Fluorescence lifetime endoscopy using TCSPC for the measurement of FRET in live cells

Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960’s. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 µm and 10 µm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications

Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with ⁸⁹Zr-oxine for Medium-Term In Vivo Cell Tracking

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with ⁸⁹Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [⁸⁹Zr]Zr(oxinate)₄ as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1–11.1 Bq ⁸⁹Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the ⁸⁹Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular ⁸⁹Zr amounts, as they failed to survive or expand in a ⁸⁹Zr-dose-dependent manner. Even at 0.1 Bq ⁸⁹Zr per Treg cell, while ⁸⁹Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of ⁸⁹Zr-Treg death after adoptive transfer in vivo. In summary, ⁸⁹Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as ⁸⁹Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario

Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-Resolution Microscopy

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intra-cluster distances (d). From this data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P50nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d<50nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells

Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-Resolution Microscopy

[Image: see text] Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ≥ 3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells

A preclinical pipeline to evaluate migrastatics as therapeutic agents in metastatic melanoma

© The Author(s) 2021.[Background]: Metastasis is a hallmark of cancer and responsible for most cancer deaths. Migrastatics were defined as drugs interfering with all modes of cancer cell invasion and thus cancers’ ability to metastasise. First anti-metastatic treatments have recently been approved. [Methods]: We used bioinformatic analyses of publicly available melanoma databases. Experimentally, we performed in vitro target validation (including 2.5D cell morphology analysis and mass spectrometric analysis of RhoA binding partners), developed a new traceable spontaneously metastasising murine melanoma model for in vivo validation, and employed histology (haematoxylin/eosin and phospho-myosin II staining) to confirm drug action in harvested tumour tissues. [Results]: Unbiased and targeted bioinformatic analyses identified the Rho kinase (ROCK)-myosin II pathway and its various components as potentially relevant targets in melanoma. In vitro validation demonstrated redundancy of several RhoGEFs upstream of RhoA and confirmed ROCK as a druggable target downstream of RhoA. The anti-metastatic effects of two ROCK inhibitors were demonstrated through in vivo melanoma metastasis tracking and inhibitor effects also confirmed ex vivo by digital pathology. [Conclusions]: We proposed a migrastatic drug development pipeline. As part of the pipeline, we provide a new traceable spontaneous melanoma metastasis model for in vivo quantification of metastasis and anti-metastatic effects by non-invasive imaging.GOF’s lab was supported by Cancer Research UK [C48390/A21153], Worldwide Cancer Research [16-1153], and King’s Health Partners [King’s Medical Research Trust Joint Research Committee studentship to A.V.]. B.F. was supported by a King’s Health Partners studentship to V.S.M. and G.O.F. V.S.M.’s lab was supported by Cancer Research UK [C33043/A12065] and [C33043/A24478] (V.S.M., E.C.M., J.L.O., L.B. and GC), the Royal Society [RG110591] (V.S.M.), The Harry J. Lloyd Charitable Trust (J.L.O. and V.S.M.), the Barts Charity (V.S.M., J.L.O., O.M., I.R.H. and E.C.M.), the Fundacion Alfonso Martin Escudero and Marie Sklodowska-Curie Action [H2020-MSCA-IF-2014-EF-ST] (I.R.H.), and Fundacion Ramon Areces (E.C.M.). F.M. was supported by an MRC Career Development Award (MR/P009417/1). This work was further supported by the Department of Health (DoH) via the National Institute for Health Research (NIHR) Comprehensive Biomedical Research Centre award to King’s Health Partners, and the Wellcome/EPSRC Centre for Medical Engineering [WT203148/Z/16/Z]. Views expressed are those of the authors and not necessarily those of the NHS, NIHR or DoH

Spatiotemporal quantitative microRNA-155 imaging reports immune-mediated changes in a triple-negative breast cancer model

