Iain Tullis

A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening

Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.

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.

ErbB-2 Blockade and Prenyltransferase Inhibition Alter Epidermal Growth Factor and Epidermal Growth Factor Receptor Trafficking and Enhance 111 In-DTPA-hEGF Auger Electron Radiation Therapy

The intracellular distribution of Auger electron–emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of 111In-labeled human epidermal growth factor (111In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of 111In-DTPA-hEGF. Methods: The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. 111In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2–blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of 111In-DTPA-hEGF in MDA-MB-468 (1.3 × 106 EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 106 EGFR per cell; 0.4 × 105 ErbB-2 per cell) cell lines. The cytotoxicity of 111In-DTPA-hEGF (0–64 nM) plus trastuzumab (0–50 μg/mL) or L-778,123 (0–22.5 μM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft–bearing mice treated with 111In-DTPA-hEGF plus trastuzumab or L-788,123. Results: Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand–receptor complex in MDA-MB-468 cells. Nuclear localization of 111In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to 111In-DTPA-hEGF. Nuclear localization and cytotoxicity of 111In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P < 0.0001). Conclusion: The therapeutic efficacy of 111In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.

Toward operative in vivo fluorescence imaging of the c-Met proto-oncogene for personalization of therapy in ovarian cancer.

Standard biomarker testing of a single macroscopic disease site is unlikely to be sufficient because of tumor heterogeneity. A focus on examining global biomarker expression or activity, particularly in microscopic residual chemotherapy‐resistant disease, is needed for the appropriate selection of targeted therapies. This study was aimed at establishing a technique for the assessment of biomarkers of ovarian cancer peritoneal spread.

MRI-guided radiotherapy of the SK-N-SH neuroblastoma xenograft model using a small animal radiation research platform

Objective: Neuroblastoma has one of the lowest survival rates of all childhood cancers, despite the use of intensive treatment regimens. Preclinical models of neuroblastoma are essential for testing new multimodality protocols, including those that involve radiotherapy (RT). The aim of this study was to develop a robust method for RT planning and tumour response monitoring based on combined MRI and cone-beam CT (CBCT) imaging and to apply it to a widely studied mouse xenograft model of neuroblastoma, SK-N-SH. Methods: As part of a tumour growth inhibition study, SK-N-SH xenografts were generated in BALB/c nu/nu mice. Mice (n = 8) were placed in a printed MR- and CT-compatible plastic cradle, imaged using a 4.7-T MRI scanner and then transferred to a small animal radiation research platform (SARRP) irradiator with on-board CBCT. MRI/CBCT co-registration was performed to enable RT planning using the soft-tissue contrast afforded by MRI prior to delivery of RT (5 Gy). Tumour response was assessed by serial MRI and calliper measurements. Results: SK-N-SH xenografts formed soft, deformable tumours that could not be differentiated from surrounding normal tissues using CBCT. MR images, which allowed clear delineation of tumours, were successfully co-registered with CBCT images, allowing conformal RT to be delivered. MRI measurements of tumour volume 4 days after RT correlated strongly with length of survival time. Conclusion: MRI allowed precision RT of SK-N-SH tumours and provided an accurate means of measuring tumour response. Advances in knowledge: MRI-based RT planning of murine tumours is feasible using an SARRP irradiator.

Imaging tumour heterogeneity of the consequences of a PKCα-substrate interaction in breast cancer patients.

Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein-protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed.

An efficient and robust MRI-guided radiotherapy planning approach for targeting abdominal organs and tumours in the mouse

Introduction Preclinical CT-guided radiotherapy platforms are increasingly used but the CT images are characterized by poor soft tissue contrast. The aim of this study was to develop a robust and accurate method of MRI-guided radiotherapy (MR-IGRT) delivery to abdominal targets in the mouse. Methods A multimodality cradle was developed for providing subject immobilisation and its performance was evaluated. Whilst CT was still used for dose calculations, target identification was based on MRI. Each step of the radiotherapy planning procedure was validated initially in vitro using BANG gel dosimeters. Subsequently, MR-IGRT of normal adrenal glands with a size-matched collimated beam was performed. Additionally, the SK-N-SH neuroblastoma xenograft model and the transgenic KPC model of pancreatic ductal adenocarcinoma were used to demonstrate the applicability of our methods for the accurate delivery of radiation to CT-invisible abdominal tumours. Results The BANG gel phantoms demonstrated a targeting efficiency error of 0.56 ± 0.18 mm. The in vivo stability tests of body motion during MR-IGRT and the associated cradle transfer showed that the residual body movements are within this MR-IGRT targeting error. Accurate MR-IGRT of the normal adrenal glands with a size-matched collimated beam was confirmed by γH2AX staining. Regression in tumour volume was observed almost immediately post MR-IGRT in the neuroblastoma model, further demonstrating accuracy of x-ray delivery. Finally, MR-IGRT in the KPC model facilitated precise contouring and comparison of different treatment plans and radiotherapy dose distributions not only to the intra-abdominal tumour but also to the organs at risk. Conclusion This is, to our knowledge, the first study to demonstrate preclinical MR-IGRT in intra-abdominal organs. The proposed MR-IGRT method presents a state-of-the-art solution to enabling robust, accurate and efficient targeting of extracranial organs in the mouse and can operate with a sufficiently high throughput to allow fractionated treatments to be given.

