Paul R. Barber

Use of the mouse aortic ring assay to study angiogenesis

Here we provide a protocol for quantitative three-dimensional ex vivo mouse aortic ring angiogenesis assays, in which developing microvessels undergo many key features of angiogenesis over a timescale similar to that observed in vivo. The aortic ring assay allows analysis of cellular proliferation, migration, tube formation, microvessel branching, perivascular recruitment and remodeling—all without the need for cellular dissociation—thus providing a more complete picture of angiogenic processes compared with traditional cell-based assays. Our protocol can be applied to aortic rings from embryonic stage E18 through to adulthood and can incorporate genetic manipulation, treatment with growth factors, drugs or siRNA. This robust assay allows assessment of the salient steps in angiogenesis and quantification of the developing microvessels, and it can be used to identify new modulators of angiogenesis. The assay takes 6–14 d to complete, depending on the age of the mice, treatments applied and whether immunostaining is performed.

Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein-protein interactions using global analysis

Förster resonance energy transfer (FRET) detected via fluorescence lifetime imaging microscopy (FLIM) and global analysis provide a way in which protein–protein interactions may be spatially localized and quantified within biological cells. The FRET efficiency and proportion of interacting molecules have been determined using bi-exponential fitting to time-domain FLIM data from a multiphoton time-correlated single-photon counting microscope system. The analysis has been made more robust to noise and significantly faster using global fitting, allowing higher spatial resolutions and/or lower acquisition times. Data have been simulated, as well as acquired from cell experiments, and the accuracy of a modified Levenberg–Marquardt fitting technique has been explored. Multi-image global analysis has been used to follow the epidermal growth factor-induced activation of Cdc42 in a short-image-interval time-lapse FLIM/FRET experiment. Our implementation offers practical analysis and time-resolved-image manipulation, which have been targeted towards providing fast execution, robustness to low photon counts, quantitative results and amenability to automation and batch processing.

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

Predictions regarding drug resistance mechanisms and treatment strategies in triple-negative breast cancer are confirmed in tumors from patients. From Models to Breast Cancer Treatments Patients with triple-negative breast cancer (TNBC), a particularly aggressive form, have few treatment options. Targeting either the phosphatidylinositol 3-kinase to Akt (PI3K-Akt) pathway or epidermal growth factor receptor (EGFR) inhibits tumor growth in some patients, but durable responses are rare. Modeling studies using cell lines predict that the EGFR family member HER3 (human epidermal growth factor receptor 3) may confer drug resistance. Now, Tao et al. provide evidence from patient tumors to support those predictions. Treatment with PI3K-Akt pathway inhibitors increased the abundance of both total and activated HER3 in TNBC cells in culture and TNBC xenografts in mice. Residual tumors from patients treated with EGFR inhibitors had increased abundance and activation of HER3. Combining inhibitors of the PI3K-Akt pathway with a dual inhibitor of EGFR and HER3 substantially suppressed tumor growth in mice with TNBC xenografts derived from either cell lines or patients, suggesting that this combined strategy may improve therapeutic outcome in TNBC patients. Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation

The RAD51 paralogues act in the homologous recombination (HR) pathway of DNA repair. Human RAD51C (hRAD51C) participates in branch migration and Holliday junction resolution and thus is important for processing HR intermediates late in the DNA repair process. Evidence for early involvement of RAD51 during DNA repair also exists, but its function in this context is not understood. In this study, we demonstrate that RAD51C accumulates at DNA damage sites concomitantly with the RAD51 recombinase and is retained after RAD51 disassembly, which is consistent with both an early and a late function for RAD51C. RAD51C recruitment depends on ataxia telangiectasia mutated, NBS1, and replication protein A, indicating it functions after DNA end resection but before RAD51 assembly. Furthermore, we find that RAD51C is required for activation of the checkpoint kinase CHK2 and cell cycle arrest in response to DNA damage. This suggests that hRAD51C contributes to the protection of genome integrity by transducing DNA damage signals in addition to engaging the HR machinery.

Biomedical Imaging: From Nano to Macro

Studying cellular protein-protein interactions in situ requires a technique such as fluorescence resonance energy transfer (FRET) which is sensitive on the nanometer scale. Observing FRET is significantly simplified if the fluorescence lifetime of the donor can be monitored. Results from live cells and tissue micro arrays are presented from an automated microscope incorporating time-domain TCSPC fluorescence lifetime imaging (FLIM). Novel hardware and software with a modular approach and scripting abilities allow us to work towards speed-optimized acquisition and ease of use to bring FLIM into the high-throughput regime.

The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients.

Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM

FLIM/FRET is an extremely powerful technique that can microscopically locate nanometre-scale protein-protein interactions within live or fixed cells, both in vitro and in vivo. The key to performing sensitive FRET, via FLIM, besides the use of appropriate fluorophores, is the analysis of the time-resolved data present at each image pixel. The fluorescent transient will, in general, exhibit multi-exponential kinetics: at least two exponential components arise from both the interacting and non-interacting protein. We shall describe a novel method and computer program for the global analysis of time resolved data, either at the single level or through global analysis of grouped pixel data. Kinetic models are fitted using the Marquardt algorithm and iterative convolution of the excitation signal, in a computationally-efficient manner. The fitting accuracy and sensitivity of the algorithm has been tested using modelled data, including the addition of simulated Poisson noise and repetitive excitation pulses which are typical of a TCSPC system. We found that the increased signal to noise ratio offered by both global and invariance fitting is highly desirable. When fitting mono-exponential data, the effects of a ca. 12.5 ns (ca. 80 MHz) repetitive excitation do not preclude the accurate extraction of populations with lifetimes in the range 0.1 to 10 ns, even when these effects are not represented in the fitting algorithm. Indeed, with global or invariance fitting of a 32x32 pixel area, the error in extracted lifetime can be lower than 0.4% for signals with a peak of 500 photon counts or more. In FRET simulations, modelling GFP with a non-interacting lifetime of 2.15 ns, it was possible to accurately detect a 10% interacting population with a lifetime of 0.8 ns.

How Forster Resonance Energy Transfer Imaging Improves the Understanding of Protein Interaction Networks in Cancer Biology

Herein we discuss how FRET imaging can contribute at various stages to delineate the function of the proteome. Therefore, we briefly describe FRET imaging techniques, the selection of suitable FRET pairs and potential caveats. Furthermore, we discuss state-of-the-art FRET-based screening approaches (underpinned by protein interaction network analysis using computational biology) and preclinical intravital FRET-imaging techniques that can be used for functional validation of candidate hits (nodes and edges) from the network screen, as well as measurement of the efficacy of perturbing these nodes/edges by short hairpin RNA (shRNA) and/or small molecule-based approaches.

Estimation of Apparent Tumor Vascular Permeability from Multiphoton Fluorescence Microscopic Images of P22 Rat Sarcomas In Vivo

Objective: To develop an image processing‐based method to quantify the rate of extravasation of fluorescent contrast agents from tumor microvessels, and to investigate the effect of the tumor vascular disrupting agent combretastatin A‐4‐P (CA‐4‐P) on apparent tumor vascular permeability to 40 kDa fluorescein isothiocyanate (FITC) labeled dextran.