Recent Advances in Angiogenesis and ... - Bentham Science
Recent Advances in Angiogenesis and ... - Bentham Science
Recent Advances in Angiogenesis and ... - Bentham Science
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Zebrafish as a Tool to Study Tumor <strong>Angiogenesis</strong> <strong>Recent</strong> <strong>Advances</strong> <strong>in</strong> <strong>Angiogenesis</strong> <strong>and</strong> Antiangiogenesis, 2009 55<br />
angiogenic stimulus is represented by a well-def<strong>in</strong>ed,<br />
topically delivered exogenous agent that leads to the<br />
growth of ectopic blood vessels. This allows the<br />
screen<strong>in</strong>g of low <strong>and</strong> high molecular weight<br />
antagonists target<strong>in</strong>g a specific angiogenic growth<br />
factor <strong>and</strong>/or its receptor(s) [13].<br />
However, the study of vascular development <strong>and</strong> on<br />
the effects of positive or negative modulators of the<br />
embryonic angiogenic process may have important<br />
limitations when translated to cancer research. Indeed,<br />
tumor-<strong>in</strong>duced vessels show profound morphofunctional<br />
alterations when compared to the normal<br />
vasculature [1]. This is reflected by significant<br />
differences <strong>in</strong> gene expression profil<strong>in</strong>g between normal<br />
<strong>and</strong> tumor-derived endothelium [14, 15]. Thus, the<br />
identification of therapeutic targets <strong>and</strong> the assessment<br />
of the efficacy of anti-angiogenic compounds require<br />
the development of appropriate animal models <strong>in</strong><br />
which tumor vasculature can be <strong>in</strong>vestigated. To this<br />
respect, tumor models have been established <strong>in</strong><br />
zebrafish embryos, juveniles, <strong>and</strong> adults (reviewed <strong>in</strong><br />
[16, 17]) that may be suitable for study<strong>in</strong>g the tumor<br />
angiogenesis process <strong>and</strong> its modulators. The<br />
availability of imbred, transgenic, gene knockout/knock-<strong>in</strong><br />
animals, of a wide array of antibodies, as<br />
well as of bio<strong>in</strong>formatic genomic, transcriptomic <strong>and</strong><br />
proteomic <strong>in</strong>formation represent important tools for<br />
tumor angiogenesis studies. Several of these tools<br />
have been becom<strong>in</strong>g available also for zebrafish.<br />
This review focuses on the recently developed tumor<br />
angiogenesis models <strong>in</strong> zebrafish, with particular<br />
emphasis to tumor engraft<strong>in</strong>g <strong>in</strong> zebrafish embryos.<br />
2. TUMOR ANGIOGENESIS MODELS<br />
IN ZEBRAFISH ADULTS<br />
Zebrafish spontaneously develops almost any type of<br />
tumor. Also, several approaches have been developed<br />
to <strong>in</strong>duce cancer <strong>in</strong> zebrafish. They <strong>in</strong>clude treatment<br />
with chemical carc<strong>in</strong>ogens, forward genetic screen<strong>in</strong>g,<br />
target-selected <strong>in</strong>activation of tumor suppressor genes,<br />
<strong>and</strong> expression of mammalian oncogenes. An overview<br />
of these approaches <strong>and</strong> of their ma<strong>in</strong> advantages <strong>and</strong><br />
disadvantages has been published recently [16]. Also,<br />
transplantable tumor cell l<strong>in</strong>es have been generated <strong>in</strong><br />
clonal zebrafish <strong>and</strong> ma<strong>in</strong>ta<strong>in</strong>ed for several passages <strong>in</strong><br />
syngeneic <strong>and</strong> isogeneic adults [18]. Interest<strong>in</strong>gly,<br />
microarray analysis has shown that gene expression<br />
signatures are conserved <strong>in</strong> fish tumors when compared<br />
to their human counterpart [3]. Relevant to tumor<br />
angiogenesis studies <strong>in</strong> adults, a transparent casper<br />
zebrafish l<strong>in</strong>e that lacks all types of pigments has been<br />
