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In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Published: September 11th, 2013



1Université Paris Descartes Sorbonne Paris Cité, INSERM UMR-S970, 2Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Radiologie

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.

We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 μm in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.

The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

Angiogenesis is a complex process 1 that involves the formation of new blood vessels from pre-existing vessels. Pathological changes in tissue microcirculation, composed of arterioles, capillaries, and venules, are implicated in a large range of diseases such as cancer, inflammation, or diabetes. It is therefore essential to develop methods to quantitatively assess microvessel structure and function. Imaging enables the study of microvessels in a non- or micro-invasive manner, in real-time and in vivo, and repeated measures over time in the same animal 2.

Currently, dynamic contrast-enhanced (DCE) imaging

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1. Preparation of the Contrast Agent

  1. For FITC-dextran 70 kDa, the dose injected is 500 mg/kg (10 mg of FITC-dextran diluted in 0.1 ml of saline for a mouse weighing 20 g).
  2. The agent should not be exposed too long to light. To avoid bleaching, it is recommended to cover the tube with aluminum foil.

2. Anesthesia

  1. Mice were anesthetized by an intraperitoneal injection of a mixture of 1:4 of xylazine (Rompun 2%, Bayer, Puteaux, France) and Ketamine (Ké.......

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Using the data collected, we could quantitatively analyze different parameters reflecting microcirculation.

We studied in vivo the peripheral vascular network of a colon tumor implanted in balb-c mice using a fibered confocal fluorescence videomicroscopy system (Cellvizio, Maunakea Technology, Paris, France 2), after injection of a macromolecular fluorescent contrast agent Fluorescein IsoThioCyanate-dextran (FITC-dextran) with a molecular weight of 70 kDa (Sigma-Aldrich, Sa.......

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The study of tumor microcirculation has become essential in understanding the pathophysiology of tumor growth, dissemination and response to therapy 1. Optical imaging is one of the techniques that can be used to observe the capillaries using a fluorescent contrast agent and to quantify morphological (Functional Capillary Density) and functional (index leakage) parameters.

The fluorescence microscopy imaging we used in this study has both advantages and limits. One advantage is bein.......

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Name Company Catalog Number Comments
Name Company Catalogue number Comments (optional)
Insulin serynge
Myjector 1ml
Terumo Europe BS-05M2913
Fluorescein isothiocyanate-dextran 70 kDa Sigma-Aldrich 01619HH 100 mg/mL
diluted in saline
Fibered confocal videomicroscopy Cellvizio - MaunaKea Technologies
Calibration and Cleaning Kit for LEICAFCM1000 Leica Microsystems LSU-488 Store at 4 °C
Probe ProFlexTM Z MaunaKea Technologies
Mosaicing software MaunaKea Technologies
Vessel detection software MaunaKea Technologies

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