IntroductionMicroRNAs are small non-coding RNAs and represent key players in physiology and disease. Aberrant microRNA expression is central to the development and progression of cancer, with various microRNAs proposed as potential cancer biomarkers and drug targets. There is a need to better understand dynamic microRNA expression changes as cancers progress and their tumor microenvironments evolve. Therefore, spatiotemporal and non-invasive in vivo microRNA quantification in tumor models would be highly beneficial.MethodsWe developed an in vivo microRNA detector platform in which the obtained signals are positively correlated to microRNA presence, and which permitted stable expression in cancer cells as needed for long-term experimentation in tumor biology. It exploits a radionuclide-fluorescence dual-reporter for quantitative in vivo imaging of a microRNA of choice by radionuclide tomography and fluorescence-based downstream ex vivo tissue analyses. We generated and characterized breast cancer cells stably expressing various microRNA detectors and validated them in vitro.ResultsWe found the microRNA detector platform to report on microRNA presence in cells specifically and accurately, which was independently confirmed by real-time PCR and through microRNA modulation. Moreover, we established various breast tumor models in animals with different levels of residual immune systems and observed microRNA detector read-outs by imaging. Applying the detector platform to the progression of a triple-negative breast cancer model, we found that miR-155 upregulation in corresponding tumors was dependent on macrophage presence in tumors, revealing immune-mediated phenotypic changes in these tumors as they progressed.ConclusionWhile applied to immunooncology in this work, this multimodal in vivo microRNA detector platform will be useful whenever non-invasive quantification of spatiotemporal microRNA changes in living animals is of interest

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells reveals a role for innate immune cells in Treg transplant recruitment

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs was shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive which hampers clinical translation. Here, we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behaviour, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nanoSPECT/CT for up to 40 days and results validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1⁺ innate immune cells. We demonstrated the utility of radionuclide reporter gene afforded quantitative Treg in vivo tracking thereby addressing a fundamental need in Treg therapy development and offering clinically compatible methodology for future Treg therapy imaging in humans

WNT11-FZD7-DAAM1 signalling supports tumour initiating abilities and melanoma amoeboid invasion

Melanoma is a highly aggressive tumour that can metastasize very early in disease progression. Notably, melanoma can disseminate using amoeboid invasive strategies. We show here that high Myosin II activity, high levels of ki-67 and high tumour-initiating abilities are characteristic of invasive amoeboid melanoma cells. Mechanistically, we find that WNT11-FZD7-DAAM1 activates Rho-ROCK1/2-Myosin II and plays a crucial role in regulating tumour-initiating potential, local invasion and distant metastasis formation. Importantly, amoeboid melanoma cells express both proliferative and invasive gene signatures. As such, invasive fronts of human and mouse melanomas are enriched in amoeboid cells that are also ki-67 positive. This pattern is further enhanced in metastatic lesions. We propose eradication of amoeboid melanoma cells after surgical removal as a therapeutic strategy. Amoeboid cells are associated with melanoma invasive capacity. Here, the authors show that the WNT11-FZD7-DAAM1 pathway regulates tumour-initiating potential, invasion and metastasis lead by amoeboid cells in the invasive front of melanoma tumours

The T2K Side Muon Range Detector

The T2K experiment is a long baseline neutrino oscillation experiment aiming to observe the appearance of {\nu} e in a {\nu}{\mu} beam. The {\nu}{\mu} beam is produced at the Japan Proton Accelerator Research Complex (J-PARC), observed with the 295 km distant Super- Kamiokande Detector and monitored by a suite of near detectors at 280m from the proton target. The near detectors include a magnetized off-axis detector (ND280) which measures the un-oscillated neutrino flux and neutrino cross sections. The present paper describes the outermost component of ND280 which is a side muon range detector (SMRD) composed of scintillation counters with embedded wavelength shifting fibers and Multi-Pixel Photon Counter read-out. The components, performance and response of the SMRD are presented.Comment: 13 pages, 19 figures v2: fixed several typos; fixed reference

Measurement of Through-Going Particle Momentum By Means Of Multiple Scattering With The ICARUS T600 TPC

The ICARUS collaboration has demonstrated, following the operation of a 600 ton (T600) detector at shallow depth, that the technique based on liquid Argon TPCs is now mature. The study of rare events, not contemplated in the Standard Model, can greatly benefit from the use of this kind of detectors. In particular, a deeper understanding of atmospheric neutrino properties will be obtained thanks to the unprecedented quality of the data ICARUS provides. However if we concentrate on the T600 performance, most of the $\nu_\mu$ charged current sample will be partially contained, due to the reduced dimensions of the detector. In this article, we address the problem of how well we can determine the kinematics of events having partially contained tracks. The analysis of a large sample of atmospheric muons collected during the T600 test run demonstrate that, in case the recorded track is at least one meter long, the muon momentum can be reconstructed by an algorithm that measures the Multiple Coulomb Scattering along the particle's path. Moreover, we show that momentum resolution can be improved by a factor two using an algorithm based on the Kalman Filtering technique