generated, allow<strong>in</strong>g the rapid identification of<br />
transplanted tumor cells [19]. Also, cross<strong>in</strong>g of the<br />
casper mutant with transgenic l<strong>in</strong>es that label<br />
vasculature or <strong>in</strong>ternal organs with fluorescent tags<br />
may represent an useful approach to study tumor-host<br />
<strong>in</strong>teractions <strong>in</strong> zebrafish by epifluorescence<br />
stereomicroscopy, confocal microscopy, <strong>and</strong> dualphoton<br />
confocal microscopy.<br />
Non<strong>in</strong>vasive imag<strong>in</strong>g <strong>in</strong> non-transparent zebrafish<br />
adults has been attempted. Ultrasound biomicroscopy<br />
has been used to follow the growth of liver tumors,<br />
their vascularity, <strong>and</strong> response to treatment [20]. Other<br />
imag<strong>in</strong>g techniques, <strong>in</strong>clud<strong>in</strong>g microcomputerized<br />
axial tomography, micromagnetic resonance imag<strong>in</strong>g,<br />
<strong>and</strong> optical projection tomography are beg<strong>in</strong>n<strong>in</strong>g to be<br />
applied <strong>in</strong> zebrafish <strong>and</strong> will help to <strong>in</strong>vestigate tumor<br />
growth <strong>and</strong> vascularization <strong>in</strong> adult zebrafish [21].<br />
3. TUMOR ANGIOGENESIS MODELS<br />
IN ZEBRAFISH JUVANILES<br />
Human cancer cells have been successfully transplanted<br />
<strong>in</strong> the peritoneal cavity of 30 day-old zebrafish [22].<br />
This has allowed the study of the dynamics of<br />
microtumor formation <strong>and</strong> neovascularization us<strong>in</strong>g<br />
high resolution imag<strong>in</strong>g techniques, lead<strong>in</strong>g to a<br />
detailed description of the <strong>in</strong>teraction among<br />
fluorescent tumor cells <strong>and</strong> the green fluorescent<br />
prote<strong>in</strong> (GFP)-labeled vasculature of the host by threedimensional<br />
reconstruction of confocal microscopy<br />
images. The results of these studies have shown that<br />
tumor cells secret<strong>in</strong>g human VEGF promote fish<br />
vessel remodel<strong>in</strong>g <strong>and</strong> angiogenesis <strong>and</strong> that the<br />
human metastatic gene RhoC drives the <strong>in</strong>itial steps of<br />
the metastatic process.<br />
Due to the fact that juvenile zebrafish has a functional<br />
immune system, dexamethasone adm<strong>in</strong>istration is<br />
required to prevent the rejection of the tumor<br />
engraftment. Also, at variance with zebrafish embryos<br />
(see below), the MO gene target<strong>in</strong>g approach is<br />
unfeasible <strong>in</strong> zebrafish juveniles. On the other h<strong>and</strong>,<br />
the impact of the tumor graft on the mature vasculature<br />
of juvenile fishes may recapitulate more closely the<br />
events that occur dur<strong>in</strong>g tumor angiogenesis <strong>in</strong> adult<br />
animals <strong>and</strong> cancer patients. Indeed, develop<strong>in</strong>g<br />
vessels of zebrafish embryos may respond differently to<br />
tumor grafts compared to the fully developed<br />
vasculature of juvenile animals [17].<br />
4. TUMOR ANGIOGENESIS MODELS<br />
IN ZEBRAFISH EMBRYOS<br />
The optical transparency <strong>and</strong> ability to survive for 3-4<br />
days without function<strong>in</strong>g circulation make the<br />
zebrafish embryo amenable for vascular biology<br />
studies. Also, because of the immaturity of the<br />
immune system <strong>in</strong> zebrafish embryos, no xenograft<br />
rejection occurs at this stage [8]. Moreover, transient<br />
gene <strong>in</strong>activation via MO <strong>in</strong>jection represents a<br />
powerful tool for the identification of target genes <strong>in</strong><br />
zebrafish embryo [5].<br />
<strong>Recent</strong> studies have shown the feasibility of <strong>in</strong>ject<strong>in</strong>g<br />
human melanoma cells <strong>in</strong> zebrafish embryos to follow