Utilizzo di zebrafish come modello animale nella ricerca oncologica
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FORMAZIONE E TRAINING DEL PERSONALE COINVOLTO
NELLE ATTIVITA’ DI SPERIMENTAZIONE ANIMALE
MAGGIO 2020
Utilizzo di zebrafish come modello animale
nella ricerca oncologica
Paolo Macor
Dept. of Life Sciences
University of Trieste
Office: +39 040 5588683
Skype: zymosan2000Zebrafish: può un pesce teloseo essere un valido
modello di patologie umane?
Nonostante le ovvie differenze, come vertebraU, i pesci possiedono molte
caraVerisUche anatomiche e fisiologiche dei mammiferi.What is a zebrafish? The Zebrafish is named for the five uniform, pigmented, horizontal, blue stripes on the side of the body, which are reminiscent of a Zebra's stripes, and which extend to the end of the caudal fin. It is laterally compressed with its mouth directed upwards. The Zebrafish is naUve to the streams of the South- Eastern Himalayan region. The species arose in the Ganges region in eastern India and commonly inhabits streams, canals, ditches, ponds and slow-moving or stagnant
Why use zebrafish?
- Small size.
- Short
generation time (3-4
months).
- Produces 300-400 eggs every
2 weeks.General Features Benefits
Appearance
Large number can be kept easily and cheaply in
-Dimension ~4 cm
lab
-Salient disUnguishable features of male and
female Good model for visualizaUon of cellular acUvity
-Oaen transparent adult bodies
Habitat
-Fresh water fish Universally available
- Tropical fish
Feeding
Low cost of maintenance
-Omnivorous
ReproducFon
-Female spawns every 2-3 days
Large number of offspring- good for batch
-Breeds all year round variaUon studies
-Several hundreds of eggs produced in single
Easy availability of eggs
clutch
-External ferUlizaUonWhy use zebrafish?
- Small size.
- Short
generation time (3-4
months).
- Produces 300-400 eggs every
2 weeks.
- All
major organs present within
5 days post fertilization.
- Translucent embryosLife cycle • Total life span: 42-66 months • Good model for developmental studies because of transparency at early stages
Why use zebrafish?
- Small size.
- Short generation time (3-4 months).
- Produces 300-400 eggs every
2 weeks.
- All
major organs present within
5 days post fertilization.
- Translucent embryos
- Lots
of genome resources available
(completely sequenced genome).Genome Sequencing The Wellcome Trust Sanger InsFtute, U.K. was the first to start the Zebrafish Genome Sequencing Project. In 2009, InsFtute of Genomics & IntegraFve Biology, New Delhi reported sequenced genes in Zebrafish. The paper “The zebrafish reference genome sequence and its relaFonship to human genome” was published in Nature in 17th April, 2013. Its genome (1.4 x 109 base pairs) has been sequenced revealing 26,606 protein-coding genes. The Zebrafish genome has been fully sequenced to a very high quality. This has enabled scienUsts to create mutaUon in more than 14,000 genes to study their funcUon.
GENETICS
1.70% of protein-coding human genes are related to genes
found in the Zebrafish.
2.84% of genes known to be associated with human disease
have a Zebrafish counterpart.
5505 12897 3909Advantages of Zebrafish as a model organism 1. OpUcally translucent embryos 2. Rapid hatching of eggs 3. Maintenance cost is significantly lower than those for mammals. 4. Completely sequenced genome 5. Amenable for molecular and geneUc analysis. 6. As Zebrafish eggs are ferUlized and develop outside the mother’s body it is an ideal model organism for studying early development.
Development
Disadvantages of Zebrafish as a model organism Physiology: - Lack of cell markers/anUbodies - Lack of hematopoieUc cell lines - Lack of biochemical reagents, eg, purified cytokines - Lack of in vitro differenUaUon system (hematopoieUc cell culture assays) - Lack of inbred strains
ZEBRAFISH AS A MODEL
On 22nd July, 1976, the Space StaUon, Salyut 5 was launched in which Zebrafish was one of the crew members.
