Application Note: Angiogenesis

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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.

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Application Note: Angiogenesis

  1. 1. Application Note AngiogenesisAngiogenesis as Anti-Cancer capillaries divided by the size of Morphological and functionalTherapy the tumor) increases. parameters (such as vascular density, vessel length andRadiotherapy and chemotherapy Another hurdle to anti-angiogenic diameter, vessel permeability andaim at killing tumor cells or at least therapy development is its vasomotion) need to be studied instopping their multiplication. Those difference in the nature from the living animal.therapies have strong limitations: traditional therapies. The aim isfirst, their inherent toxicity is not not to kill tumoral cells directly, The need for in vivo imaging islimited to tumoral cells, but also making the traditional definition obvious in this field. Intravitalaffects healthy tissue; second, only of appropriate dose - maximal microscopy, on tumors growingthe strongest and most resistant supported dose - irrelevant. In in window chambers or implantedtumoral cells are able to survive, anti-angiogenic therapies, the subcutaneously, yields valuableleading to increasingly aggressive highest dose may not be the most information, yet is limited by itstumors. efficient on tumor vasculature. access capabilities and by the Therefore, to determine the preparation required for imagingAngiogenesis inhibition could optimal dose, one needs a with a microscope.help avoid these problems. thorough assessment of theAngiogenesis is the formation of therapy’s impact on the tumor The Cellvizio offers a new solutionnew blood vessels; it takes place vascularization. without these limitations.during embryonic development,wound healing and tumor growth.Compared to traditional therapies,angiogenesis inhibition has thefollowing 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 effectsInterest for angiogenesis startedin the 1960s and kept growing Figure 1: Effects of anti-angiogenic therapiesas new growth factors for bloodvessels were identified. However, The Cellvizio® LAB by a dedicated software thatthis enthusiasm for angiogenesis reconstructs images in real-timetherapies was hindered by the The Cellvizio is a fibered (up to 200 frames per second).difficulties researchers met fluorescence imaging system.in imaging and characterizing The ProFlex, flexible imaging A range of ProFlex microprobesangiogenesis. Indeed, assessing microprobes with a diameter are available with various opticalthe efficiency of the therapy is compatible with endoscopic parameters. The images shownnot straightforward as opposite procedures in small animals, in this document were made witheffects can be observed with anti- are specially designed for in situ two different types: the S-0650-angiogenic therapies. An initial and in vivo imaging. The system 5.0 and the S-1500-5.0 with outerdecrease of the tumor vascular also includes a 488 nm laser diameters of 650 μm and 1.5 mm,density results in tumoral cell (compatible with fluorescent dyes respectively. Both have a maximumdeath at the periphery, and thus in used in vivo) that is injected in field of view of 600 x 500 μm, aa shrinkage of the tumor size. the fibers of the microprobe to lateral resolution of 5 μm, an axial illuminate the tissue. The resulting resolution of 15 μm and a workingAs a consequence, the vascular fluorescence emission signal distance of 0 μm.density (number of vessels and is collected back and analyzedApplication Note: Angiogenesis 1
  2. 2. The system is controlled by anApple computer running OS X.ImageCell, the sophisticated imageprocessing software developed byMauna Kea Technologies, controlsimage acquisition, processing and ProFlex microprobes withdisplay. Quantification and file different orientationsexport tools are included in thepackage.Suitability of the Cellvizio forAngiogenesis StudiesThe Cellvizio offers a new way toimage and characterize tumoral Figure 2: Illustration of the facilitated access with ProFlex microprobes. Aangiogenesis with numerous conventional lens has a diameter of a few millimeters and can only move in the X, Yadvantages: and Z directions. ProFlex microprobes, with an outer diameter inferior to 1 mm, can also be oriented with theta and phi movements.Easy Imaging Longitudinal Studies Made PossibleIntravital microscopy imaging mostoften requires a long and tedious The new type of access offered by the Cellvizio enables longitudinalpreparation of the animal: studies. The efficiency of a drug can now be studied on the same animal• Preparation of a window chamber during days through the observation of their tumoral vascularization on the organ development. Less animals are required and the physiological relevance of• Perfusion of tissues with the results is improved. physiological solutions• A half an hour pause to reach physiological stabilityWith the Cellvizio, there is no need Image Procedures - A Range of Possibilitiesfor time consuming preparation: The Cellvizio can be used with a wealth of different protocols when itimaging is immediate, as soon comes to angiogenesis imaging. Below are a few examples.as a microprobe is put in contactwith the tissue, with real time Plasma Labelingacquisition. The procedure for vascularizationImproved Physiological Relevance imaging with the Cellvizio is noDue to Limited Disturbance longer than two lines: inject a fluorescent plasma-labelingSince a small 2 mm incision of dye intravenously and put thethe skin is enough for imaging microprobe in direct contact withwith the Cellvizio, the tissue is the desired organ after a smallnot physiologically disturbed. incision of the skin.Plasmatic staining reveals thereal vascularization system of the With this simple protocol, theanimal. Labeled blood flows into entire circulatory system isvessels and capillaries. revealed: vessels and capillaries in all organs, sinusoids in the liver, Figure 3: Access to a subcutaneousQuantification in Real Time tubules in the kidneys, capillaries tumor with ProFlex microprobesImageCell provides many in the lungs and in the brain, etc.quantification features in real Note: Angiogenesis imaging requires the use of a dye with a hightime, like measure of fluorescence molecular weight, such as FITC-Dextran 250 kDa, Sigma Aldrich, to avoidintensities with complex ROI leakage associated with windowed vessels in tumors.management, histogram displayand distance measurements. Theseopen the door to instant vesselpermeability and vasodynamicsmeasurement.Application Note: Angiogenesis 2
