Boris Vojnovic

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.

A plethysmographic method for measuring function in locally irradiated mouse lung

A plethysmograph has been developed to measure pulmonary function in mice after single doses of X rays to both lungs. The apparatus consists of a whole-body airtight chamber fitted with a Lavalier microphone. The microphone acts as a sensitive electrical capacitance manometer converting pressure changes in the chamber into an electrical signal which is electronically processed and recorded on a pen recorder. Two parameters of lung function were simultaneously monitored, breathing rate and amplitude. Lung function has been tested in male CBA mice aged two to six months and in animals which have received graded X-ray doses to both lungs. No diurnal rhythm or agerelated increase has been observed up to six months in control mice. The two lung-function parameters exhibited a dose-dependent response in irradiated lungs tested 16 weeks after irradiation; the response was reproducible in successive experiments. Respiration rate was increased above a threshold dose of 11 Gy (1100 rad), while amplitude decreased, also with a threshold at 11 Gy. These changes were observed before histological evidence of fibrosis became apparent and before pulmonary insufficiency led to deaths in the higher dose groups. The measurement of lung function by plethysmography is an alternative to lethality for assessing radiation damage in the lungs of small animals. The technique is non-destructive, responding to lower doses than LDso, and allows quantitative assessment of sequential changes in the lungs in each mouse over long post-irradiation times. New quantitative endpoints for the assessment of normal tissue injury after irradiation are being developed by an increasing number of investigators (Chauser et al., 1976; Glatstein et al., 1975; Hayashi and Suit, 1971; Hirst et al., 1977; Hopewell, 1975; Stewart, 1977; Van der Kogel et al., 1977; Vatistas and Hornsey, 1966; White and Hornsey, 1977; Withers, 1967; Withers and Elkind, 1969). Because radiation damage to normal tissues is the doselimiting factor in radiotherapy it is important that endpoints should be established which enable injury, particularly late injury, to be quantitated. Many of the techniques presently available are invasive or involve sacrifice of the animal. Functional assays which permit sequential assessment of injury in the same groups of animals over a period of time are available for only a few organs in small laboratory animals. The present study reports a new non-invasive technique of measuring changes in pulmonary function after irradiation of both lungs of mice. MATERIALS AND METHODS Experimental animals and radiation procedures Lung function was tested in mice in which both lungs were irradiated and in sham-irradiated controls. Male CBA/Ht mice, 10-12 weeks old, were given single doses of X rays ranging from 8 to 20 Gy. Radiation was delivered with a 250 kV X-ray unit, operated at 240 kV with an HVT of 1.3 mm Cu at a target skin distance of 20 cm. The dose rate was 3.65 Gy per min. Four mice were irradiated simultaneously on a specially constructed jig. The total dose was delivered within four to ten minutes in four equal increments with the jig rotated 90 deg after each increment to allow for a constant 10% dose-difference between the four irradiation positions. Both lungs were irradiated through two anterior 20x15 mm apertures in a 3.0 mm thick lead shield which protected the remainder of the body. Structures in the thoracic mid-line were also protected by a 1.0 mm wide strip in the lead shield separating the two lung fields. Perspex pillars fixed to the base of the jig kept the mouse in a supine nonrotated position and held the rib cage in position with respect to the radiation fields. This positioning was checked in some animals by radiographs which showed that less than 5% of the total volume of lung was protected by the mid-line shield. Dose distribution in the lung was checked using thermoruminescent dosimeters in dead mice. The mice were anaesthetized before irradiation with sodium pentobarbital (60 mg/kg) given intraperitoneally. Megimide (15 mg) was given to each mouse after irradiation to speed recovery from the anaesthetic.

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.

Tumour vascular disrupting agents: combating treatment resistance

A large group of tubulin-binding microtubule-depolymerizing agents act as tumour vascular disrupting agents (VDAs). Several members of this group are now in clinical trials in combination with conventional anticancer drugs and radiotherapy. Here we briefly update on the development of tubulin-binding combretastatins as VDAs, summarize what is known of their mechanisms of action and address issues relating to treatment resistance, using disodium combretastatin A-4 3-O-phosphate (CA-4-P) as an example. Characteristically, VDAs cause a rapid shutdown of blood flow to tumour tissue with much less effect in normal tissues. However, the tumour rim is relatively resistant to treatment. Hypoxia (or hypoxia reoxygenation) induces upregulation of genes associated with angiogenesis and drug resistance. It may be possible to take advantage of treatment-induced hypoxia by combining with drugs that are activated under hypoxic conditions. In summary, VDAs provide a novel approach to cancer treatment, which should effectively complement standard treatments, if treatment resistance is addressed by judicious combination treatment strategies.