Replacement alternatives refers to methods which avoid or replace the use of animals in an area where animals would otherwise have been used. This includes both absolute replacements (i.e. replacing animals with inanimate systems, such as computer programs) and relative replacements (i.e. replacing more sentient animals, such as vertebrates, with animals that current scientific evidence indicates have a significantly lower potential for pain perception, such as some invertebrates). Russell and Burch, 1959
Pesci: modelli di replacement relativo
Il presente decreto si applica ai seguenti animali: a) animali vertebrati vivi non umani, comprese: 1) forme larvali capaci di alimentarsi autonomamente; 2) forme fetali di mammiferi a partire dall’ultimo terzo del loro normale sviluppo; b) cefalopodi vivi.
Art. 4 - Anestesia
Immersione in....(=inalazione nei vertebrati terrestri)
Tricaine methanesulphonate (MS222; 200mg/L)
O
etomidate 10 mg/L, pH 7.0-7.5
Graduale raffreddamento
(sedazione ed immobilizzazione in procedure non gArt. 6 - Metodi di Soppressione
MS222 (Tricaina 900 mg/L), eugenolo (1500 μL/L), and shock ipotermico (acqua e ghiaccio ≤4°C)
Cessazione batto cardiaco Percentuale di ripresaZebrafish e benessere FonF di stress: trasporto avviene in buste di plasUca contenenU pillole di ossigeno, con una densità di 10/2 litri manipolazione l’impiego di reUni durante il cambi di vascheVa, o nel passaggio da una vascheVa all’altra. Un’errata manipolazione causa stress, ma anche possibili lesioni al muco di rivesUmento superficiale esponendo l’animale a possibili infezioni
Zebrafish e benessere FonF di stress: - sovraffollamento (comporta un notevole aumento di stress, con conseguente immunodepressione, aumento di cataboliU nell’acqua, ridoVa ferUlità, esposizione ad infezioni) - malnutrizione (immunodepressione, scarsa o assente ferUlità, esposizione ad infezioni e malate) - modifiche dei parametri dell’acqua (temperatura, ossigeno e salinità)
Zebrafish e benessere Come riconoscere lo stress: osservare gli animali, il loro comportamento e l’andamento della colonia , calo nell'ovodeposizione, ristagno di cibo causato da ridoda assunzione.
Zebrafish e benessere Segni clinici di malaea o di stress: CambiamenF comportamentali Perdita dell’appeUto Letargia Tendenza all’isolamento Animali tendono a nascondersi Respirazione in superficie Pinne bloccate Alterazioni nell’equilibrio Atvità natatoria alterata Cambi della frequenza respiratoria
Zebrafish e benessere Segni clinici di malaea o di stress: CambiamenF dell’aspedo Lesioni della cute (ulcere, macchie, arrossamenU) Modifiche del colore Perdita di scaglie Protrusione di scaglie Ascite
ZEBRAFISH AS A MODEL IS USED TO STUDY: ! genetica dello sviluppo ! neurobiologia ! malattie neurodegenerative ! cancerogenesi ! tossicologia ! medicina rigenerativa/rigenerazione tissutale ! immunologia ! malattie infettive ! malattie metaboliche
Myocardial infarcFon Mammals respond to a myocardial infarcUon by irreversible scar formaUon. By contrast, the Zebrafish are able to resolve the scar and to regenerate funcUonal cardiac muscle. The reparaUve and regeneraUve process is achieved through Smad3- dependent TGFβ signaling.
TAIL FIN REGENERATION 1. Zebrafish fins are complex appendages that quickly and reliably regenerate aaer amputaUon, restoring both size and shape. 2. The key regeneraUve units are their many rays of dermal bone, which are segmented and lined by osteoblasts. 3. An amputated fin ray is covered within the first several hours by epidermis, and within one to two days, a regeneraUon blastema forms. The blastema is a proliferaUve mass of morphologically similar cells, formed through disorganizaUon and distal migraUon of fibroblasts and osteoblasts. 4. Blastema formaUon is the only one step in zebrafish tail fin regeneraUon. 5. Wnt signaling posiUvely regulate blastemal proliferaUon and outgrowth.