  3. 3. Transgenic animals imaging Injection of fluorescent endothelial Visualization of Cells cellsTie2 mice and beta-actin GFP Blood cells labeling can givemice have fluorescent endothelial Angiogenesis progression can critical information about thecells, making them interesting for also be imaged and studied after cell-wall interaction. For example,vascularization visualization. injection of fluorescent endothelial leukocytes can be imaged by cells. The cells are recruited to intravenous injection of Rhodamine build up new vessels and reveal 6G. sites were angiogenesis is active. FOV: 415 x 300 µmFigure 4: Capillaries of a mousesubcutaneous prostate tumor stained FOV: 400 x 280 µmwith FITC-Albumin Figure 6: Rolling leukocytes in a FOV: 400 x 280 µm venule of the cremaster muscle of a mouse Figure 5: Mouse tumoral arterioles and venulesBeyond Imaging: Quantification Examples with the Cellvizio Permeability MeasurementFunctional Capillary Density (FCD) Blood vessel leakiness is a well documented characteristic ofAssessment of the vascular density tumor vessels. It contributes tois a standard approach to quantify high interstitial pressure and mayangiogenesis. It can also be used facilitate angiogenesis; in addition,as critical information in making a increased endothelial permeabilityprognosis for the tumor evolution. enables efficient drug delivery fromHistological sections labeled with the bloodstream to tumor cells.reliable immunochemistry markerscan help identify vessels, but The extravasation, resultingcannot differentiate vessels where from blood leakiness, is veryblood flows effectively from the easy to record and quantifyones without blood flow. Since with the Cellvizio. Using a bloodour in vivo labeling method for FCD = 3.6 mm / 0.124 mm2 plasma labeling procedure, thevascularization studies is based Figure 7: Estimation of blood vessel fluorescence intensity of anon fluorescent blood plasma, only length per area unit on a Cellvizio image. avascular zone will be directlyfunctional vessels are revealed. linked with the extravasation. Thus, studying the histogram or simplyThe Cellvizio images enable the assessment of the functional capillary the mean intensity of a givendensity of the observed tissue. ImageCell enables an automatic region of interest (ROI) revealssegmentation of the images in order to recognize vessels and capillaries. the evolution of the amount ofTotal vessel length and ratio vessel length per tissue surface unit can then plasma that has leaked to the ROI.be used as an index for functional capillary density. Vascularization of With ImageCell functionalities, thisdifferent areas within a tumor of the same area at different time points or analysis is immediate.of different tumors can thus be compared.Application Note: Angiogenesis 3
  4. 4. Figure 8 shows an example of such a study. The extravasation caused by histamine is measured on a mousemuscular tissue. On the first image, a region of interest excluding vessels and capillaries is defined. Thecorrespondent histogram shows therefore a very low level of signal. The second and third images show a dramaticincrease of fluorescence intensity of the ROI, the curve of the signal is shifted to the right. Since the protocol callsfor fluorescently labeled blood plasma, the observed phenomenon enables the quantification of the extravasationat the studied site. FOV: 400 x 280 µm FOV: 400 x 280 µm FOV: 400 x 280 µmFigure 8: Effect of histamine on vessel permeability: Extravasation of mouse muscular vessels. The histograms show thefluorescence intensity of the regions of interest (in red on the images)Studying Vasodynamics FOV: 400 x 280 µm 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 4
  5. 5. DiscussionCompared to intravital microscopy, the Cellvizio offersnew access possibilities and new opportunities forminimally invasive protocols, immediate and intuitiveuse and easy quantification capabilities. The real-timeimaging capacity of the system makes it perfectlyadapted to record dynamic events associated withblood flow.The Cellvizio is a valuable tool for more efficientstudies of angiogenesis development and effects ofanti-angiogenic therapies.CourtesyAnne-Carole Duconseille, Nathalie Faye, LaureFournier, Charles A. Cuenod and Olivier Clément,Descartes Image, Small Animal Imaging Facility,Université Paris V, Paris, FrancePr Vicaut, Microcirculation Lab, Hopital Fernand Widal,Paris, FranceElisabeth Laemmel and Eric Vicaut, LEM, Paris, FranceBibliography1. Donald M MacDonald, Peter L Choyke: Imaging of angiogenesis: from microscope to clinic Nat Med. 2003 Jun;9(6):713-252. 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):135-523. 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 VisualSonics Inc. Sep 30. T.1.416.484.5000 Toll Free (North America) 1.866.416.4636 Toll Free (Europe) +800.0751.2020 E. info@visualsonics.com www.visualsonics.com VisualSonics®, Vevo®, MicroMarker™, VevoStrain™, DEPO®, SoniGene™, RMV™, EKV® and Insight through In Vivo Imaging are trademarks™ of VisualSonics Inc.Application Note: Angiogenesis 5

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