The Development of Technology for Effective Respiratory-Gated Irradiation Using an Image-Guided Small Animal Irradiator

The development of image-guided small animal irradiators represents a significant improvement over standard irradiators by enabling preclinical studies to mimic radiotherapy in humans. The ability to deliver tightly collimated targeted beams, in conjunction with gantry or animal couch rotation, has the potential to maximize tumor dose while sparing normal tissues. However, the current commercial platforms do not incorporate respiratory gating, which is required for accurate and precise targeting in organs subject to respiration related motions that may be up to the order of 5 mm in mice. Therefore, a new treatment head assembly for the Xstrahl Small Animal Radiation Research Platform (SARRP) has been designed. This includes a fast X-ray shutter subsystem, a motorized beam hardening filter assembly, an integrated transmission ionization chamber to monitor beam delivery, a kinematically positioned removable beam collimator and a targeting laser exiting the center of the beam collimator. The X-ray shutter not only minimizes timing errors but also allows beam gating during imaging and treatment, with irradiation only taking place during the breathing cycle when tissue movement is minimal. The breathing related movement is monitored by measuring, using a synchronous detector/lock-in amplifier that processes diffuse reflectance light from a modulated light source. After thresholding of the resulting signal, delays are added around the inhalation/exhalation phases, enabling the “no movement” period to be isolated and to open the X-ray shutter. Irradiation can either be performed for a predetermined time of X-ray exposure, or through integration of a current from the transmission monitor ionization chamber (corrected locally for air density variations). The ability to successfully deliver respiratory-gated X-ray irradiations has been demonstrated by comparing movies obtained using planar X-ray imaging with and without respiratory gating, in addition to comparing dose profiles observed from a collimated beam on EBT3 radiochromic film mounted on the animals chest. Altogether, the development of respiratory-gated irradiation facilitates improved dose delivery during animal movement and constitutes an important new tool for preclinical radiation studies. This approach is particularly well suited for irradiation of orthotopic tumors or other targets within the chest and abdomen where breathing related movement is significant.

A novel multiwavelength fluorescence image-guided surgery imaging system

We describe the development and performance analysis of two clinical near-infrared fluorescence image-guided surgery (FIGS) devices that aim to overcome some of the limitations of current FIGS systems. The devices operate in a widefield-imaging mode and can work (1) in conjunction with a laparoscope, during minimally invasive surgery, and (2) as a hand-held, open surgery imaging system. In both cases, narrow-band excitation light, delivered at multiple wavelengths, is efficiently combined with white reflectance light. Light is delivered to ~100 cm2 surgical field at 1-2 mW/cm2 for white light and 3-7 mW/cm2 (depending on wavelength) of red - near infrared excitation, at a typical working distance of 350 mm for the hand-held device and 100 mm for the laparoscope. A single, sensitive, miniaturized color camera collects both fluorescence and white reflectance light. The use of a single imager eliminates image alignment and software overlay complexity. A novel filtering and illumination arrangement allows simultaneous detection of white reflectance and fluorescence emission from multiple dyes in real-time. We will present both fluorescence detection sensitivity modeling and practical performance data. We have demonstrated the efficiency and the advantages of the devices both pre-clinically and during live surgery on humans. Both the hand-held and the laparoscopic systems have proved to be reliable and beneficial in an ongoing clinical trial involving sentinel lymph node detection in gynecological cancers. We will show preliminary results using two clinically approved dyes, Methylene blue and indocyanine green. We anticipate that this technology can be integrated and routinely used in a larger variety of surgical procedures.

Abstract 2235: FRET assay to assess EGFR/HER2 dimerization in cancer cell lines

Expression level of the HER family is unreliable as a predictive marker for the outcome of targeted therapies in cancer. Only a third of HER2 positive breast cancer patients respond to trastuzumab monotherapy. Thus there is significant need for the development of new biomarkers and the role of dimerization may prove a more effective predictor than expression itself. It has been shown that specific HER dimerization pairs are important. However, there has been no protocol proposed which can accurately assess dimerization to facilitate the development of an assay viable for use in a clinical setting. Previous attempts have either been based upon transfection of proteins whose interactions can be monitored which would not seem to be directly applicable to in-situ monitoring or do not explicitly separate true dimerization from mere colocalization. Our approach also allows intracellular localization to be visualized, this is significant as nuclear translocation of EGFR can lead to a number of signaling events. Utilizing measurement of Foerster Resonance Energy Transfer (FRET) to assess distances of the order 7 nm between receptors we can quantify dimerization. Our secondary conjugation technique uses anti-mouse and anti-rabbit primary antibodies and species specific dye-conjugated secondary antibodies, with Alexa488 as the donor and Alexa546 as the acceptor dye. An increase in dimerization results in increased occurrence of FRET which can be measured as a reduction in donor fluorescence lifetime. We have validated our assay for dimerization of EGFR and HER2 in 3 cell lines, BT474, SKBr3 and A431, and furthermore in paraffin embedded cell pellets. Comparison of preparations with donor antibodies alone with those containing donor and acceptor we demonstrate basal EGFR/HER2 dimerization levels are present (p We then applied the assay to monitor response to targeted therapies, the HER2 specific monoclonal antibodies, trastuzumab and pertuzumab. Trastuzumab is seen to decrease lifetime while dimerization inhibitor pertuzumab produces an increase. This is consistent with findings using biochemical methods. Finally, we applied and validated our assay in paraffin embedded cell pellets. These data demonstrate the robustness of our assay and the potential clinical application to paraffin-embedded tissue microarrays in order to assess the contribution of HER signaling in-situ. The assay represents a proof of principle, from here this same technique could be applied to other dimerization pairs. This will allow the prognostic potential of dimerization to be assessed and used to inform treatment regimes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2235. doi:10.1158/1538-7445.AM2011-2235