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.

Operable Non–Small Cell Lung Cancer: Correlation of Volumetric Helical Dynamic Contrast-enhanced CT Parameters with Immunohistochemical Markers of Tumor Hypoxia

PURPOSE To assess the relationship between helical dynamic contrast material-enhanced (DCE) computed tomographic (CT) parameters and immunohistochemical markers of hypoxia in patients with operable non-small cell lung cancer (NSCLC). MATERIALS AND METHODS After institutional review board approval was obtained, 20 prospective patients who were suspected of having NSCLC underwent whole-tumor DCE CT with kinetic modeling (Patlak analysis) 24 hours before scheduled surgery. Flow-extraction product (in milliliters per 100 milliliters per minute) and blood volume (in milliliters per 100 milliliters) were derived. After surgery, matched whole-tumor sections were stained for exogenous and endogenous markers of hypoxia (pimonidazole infused intravenously 24 hours before surgery, immediately after DCE CT; glucose transporter protein). Correlation between DCE CT parameters and immunohistochemical markers was assessed by using the Spearman rank correlation. DCE CT parameters and immunohistochemical markers were also compared according to pathologic subtype, grade, stage, and nodal status by using the Mann-Whitney test. P values less than .05 indicated a statistically significant difference. RESULT Fourteen patients with confirmed primary NSCLC underwent resection. There were negative correlations between blood volume and pimonidazole staining (r = -0.48, P = .004), and between flow-extraction product and glucose transporter protein expression (r = -0.50, P = .002). Flow-extraction product was significantly higher in adenocarcinomas than in squamous cell tumors (17.73 vs 11.46; P = .043). Glucose transporter protein expression was significantly lower for adenocarcinomas than for squamous tumors (14.07 vs 33.03; P < .001) and in node negative than in node positive tumors (15.63 vs 23.85; P = .005). CONCLUSION Blood volume and flow-extraction product derived at DCE CT correlated negatively with pimonidazole and glucose transporter protein expression, indicating the potential of these CT parameters as imaging biomarkers of hypoxia.

Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays : evidence for chromatin movement during repair

Abstract Hamada, N., Schettino, G., Kashino, G., Vaid, M., Suzuki, K., Kodama, S., Vojnovic, B., Folkard, M., Watanabe, M., Michael, B. D. and Prise, K. M. Histone H2AX Phosphorylation in Normal Human Cells Irradiated with Focused Ultrasoft X Rays: Evidence for Chromatin Movement during Repair. Radiat. Res. 166, 31–38 (2006). DNA repair within the cell nucleus is a dynamic process involving a close interaction between repair proteins and chromatin structure. Recent studies have indicated a quantitative relationship between DNA double-strand break induction and histone H2AX phosphorylation. The dynamics of this process within individual cell nuclei is unknown. To address this, we have used a novel focused ultrasoft X-ray microprobe that is capable of inducing localized DNA damage within a subnuclear area of intact cells with a 2.5-μm-diameter beam spot. The present investigation was undertaken to explore the influence of focused irradiation of individual nuclei with 1.49 keV characteristic aluminum K-shell (AlK) X rays on H2AX phosphorylation in normal human cells. Immunofluorescence analyses revealed that significant diffusion of the initial spots of clustered foci of phosphorylated H2AX occurred in a time-dependent fashion after exposure to AlK X rays. Irradiation under cooled conditions resulted in a reduction in the size of spots of clustered foci of phosphorylated H2AX as well as of individual phosphorylated H2AX foci. These findings strongly suggest that diffusion of the chromatin microenvironment occurs during the repair of DNA damage. We also found that AlK ultrasoft X rays (71 foci per gray) were 2.2-fold more effective at the initial formation of phosphorylated H2AX foci than with conventional X rays (32 foci per gray), and that the time required to eliminate 50% of the initial number of foci was 3.4-fold longer in AlK-irradiated cells than that in cells exposed to conventional X rays. For conventional X rays, we also report significant accumulation of larger-sized foci at longer times after irradiation.

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.

Two approaches for irradiating cells individually: a charged-particle microbeam and a soft X-ray microprobe

Abstract We are developing two independent, but complementary microbeams for irradiating cells individually in vitro. Firstly, a charged-particle microbeam that uses a fine-bore glass capillary, combined with a transmission detector to precisely irradiate cells with exact numbers of energetic charge-particles and secondly, a soft X-ray microprobe that produces a very fine beam of carbon-K (278 eV) ultrasoft X-rays, focused to a spot size

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.