In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Summary

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.

Abstract

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.

Introduction

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 ³ is commonly used to assess tissue microcirculation. Dynamic contrast-enhanced imaging is a technique which follows over time the biodistribution of a tracer injected intravenously. From this acquisition, quantitative parameters can be extracted reflecting tissue vascularization. DCE imaging has been most often used with CT, MRI or ultrasound. However, these imaging techniques do not allow direct viewing of the microvessels, since their resolution, other than with the use of specific experimental devices, most often remains macroscopic.

In this paper, we propose to study the tumor vasculature at the microscopic scale and in vivo using dynamic contrast-enhanced optical imaging, with fibered confocal videomicroscopy. We used a macromolecular contrast agent (FITC-dextran) which remains exclusively within vessels or leaks through the endothelial barrier into the interstitium, according to its molecular weight and the characteristics of the endothelium of the tissue studied ⁴. This allowed the study of both microvessel structure, by correctly delineating vessels, and capillary permeability, by leaking and accumulating in the interstitium.

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Protocol

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 (Kétamine 500, Virbac, Carros, France), respectively 66 mg/kg and 264 mg/kg for a 20 g mouse.

3. Preparation of the Organ of Interest

1. We shaved the mice at the location of interest (for example, over a subcutaneous tumor). Animal hair is often auto-fluorescent when white. When black, it absorbs light.
2. The skin facing the organ to be imaged was incised. It is important to wait until the bleeding has stopped before injecting the contrast agent, otherwise it will leak in the blood and contaminate the image.

4. Acquisition

1. The contrast agent was injected through either the jugular vein or the caudal vein. There is no or little background signal in the organ observed in the absence of a fluorescent contrast agent.
2. The probe was placed in front of the organ to be imaged. In our study, this was the tumor.
3. The laser was turned on to illuminate the tumor and see the fluorescence in the capillaries.
4. The tumor was explored manually by moving the probe in a very slow movement while recording to visualize the capillary network. It is important to maintain a steady hand, and this technique requires a little experience. In our study, this first step allowed quantification of functional capillary density.
5. The second step was the dynamic acquisition over time. For this study, we used a 70 kDa FITC-dextran. There is no interstitial leakage in most normal organs but there is in tumors. To acquire images of the same location over time (as in our case), it is important to set up a system to maintain the probe on the area of interest. This was done by using a handmade support to hold the probe, and by placing a bit of ultrasound gel on the tip of the probe. Before recording, time was spent to stabilize the probe placed in contact with the tumor. Once the position was secured, there was only minimal motion due to the mouse's breathing. The laser was turned on to record 3 images every 30 sec for 20 min to detect the presence of capillary leakage. It was turned off between each recording to reduce contrast agent bleaching.
6. In our experiment, mice were sacrificed at the end of the procedure for histological analysis of tumors.

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Results

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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Discussion

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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Materials

Name	Company	Catalog Number	Comments
Insulin serynge Myjector 1ml 29G	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