ZEBRAFISH AS A MODEL IN CANCER RESEARCH 1. Zebrafish have been used to make several transgenic models of cancer, including melanoma, leukemia, pancreaUc cancer, colon cancer. 2. Researchers have created a model of cancer in Zebrafish that allows them to capture live images of tumors forming and growing.
ways. Although cancer is primarily a disease of adults, mutage- lished data). Chemical screens using embryos would select for
ZEBRAFISH AS A MODEL IN CANCER RESEARCH
nesis screens could be designed to examine cell-cycle pheno- drugs active in a multicellular organism, an advantage over tra-
Figure 1. Histology of cholangiocarcinoma in
human and zebrafish
Cholangiocarcinoma is a malignant bile duct
neoplasm that occurs in both humans and
zebrafish. The histologic appearance, including
atypical nuclei, haphazard arrangement of
irregularly shaped glands, and increased mitot-
ic activity, is very similar in the two organisms.
Bar is 50 µm.
P R I M E R
CANCER CELL : APRIL 2002 * VOL. 1 * COPYRIGHT © 2002 CELL PRESS 229
Figure 2. Strengths of the zebrafish system
The zebrafish is an ideal complement to existing
genetic systems. Like flies and worms, the trans-
parent embryos are produced in large numbers
and are accessible for rapid screening and
experimental manipulation. Like mice, zebrafish
have vertebrate anatomy, physiology, and
tumor biology.
evaluate cellular processes related to
cancer biology and determine if the path-
ways found in mammals are present in
the fish.
To facilitate the use of zebrafish as a
forward genetic tool, the speed and effi-
ciency of mutant screening and gene
cloning needs to be improved. For exam-
ple, while ethylnitrosourea (ENU) muta-
Amatruda, Cancer Cell, 2002
genesis is relatively efficient, recovery of
mutations is still time-consuming. The
Sanger Center sequence of theCancer Research
• Shares most of their organs with mammalian
counterparts
• Differently aged animals each offers disUnct
advantages for cancer-relevant phenotypesCancer Research
A
Adult Embryo
Cancer Research Mutagenesis Transplant Mutagenesis Transgenesis Transplant
Dexamethasone
Gamma-irradiation
Immunosuppressed Immunosuppressed
zebrafish zebrafish
Chemical Genetic Exogenous
treatment mutagenesis DNA
Chemical Tumor cells Tumor cells
treatment
Studies
Compound screening Compound testing Drug reprofiling
B
Allograft Chemical treatment Transplant
Tumor cells
Xenograft
Intraperitoneal
transplant
Biopsy
Lung tumor Culture tumor cells
Orthograft
Liver transplant
Biopsy
Liver tumor cells
Liver tumor
© 2018 American Association for Cancer Research
Figure 1.
A, Methods of cancer generation in adult and embryo zebrafish. B, Transplant assays in zebrafish.Cancer Research
Feng H
Cancer types
p53
ptena
ptenb
apc
nf1a
nf1b
separase
bmyb
TEL-AML1
Myc
MYC
Akt2
NOTCH1
MY5T3-NCOA2
NUP98-HOXA9 MPN
KRASG12D
BRAF-V600E
HRASG12V
HRASG12V
MYCN
Xmrk, kras, Myc
most common locations for this spontaneous neoplasia to arise Interspaced Short Palindromic Repeats/CRISPR associated (CRISPR/
include gut, thyroid, and liver. Lower levels of spontaneous neoplasia Cas) technologies [12].
occur in blood vessels, brains, and gills. In light of spontaneous
In forward genetic screens, mutations are introduced to the adult
tumor acquisition, detailed chemical approaches to induce cancer
zebrafish’s genome through chemical, viral, or transposon-based
have been developed [10]. To chemically induce cancer, zebrafish
Feng et al, 2015
are soaked in water dissolved with carcinogens for varied periods of mutagenesis. The progeny of these mutagenized adult zebrafish
time. Advantageously, zebrafish can endure treatments at a variety are screened for abnormal phenotypes. Genes that harbor genetic
of chemical concentrations and durations. For instance, smaller mutations are then identified through gene mapping, sequenceTable 1. Genetic models of cancer in the zebrafish.
