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.

1. Application Note
Angiogenesis
Angiogenesis as Anti-Cancer capillaries divided by the size of Morphological and functional
Therapy the tumor) increases. parameters (such as vascular
density, vessel length and
Radiotherapy and chemotherapy Another hurdle to anti-angiogenic diameter, vessel permeability and
aim at killing tumor cells or at least therapy development is its vasomotion) need to be studied in
stopping their multiplication. Those difference in the nature from the living animal.
therapies have strong limitations: traditional therapies. The aim is
first, their inherent toxicity is not not to kill tumoral cells directly, The need for in vivo imaging is
limited to tumoral cells, but also making the traditional definition obvious in this field. Intravital
affects healthy tissue; second, only of appropriate dose - maximal microscopy, on tumors growing
the strongest and most resistant supported dose - irrelevant. In in window chambers or implanted
tumoral cells are able to survive, anti-angiogenic therapies, the subcutaneously, yields valuable
leading to increasingly aggressive highest dose may not be the most information, yet is limited by its
tumors. efficient on tumor vasculature. access capabilities and by the
Therefore, to determine the preparation required for imaging
Angiogenesis inhibition could optimal dose, one needs a with a microscope.
help avoid these problems. thorough assessment of the
Angiogenesis is the formation of therapy’s impact on the tumor The Cellvizio offers a new solution
new blood vessels; it takes place vascularization. without these limitations.
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 Figure 1: Effects of anti-angiogenic therapies
as new growth factors for blood
vessels were identified. However, The Cellvizio® LAB by a dedicated software that
this enthusiasm for angiogenesis reconstructs images in real-time
therapies 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 microprobes
angiogenesis. Indeed, assessing microprobes with a diameter are available with various optical
the efficiency of the therapy is compatible with endoscopic parameters. The images shown
not straightforward as opposite procedures in small animals, in this document were made with
effects 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 outer
decrease 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 maximum
death at the periphery, and thus in used in vivo) that is injected in field of view of 600 x 500 μm, a
a 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 working
As a consequence, the vascular fluorescence emission signal distance of 0 μm.
density (number of vessels and is collected back and analyzed
Application Note: Angiogenesis 1

2. 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
ProFlex microprobes with
display. Quantification and file
different orientations
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
Figure 2: Illustration of the facilitated access with ProFlex microprobes. A
angiogenesis with numerous
conventional lens has a diameter of a few millimeters and can only move in the X, Y
advantages: 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 Possible
Intravital microscopy imaging most
often requires a long and tedious The new type of access offered by the Cellvizio enables longitudinal
preparation 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 stability
With the Cellvizio, there is no need Image Procedures - A Range of Possibilities
for time consuming preparation:
The Cellvizio can be used with a wealth of different protocols when it
imaging is immediate, as soon
comes to angiogenesis imaging. Below are a few examples.
as a microprobe is put in contact
with the tissue, with real time Plasma Labeling
acquisition.
The procedure for vascularization
Improved Physiological Relevance imaging with the Cellvizio is no
Due to Limited Disturbance longer than two lines: inject
a fluorescent plasma-labeling
Since a small 2 mm incision of
dye intravenously and put the
the skin is enough for imaging
microprobe in direct contact with
with the Cellvizio, the tissue is
the desired organ after a small
not physiologically disturbed.
incision of the skin.
Plasmatic staining reveals the
real vascularization system of the With this simple protocol, the
animal. Labeled blood flows into entire circulatory system is
vessels and capillaries. revealed: vessels and capillaries
in all organs, sinusoids in the liver, Figure 3: Access to a subcutaneous
Quantification in Real Time
tubules in the kidneys, capillaries tumor with ProFlex microprobes
ImageCell 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 high
time, like measure of fluorescence
molecular weight, such as FITC-Dextran 250 kDa, Sigma Aldrich, to avoid
intensities with complex ROI
leakage associated with windowed vessels in tumors.
management, histogram display
and distance measurements. These
open the door to instant vessel
permeability and vasodynamics
measurement.
Application Note: Angiogenesis 2

3. Transgenic animals imaging Injection of fluorescent endothelial Visualization of Cells
cells
Tie2 mice and beta-actin GFP Blood cells labeling can give
mice have fluorescent endothelial Angiogenesis progression can critical information about the
cells, 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 µm
Figure 4: Capillaries of a mouse
subcutaneous prostate tumor stained FOV: 400 x 280 µm
with 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
venules
Beyond Imaging: Quantification Examples with the Cellvizio Permeability Measurement
Functional Capillary Density (FCD) Blood vessel leakiness is a well
documented characteristic of
Assessment of the vascular density tumor vessels. It contributes to
is a standard approach to quantify high interstitial pressure and may
angiogenesis. It can also be used facilitate angiogenesis; in addition,
as critical information in making a increased endothelial permeability
prognosis for the tumor evolution. enables efficient drug delivery from
Histological sections labeled with the bloodstream to tumor cells.
reliable immunochemistry markers
can help identify vessels, but The extravasation, resulting
cannot differentiate vessels where from blood leakiness, is very
blood flows effectively from the easy to record and quantify
ones without blood flow. Since with the Cellvizio. Using a blood
our in vivo labeling method for FCD = 3.6 mm / 0.124 mm2 plasma labeling procedure, the
vascularization studies is based Figure 7: Estimation of blood vessel fluorescence intensity of an
on fluorescent blood plasma, only length per area unit on a Cellvizio image. avascular zone will be directly
functional vessels are revealed. linked with the extravasation. Thus,
studying the histogram or simply
The Cellvizio images enable the assessment of the functional capillary the mean intensity of a given
density of the observed tissue. ImageCell enables an automatic region of interest (ROI) reveals
segmentation of the images in order to recognize vessels and capillaries. the evolution of the amount of
Total 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, this
different 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. 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 µm FOV: 400 x 280 µm FOV: 400 x 280 µm
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 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. 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 Clément,
Descartes Image, Small Animal Imaging Facility,
Université 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
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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.
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Application Note: Angiogenesis 5