in vitro quantification of NIR fluorophores lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation. We apply this approach to the study of binding equilibria by Forster resonant energy transfer (FRET), using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend our demonstration to dynamic imaging of the pharmacokinetics of transferrin binding to the transferrin receptor in live mice, elucidating the kinetic of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from non-specific binding. Our method, implemented in a freely-available software package, has all the advantages of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging, and could be extended to applications as diverse as image guided-surgery or optical tomography.

bioengineering↗

bioRxiv2017

Quantitative Imaging of Receptor-Ligand Engagement in Intact Live Animals

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 whole-body macroscopic lifetime-based Forster 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.

cancer biology↗