Cancer Cancer
Peripheral nerve sheath
tumor (PNST)
Genotype
tp53M214K
brca2Q658X tp53M214K
Zebrafish Background
WT
WT or tp53M214K
Reference
[39]
[47]
PNST, angiosarcoma, CG1 syngeneic
tp53del/del [40]
Research
leukemia, germ cell tumor zebrafish strain
rag2:KRASG12D WT; ↵-actin:GFP;
Rhabdomyosarcoma (RMS) [43,44]
rag2:dsRed2 tp53M214K
BRAFV600E tp53M214K tp53M214K [45]
BRAFV600E tp53M214K crestin:EGFP; tp53M214K [50]
BRAFV600E mitfavc7 mitfavc7 [54]
Melanoma
hsp70I:GFP-HRASG12V N.A. [51,55]
kita:GalTA4,UAS:mCherry
N.A. [52,55]
UAS:eGFP-HRASGV12
kita:Gal4TA, UAS:mCherry
UAS:eGFP-HRASGV12 WT or tp53M214K [55]
UAS:eGFP-jmjd6
Thyroid cancer tg:BRAFV600E -pA;tg:TdTomato-pA WT [53]
ptf1a:eGFP-KRASG12V WT [56]
Pancreatic cancer
ptf1a:CREERT2
ubb:lox-Nuc-eCFP-stop-lox-GAL4-VP16 N.A. [57]
UAS:eGFP-KRASG12V
fabp10a: RPIA; myl7:GFP N.A. [58]
Hepatocellular cancer (HCC) fabp10:rtTA2s-M2;TRE2:eGFP-krasG12V WT or lepr+/- [60]
fabp10:TA; TRE:Myc; krt4:GFP
WT [61]
fabp10:TA; TRE:xmrk; krt4:GFP
pDs-ifabp:LexPR-Lexop:eGFP-krasV12 N.A. [59]
Intestinal tumors
5⇥UAS:EGFP-P2A-krasG12D
fabp10a:mCherry 5
WT or cyp7a1 [62]
fabp10a:mCherry-P2A-cyp7a1
+ various Gal4 lines
Testicular tumor brca2Q658X WT [48]
rag2:mMyc
rag2:GFP WT [42,43]
T-cell acute lymphoid
rag2:dsRed2
leukemia (T-ALL)
rag2:loxP-dsRED2-loxP-eGFP-mMyc WT [66]
spi1:tel-jak2a WT [72]
hsp70:AML1-ETO WT [68,69]
Acute lymphoid leukemia
spi1:MYST3/NCOA2-eGFP N.A. [70]
(AML)
pHsFLT3-WT-T2a-eGFP
pHsFLT3-ITD-T2a-eGFP WT [74]
FLT3-ITD-T2a-mRFP
Chronic myeloid leukemia
spi1:tel-jak2a WT [71,72]
Hason, Genes, 2019
(CML)
Myelodysplastic syndrome
tet2-/- cmyb:eGFP; cd41:eGFP [75]
(MDS)
WT: Wild type; N.A: Not Available.in Table 4.
Table 4. Human cancer xenograft transplantation models in zebrafish.
Transplanted Cancer Type Developmental Stage Injection Site Reference
Cancer Melanoma
Melanoma (uveal and cutaneous)
Melanoma and colorectal cancer
Blastula
Blastula
48 h post-fertilization (hpf)
Blastodisc
N.A.
