The Gray Institute 'open' high-content, fluorescence lifetime microscopes.

We describe a microscopy design methodology and details of microscopes built to this 'open' design approach. These demonstrate the first implementation of time-domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off-the-shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup. Several automated high-content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time-domain FLIM via time-correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide-field and laser scanning capabilities designed for high-content microscopy. Devices using these designs also form radiation-beam 'end-stations' at Oxford and Surrey Universities, showing the versatility and extendibility of this approach.

"Broadbeam" irradiation of mammalian cells using a vertical microbeam facility.

A "broadbeam" facility is demonstrated for the vertical microbeam at Surrey's Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651-662, 2012). Here, droplets with a diameter of about 4 mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in "broadbeam" mode with 2 MeV protons and 4 MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2 MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed.

Osimertinib and anti-HER3 combination therapy engages immune dependent tumor toxicity via STING activation in trans.

Over the past decade, immunotherapy delivered novel treatments for many cancer types. However, lung cancer still leads cancer mortality, and non-small-cell lung carcinoma patients with mutant EGFR cannot benefit from checkpoint inhibitors due to toxicity, relying only on palliative chemotherapy and the third-generation tyrosine kinase inhibitor (TKI) osimertinib. This new drug extends lifespan by 9-months vs. second-generation TKIs, but unfortunately, cancers relapse due to resistance mechanisms and the lack of antitumor immune responses. Here we explored the combination of osimertinib with anti-HER3 monoclonal antibodies and observed that the immune system contributed to eliminate tumor cells in mice and co-culture experiments using bone marrow-derived macrophages and human PBMCs. Osimertinib led to apoptosis of tumors but simultaneously, it triggered inositol-requiring-enzyme (IRE1α)-dependent HER3 upregulation, increased macrophage infiltration, and activated cGAS in cancer cells to produce cGAMP (detected by a lentivirally transduced STING activity biosensor), transactivating STING in macrophages. We sought to target osimertinib-induced HER3 upregulation with monoclonal antibodies, which engaged Fc receptor-dependent tumor elimination by macrophages, and STING agonists enhanced macrophage-mediated tumor elimination further. Thus, by engaging a tumor non-autonomous mechanism involving cGAS-STING and innate immunity, the combination of osimertinib and anti-HER3 antibodies could improve the limited therapeutic and stratification options for advanced stage lung cancer patients with mutant EGFR.

Time-lapse FRET microscopy using fluorescence anisotropy.

We present recent data on dynamic imaging of Rac1 activity in live T-cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi-functional high-content screening instrument using fluorescence anisotropy to provide a means of monitoring protein-protein activity with high temporal resolution. Specifically, the response of T-cells upon interaction of a cell surface receptor with an antibody coated multi-well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.

Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor.

Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.

Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability.

The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.

Automated counting of mammalian cell colonies.

Investigating the effect of low-dose radiation exposure on cells using assays of colony-forming ability requires large cell samples to maintain statistical accuracy. Manually counting the resulting colonies is a laborious task in which consistent objectivity is hard to achieve. This is true especially with some mammalian cell lines which form poorly defined or 'fuzzy' colonies, typified by glioma or fibroblast cell lines. A computer-vision-based automated colony counter is presented in this paper. It utilizes novel imaging and image-processing methods involving a modified form of the Hough transform. The automated counter is able to identify less-discrete cell colonies typical of these cell lines. The results of automated colony counting are compared with those from four manual (human) colony counts for the cell lines HT29, A172, U118 and IN1265. The results from the automated counts fall well within the distribution of the manual counts for all four cell lines with respect to surviving fraction (SF) versus dose curves, SF values at 2 Gy (SF2) and total area under the SF curve (Dbar). From the variation in the counts, it is shown that the automated counts are generally more consistent than the manual counts.

The challenges of integrating molecular imaging into the optimization of cancer therapy.

We review novel, in vivo and tissue-based imaging technologies that monitor and optimize cancer therapeutics. Recent advances in cancer treatment centre around the development of targeted therapies and personalisation of treatment regimes to individual tumour characteristics. However, clinical outcomes have not improved as expected. Further development of the use of molecular imaging to predict or assess treatment response must address spatial heterogeneity of cancer within the body. A combination of different imaging modalities should be used to relate the effect of the drug to dosing regimen or effective drug concentration at the local site of action. Molecular imaging provides a functional and dynamic read-out of cancer therapeutics, from nanometre to whole body scale. At the whole body scale, an increase in the sensitivity and specificity of the imaging probe is required to localise (micro)metastatic foci and/or residual disease that are currently below the limit of detection. The use of image-guided endoscopic biopsy can produce tumour cells or tissues for nanoscopic analysis in a relatively patient-compliant manner, thereby linking clinical imaging to a more precise assessment of molecular mechanisms. This multimodality imaging approach (in combination with genetics/genomic information) could be used to bridge the gap between our knowledge of mechanisms underlying the processes of metastasis, tumour dormancy and routine clinical practice. Treatment regimes could therefore be individually tailored both at diagnosis and throughout treatment, through monitoring of drug pharmacodynamics providing an early read-out of response or resistance.

A Bayesian method for single molecule, fluorescence burst analysis.

There is currently great interest in determining physical parameters, e.g. fluorescence lifetime, of individual molecules that inform on environmental conditions, whilst avoiding the artefacts of ensemble averaging. Protein interactions, molecular dynamics and sub-species can all be studied. In a burst integrated fluorescence lifetime (BIFL) experiment, identification of fluorescent bursts from single molecules above background detection is a problem. This paper presents a Bayesian method for burst identification based on model selection and demonstrates the detection of bursts consisting of 10% signal amplitude. The method also estimates the fluorescence lifetime (and its error) from the burst data.

Time-correlated single-photon counting fluorescence lifetime confocal imaging of decayed and sound dental structures with a white-light supercontinuum source.

We report the demonstration of time-correlated single-photon counting (TCSPC) fluorescence lifetime imaging (FLIM) to ex vivo decayed and healthy dentinal tooth structures, using a white-light supercontinuum excitation source. By using a 100 fs-pulsed Ti:Sapphire laser with a low-frequency chirp to pump a 30-cm long section of photonic crystal fibre, a ps-pulsed white-light supercontinuum was created. Optical bandpass interference filters were then applied to this broad-bandwidth source to select the 488-nm excitation wavelength required to perform TCSPC FLIM of dental structures. Decayed dentine showed significantly shorter lifetimes, discriminating it from healthy tissue and hard, stained and thus affected but non-infected material. The white-light generation source provides a flexible method of producing variable-bandwidth visible and ps-pulsed light for TCSPC FLIM. The results from the dental tissue indicate a potential method of discriminating diseased tissue from sound, but stained tissue, which could be of crucial importance in limiting tissue resection during preparation for clinical restorations.

Semi-automated software for the three-dimensional delineation of complex vascular networks.

The understanding of tumour angiogenesis is of great importance in cancer research, as is the tumour response to vascular-targeted drugs. This paper presents software aimed at aiding these investigations and other situations where linear or dendritic structures are to be delineated from three-dimensional (3D) data sets. This software application was written to analyse the data from 3D data sets by allowing the manual and semi-automated tracking and delineation of the vascular tree, including the measurement of vessel diameter. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to locate vessel centres and measure diameters automatically. The robustness of this algorithm to image smoothing and noise has been investigated.