Application Note

Angiogenesis

Angiogenesis as Anti-Cancer Therapy Radiotherapy and chemotherapy aim at killing tumor cells or at least stopping their multiplication. Those therapies have strong limitations: first, their inherent toxicity is not limited to tumoral cells, but also affects healthy tissue; second, only the strongest and most resistant tumoral cells are able to survive, leading to increasingly aggressive tumors. Angiogenesis inhibition could help avoid these problems. Angiogenesis is the formation of new blood vessels; it takes place during embryonic development, wound healing and tumor growth. Compared to traditional therapies, angiogenesis inhibition has the following advantages: It does not target the tumor itself, and thus does not increase tumor aggressiveness by a genetic selection of cells The toxicity is limited to new blood vessels promoted by the tumor, limiting side effects Interest for angiogenesis started in the 1960s and kept growing as new growth factors for blood vessels were identified. However, this enthusiasm for angiogenesis therapies was hindered by the difficulties researchers met in imaging and characterizing angiogenesis. Indeed, assessing the efficiency of the therapy is not straightforward as opposite effects can be observed with antiangiogenic therapies. An initial decrease of the tumor vascular density results in tumoral cell death at the periphery, and thus in a shrinkage of the tumor size. As a consequence, the vascular density (number of vessels and
Application Note: Angiogenesis

capillaries divided by the size of the tumor) increases. Another hurdle to anti-angiogenic therapy development is its difference in the nature from traditional therapies. The aim is not to kill tumoral cells directly, making the traditional definition of appropriate dose - maximal supported dose - irrelevant. In anti-angiogenic therapies, the highest dose may not be the most efficient on tumor vasculature. Therefore, to determine the optimal dose, one needs a thorough assessment of the therapys impact on the tumor vascularization.

Morphological and functional parameters (such as vascular density, vessel length and diameter, vessel permeability and vasomotion) need to be studied in the living animal. The need for in vivo imaging is obvious in this field. Intravital microscopy, on tumors growing in window chambers or implanted subcutaneously, yields valuable information, yet is limited by its access capabilities and by the preparation required for imaging with a microscope. The Cellvizio offers a new solution without these limitations.

Figure 1: Effects of anti-angiogenic therapies

The Cellvizio LAB The Cellvizio is a fibered fluorescence imaging system. The ProFlex, flexible imaging microprobes with a diameter compatible with endoscopic procedures in small animals, are specially designed for in situ and in vivo imaging. The system also includes a 488 nm laser (compatible with fluorescent dyes used in vivo) that is injected in the fibers of the microprobe to illuminate the tissue. The resulting fluorescence emission signal is collected back and analyzed

by a dedicated software that reconstructs images in real-time (up to 200 frames per second). A range of ProFlex microprobes are available with various optical parameters. The images shown in this document were made with two different types: the S-06505.0 and the S-1500-5.0 with outer diameters of 650 m and 1.5 mm, respectively. Both have a maximum field of view of 600 x 500 m, a lateral resolution of 5 m, an axial resolution of 15 m and a working distance of 0 m.

The system is controlled by an Apple computer running OS X. ImageCell, the sophisticated image processing software developed by Mauna Kea Technologies, controls image acquisition, processing and display. Quantification and file export tools are included in the package. Suitability of the Cellvizio for Angiogenesis Studies The Cellvizio offers a new way to image and characterize tumoral angiogenesis with numerous advantages: Easy Imaging Intravital microscopy imaging most often requires a long and tedious preparation of the animal: Preparation of a window chamber on the organ Perfusion of tissues with physiological solutions A half an hour pause to reach physiological stability With the Cellvizio, there is no need for time consuming preparation: imaging is immediate, as soon as a microprobe is put in contact with the tissue, with real time acquisition. Improved Physiological Relevance Due to Limited Disturbance Since a small 2 mm incision of the skin is enough for imaging with the Cellvizio, the tissue is not physiologically disturbed. Plasmatic staining reveals the real vascularization system of the animal. Labeled blood flows into vessels and capillaries. Quantification in Real Time ImageCell provides many quantification features in real time, like measure of fluorescence intensities with complex ROI management, histogram display and distance measurements. These open the door to instant vessel permeability and vasodynamics measurement.
Application Note: Angiogenesis

ProFlex microprobes with different orientations

Figure 2: Illustration of the facilitated access with ProFlex microprobes. A conventional lens has a diameter of a few millimeters and can only move in the X, Y and Z directions. ProFlex microprobes, with an outer diameter inferior to 1 mm, can also be oriented with theta and phi movements.

Longitudinal Studies Made Possible The new type of access offered by the Cellvizio enables longitudinal studies. The efficiency of a drug can now be studied on the same animal during days through the observation of their tumoral vascularization development. Less animals are required and the physiological relevance of the results is improved.

Image Procedures - A Range of Possibilities The Cellvizio can be used with a wealth of different protocols when it comes to angiogenesis imaging. Below are a few examples. Plasma Labeling The procedure for vascularization imaging with the Cellvizio is no longer than two lines: inject a fluorescent plasma-labeling dye intravenously and put the microprobe in direct contact with the desired organ after a small incision of the skin. With this simple protocol, the entire circulatory system is revealed: vessels and capillaries in all organs, sinusoids in the liver, tubules in the kidneys, capillaries in the lungs and in the brain, etc.

Figure 3: Access to a subcutaneous tumor with ProFlex microprobes

Note: Angiogenesis imaging requires the use of a dye with a high molecular weight, such as FITC-Dextran 250 kDa, Sigma Aldrich, to avoid leakage associated with windowed vessels in tumors.