Yolk sac; hindbrain ventricle;
circulation
[134]
[135]
[136]
Uveal melanoma 48 hpf Yolk sac [152]
Melanoma 48 hpf Yolk sac [146]
Research
Colorectal cancer 48 hpf Yolk sac [139]
Colorectal cancer 48 hpf Yolk sac [27,176,177]
Pancreatic cancer 48 hpf Yolk sac [140]
Melanoma, adenocarcinoma, triple
Yolk sac, proximity of
negative breast cancer (TNBC) and 48 hpf [141,142]
subintestinal veins (SIV)
Cell lines
ovarian cancer
Colorectal cancer, melanoma (both
48 hpf Yolk sac [143]
murine)
Prostate cancer 48 hpf Yolk sac [144,167]
Prostate cancer, androgen dependent
48 hpf Yolk sac [168]
and independent
Subcutaneous, above yol
Prostate cancer 48 hpf [169]
sack
Breast, prostate, colon, pancreatic
48 hpf Yolk sac [153]
cancer, fibrosarcoma
Breast cancer 48 hpf Yolk sac [25]
Breast, prostate, colorectal cancer 48 hpf Yolk sac [156]
Breast cancer, non-invasive and
48 hpf Duct of Cuvier [157]
metastatic
Breast cancer 48 hpf Duct of Cuvier [158]
Breast cancer 48 hpf Yolk sac [159]
Breast adenocarcinoma and TNBC 48 hpf Duct of Cuvier [161]
TNBC
Genes 2019, 10, 935 and prostate cancer 48 hpf Duct of Cuvier [162]18 of 30
Breast cancer 48 hpf Yolk sac [165]
Breast cancer and TNBC 48 hpf Duct of Cuvier [166]
TNBC 48 hpf Duct of Cuvier [165]
Table 4. Cont.
AML, CML 48 hpf Yolk sac [147]
Transplanted Cancer Type Developmental Stage Injection Site Reference
Posterior cardinal vein
AML, T-ALL 48 hpf [148]
(PCV)
T-ALL 48 hpf Yolk sac [149]
Multiple myeloma (MM) 48 hpf Yolk sac [150]
MM, Waldenstrom’s
48 hpf Pericardium [151]
macroglobulinemia, TNBC
CML, HCC, prostate cancer (sorted 48 hpf Yolk sac
[184]
for cancer stem cells) Adult Trunk near dorsal aorta
Cell lines
Yolk sac
48 hpf
AML, HCC Trunk near dorsal aorta; [185]
Adult
heart
Retinoblastoma 48 hpf Vitreous cavity [170]
Glioblastoma 52 hpf Yolk sack; brain [154]
Glioblastoma 36 hpf Hindbrain [171]
Glioblastoma 72 hpf Brain [172]
Glioblastoma and colon cancer Blastula Blastoderm [174]
Gastrointestinal tumors – pancreas,
48 hpf Yolk sac; liver [140]
stomach, colon
Gastric cancer 48 hpf Yolk sac [178,179]
Oral squamous cell carcinoma 48 hpf Yolk sac [180]
Non-small-cell lung cancer (NSCLC) 48 hpf Yolk sac [181]
NCSLC 48 hpf N.A. [182]
48 hpf Yolk sac
Ewing sarcoma (EWS) [183]
Juvenile (35 dpf) Eye vessels
Intraperitoneal cavity
Various types of human cancer Adult [186]
Peri-ocular muscle
AML blast cells 48 hpf PCV [148]
T-ALL from bone marrow 48 hpf Yolk sac [149]
Hason, Genes, 2019
MM cells from plasma 48 hpf Yolk sac [150]
MM cells from bone marrow 48 hpf Pericardium [151]
PDX
Glioblastoma 36 hpf Brain [173]
Glioblastoma blastula Blastoderm [174]
Gastric cancer 48 hpf Yolk sac [178]
Glioblastoma, melanoma, breast
Adult Peri-ocular muscle [186]
cancer, RMSLetrado et al.
Cancer Research A 1%
7%
Compound activity testing
Compound screening
Drug reprofiling
92%
B Rhabdomyosarcoma, 3
Ewing sarcoma, 2 Ovarian cancer, 3
Gastric cancer, 2 Myeloma, 3
Osteosarcoma, 2
Oral cancer, 4
Retinoblastoma, 2
Head and neck
Anti- squamous cell
lymphagenic carcinoma (HNSCC), 4
Anti-tumoral activity 1%
activity 7% Colon cancer, 4
Pancreatic cancer, 5
Prostate cancer, 7
Many cancer types, 49
Other cancer
types, 10
Anti-
angigogenic Hepatocarcinoma, 9
activity 26%
Breast cancer, 32
Specific anti-tumoral Colorectal, 11
activity 66%
Glioblastoma, 12
Leukemia, 27 Melanoma, 21
Lung
cancer, 21
© 2018 American Association for Cancer Research
Figure 2.
A, Classification of the 355 reported case studies in zebrafish, cancer drug discovery projects, according to the aim of the study. B, Left graph represents studies
reported in literature classified by the subject matter. Right graph shows cancer types studied in cases encompass in "Specific anti-tumoral activity." All details about
the 355 case studies are described in Supplementary Table S10.
phenotypically as developmental disruptions. Other case studies costs, work feasibility, and simplicity to obtain cancer pheno-
identified compounds using mutant or transgenic zebrafish to types, zebrafish has recently become a meaningful tool in science.melanoma pathogenesis and inhibition. ZMEL was derived from melanomas of the mitfa-BRAF
tp53-/- transgenic fish [131]. ZMELs have been since used for transplantation studies to assess melanoma
pathology and metastatic behavior in zebrafish [132]. Hyenne et al. have recently published a paper
focusing on the fate of tumor extracellular vesicles (EVs) derived from ZMELs. They show that EVs
can be tracked in vivo in zebrafish and that they activate macrophages and promote metastases [133].
Cancer Research Zebrafish models of allogeneic transplantation are summarized in Table 3.
Table 3. Cancer allograft transplantation models in zebrafish.
Transplanted Cancer Type Developmental Stage Injection Site Reference
Genes 2019, 10, 935 8 of 30 T-ALL Adult Intraperitoneal cavity [42,66,124,127]
Genes 2019, 10, 935 8 of 31 RMS Adult Intraperitoneal cavity [124,127]
Primary cells
Melanoma Adult Intraperitoneal cavity [124]
T-ALL, RMS, Intraperitoneal cavity,
melanoma, Adult retro-orbital, [129,130]
neuroblastoma intramuscular
Melanoma Adult N.A. [131]
Adult Subcutaneous
48 h post-fertilization Circulation (duct of [131]
Melanoma (hpf) Cuvier)
ZMELs
Retro-orbital
Adult Intravenous (cardinal [132]
vein)
48 hpf Circulation [133]
3.2. Zebrafish Xenotransplantation Model for the Evaluation of Cancer Progress and Metastasis
Zebrafish as a tool in human cancer xenotransplantation studies could overcome some of the
drawbacks of the murine model. The main benefits of zebrafish are most prominent when using
embryonal stages for xenotransplantation. With the small-sized transparent embryos lacking a
mature immune system, it is possible to transplant and track high numbers of animals. This fact is a
powerful reason for the utilization of zebrafish as a pre-clinical screening model which could lead to
patient-derived cancer cell xenotransplantation and to new options for personalized medicine [19].
Most of the recent transplantation studies in zebrafish use embryonal stages of 48 hours post fertilization
(hpf) as the stage for transplantation. However, some of the first zebrafish xenograft studies were
done in the blastula stage of the embryo. Transplanted melanoma cells survived, divided, stayed in
de-di↵erentiated stage but did not form tumors in zebrafish embryos. This was the first observation
of human melanoma cells in zebrafish [134]. In a study utilizing the same type of melanoma
xenotransplantation into zebrafish blastula, the authors compared di↵erent types of human cutaneous
and uveal melanoma cancer cell lines. They found out that aggressive melanoma cells secrete Nodal.