Transgenic animals imaging Tie2 mice and beta-actin GFP mice have fluorescent endothelial cells, making them interesting for vascularization visualization.

Injection of fluorescent endothelial cells Angiogenesis progression can also be imaged and studied after injection of fluorescent endothelial cells. The cells are recruited to build up new vessels and reveal sites were angiogenesis is active.

Visualization of Cells Blood cells labeling can give critical information about the cell-wall interaction. For example, leukocytes can be imaged by intravenous injection of Rhodamine 6G.

FOV: 415 x 300 m Figure 4: Capillaries of a mouse subcutaneous prostate tumor stained with FITC-Albumin FOV: 400 x 280 m Figure 5: Mouse tumoral arterioles and venules FOV: 400 x 280 m Figure 6: Rolling leukocytes in a venule of the cremaster muscle of a mouse

Beyond Imaging: Quantification Examples with the Cellvizio Functional Capillary Density (FCD) Assessment of the vascular density is a standard approach to quantify angiogenesis. It can also be used as critical information in making a prognosis for the tumor evolution. Histological sections labeled with reliable immunochemistry markers can help identify vessels, but cannot differentiate vessels where blood flows effectively from the ones without blood flow. Since our in vivo labeling method for vascularization studies is based on fluorescent blood plasma, only functional vessels are revealed.

Permeability Measurement Blood vessel leakiness is a well documented characteristic of tumor vessels. It contributes to high interstitial pressure and may facilitate angiogenesis; in addition, increased endothelial permeability enables efficient drug delivery from the bloodstream to tumor cells. The extravasation, resulting from blood leakiness, is very easy to record and quantify with the Cellvizio. Using a blood plasma labeling procedure, the fluorescence intensity of an avascular zone will be directly linked with the extravasation. Thus, studying the histogram or simply the mean intensity of a given region of interest (ROI) reveals the evolution of the amount of plasma that has leaked to the ROI. With ImageCell functionalities, this analysis is immediate.

FCD = 3.6 mm / 0.124 mm2 Figure 7: Estimation of blood vessel length per area unit on a Cellvizio image.

The Cellvizio images enable the assessment of the functional capillary density of the observed tissue. ImageCell enables an automatic segmentation of the images in order to recognize vessels and capillaries. Total vessel length and ratio vessel length per tissue surface unit can then be used as an index for functional capillary density. Vascularization of different areas within a tumor of the same area at different time points or of different tumors can thus be compared.

Application Note: Angiogenesis

Figure 8 shows an example of such a study. The extravasation caused by histamine is measured on a mouse muscular tissue. On the first image, a region of interest excluding vessels and capillaries is defined. The correspondent histogram shows therefore a very low level of signal. The second and third images show a dramatic increase of fluorescence intensity of the ROI, the curve of the signal is shifted to the right. Since the protocol calls for fluorescently labeled blood plasma, the observed phenomenon enables the quantification of the extravasation at the studied site.

FOV: 400 x 280 m

FOV: 400 x 280 m

FOV: 400 x 280 m

Figure 8: Effect of histamine on vessel permeability: Extravasation of mouse muscular vessels. The histograms show the fluorescence intensity of the regions of interest (in red on the images)

Studying Vasodynamics

FOV: 400 x 280 m

Vasodilation upon carbogen inhalation

Vasoconstriction after carbogen removal

Figure 9 - Constriction and dilation of tumoral vessels. The vasodynamics of a tumoral vessel was observed upon inhalation of carbogen by the mouse. On the first two images, you see the evolution of the diameter of the vessel while the mouse is breathing carbogen: it is dilated. After removal of carbogen, the vessel constricts back to its original size. The diagram on the left shows the vessel response to the sequence carbogen - air carbogen.

Application Note: Angiogenesis

Discussion Compared to intravital microscopy, the Cellvizio offers new access possibilities and new opportunities for minimally invasive protocols, immediate and intuitive use and easy quantification capabilities. The real-time imaging capacity of the system makes it perfectly adapted to record dynamic events associated with blood flow. The Cellvizio is a valuable tool for more efficient studies of angiogenesis development and effects of anti-angiogenic therapies.

Courtesy Anne-Carole Duconseille, Nathalie Faye, Laure Fournier, Charles A. Cuenod and Olivier Clment, Descartes Image, Small Animal Imaging Facility, Universit Paris V, Paris, France Pr Vicaut, Microcirculation Lab, Hopital Fernand Widal, Paris, France Elisabeth Laemmel and Eric Vicaut, LEM, Paris, France

Bibliography 1. Donald M MacDonald, Peter L Choyke: Imaging of angiogenesis: from microscope to clinic Nat Med. 2003 Jun;9(6):713-25 2. Gillian M. Tozer, Simon M. Ameer-Beg, Jennifer Baker, Paul R. Barber, Sally A. Hill, Richard J. Hodgkiss, Rosalind Locke, Vivien E. Prise, Ian Wilson, Borivoj Vojnovic: Intravital imaging of tumour vascular networks using multiphoton fluorescence microscopy Adv Drug Deliv Rev. 2005 Jan 2;57(1):135-52 3. E Laemmel, M Genet, G Le Goualher, A Perchant, JF Le Gargasson, E Vicaut Fibered confocal fluorescence microscopy (Cellvizio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy. J Vasc Res. 2004 Sep-Oct;41(5):400-11. Epub 2004 Sep 30.

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Application Note: Angiogenesis