The expression of Nodal correlated with melanoma aggressiveness and progression, and caused
developmental defects of the zebrafish embryo [135]. Haldi et al. optimized the parameters for
zebrafish xenotransplantation where they propose the 48 hpf developmental stage as the best for
transplantation. At this stage, developmental cell migration is finished, therefore cancer cell migration
after injection is likely to be an active process. Human melanoma cells together with other types of
cancer cell lines, which they transplanted into zebrafish, were able to survive and formed tumors in the
embryo [136]. The site of transplantation might be variable but usually it is the yolk sac, cardinal vein,
Figure 1. Zebrafish models of cancer. Zebrafish develops cancer phenotypes similar to human cancer
Figure 1. Zebrafish models of cancer. Zebrafish develops cancer phenotypes similar to human cancer
in di↵erent tissues and organs. All of these cancer types and their zebrafish models are discussed in
in different tissues and organs. All of these cancer types and their zebrafish models are discussed in
Section 2. Genetic models of cancer. PNST—peripheral nerve sheath tumor; HCC—hepatocellular
Section 2. Genetic models of cancer. PNST—peripheral nerve sheath tumor; HCC—hepatocellular
carcinoma; RMS—rhabdomyosarcoma; ⇡—female.
carcinoma; RMS—rhabdomyosarcoma;⇢—male; —male; —female.
2.1. Zebrafish and New Methods for Cancer Modelling
Hason, Genes, 2019
2.1. Zebrafish and New Methods for Cancer Modelling
In this section, we will discuss in more detail the most popular and widely used gene manipulation
In this section, we will discuss in more detail the most popular and widely used gene
techniques which were engaged in the majority of above-discussed zebrafish cancer-modeling
manipulation techniques which were engaged in the majority of above-discussed zebrafish cancer-
studies [80–86].
modeling In[80–86].
studies zebrafish it is possible
In zebrafish to perform
it is possible forward
to perform and and
forward reverse genetic
reverse screens
genetic and
screens
directly assess assess
and directly the rolethe
ofrole
various genes genes
of various in cancer relatedrelated
in cancer phenotypes [87]. Currently,
phenotypes the most
[87]. Currently, widely
the most326 Julia Etchin et al.
Cancer
[(Fig._6)TD$IG]
Research
Fig. 6 Visualization of human tumor-induced angiogenesis. (A, B) Three-dimensional (3D) con-
focal reconstructions of VEGF-secreting MDA-435 tumor cells in the body wall of fli1:EGFP
zebrafish, 4 (A), and 5 (B) days post injection. (C, D) Single confocal optical sections (1 mm) of
microtumor in (A) and (B). (E, F) 3D reconstructions of digitally isolated tumor cells in contact with
host blood vessels from A and B. Dotted squares indicate insets showing magnified views of the
interior vessel surface at sites of vessel openings and tumor cell membrane insertion. Modified with
permission from (Stoletov et al., 2007). (See color plate.)
in vasculature formation. vhl syndrome is characterized by an elevated predisposi- Stoletov et al, 2002ZEBRAFISH IN UNITS
ZEBRAFISH IN UNITS Strains: 1. wild type (AB) 2. Casper - homozygous for the pigment-affecUng mutaUons of roya9 and nacrew2 (AB) 3. Tg(fli1:EGFP)y1 (AB)
ZEBRAFISH IN UNITS
examination euthanasia
dechorionation treatment administration examination
Fertilization 24hpf 48hpf 24hpi 48hpi 72hpi
tumor injection examination
examinationZEBRAFISH IN UNITS
Biodistribution of liposomes in healthy zebrafish embryos:
Injection of 9,2 nL/fish LIPOSOMES in the duct of Cuvier of Zebrafish embryo 72hours post-fertilization
Images: 2 hours of liposomes injection
Non injected Duct of Cuvier 9,2nL Lipo SLa Duct of Cuvier 9,2nL Lipo SLb
Red-visible merge
Red channelZEBRAFISH IN UNITS
20X TAIL images of Embryo SLb – Duct of Cuvier 9,2 nL :
Red channel Red-visible merge Red-green merge
Non injected
Injected Lipo SLbZEBRAFISH IN UNITS
3-3.5 dpf
2.5 dpf
5.5 dpf
4 dpf
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