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New horizons for animal models in neuroendocrine tumours.
Using the zebrafish as model.
Jorge Barriuso Marie-Curie Fellow Hurlstone lab University of Manchester
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Do we need animal models?
Oncogenes Tumour suppressor genes
celullarstroma Immune
system Vasculature ECM
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Do we need animal models?
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About fish and men
Howe et al 2013 Spitsbergen et al 2012
• 26000 protein coding genes.
• 70% of genes have an obvious orthologue.
• The fish develops tumours that resemble human ones.
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pNET model
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Hanahan 1985
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Cancer Oncogene Tumour suppressor
Melanoma mi#a–BRAFV600E mi#a:EGFP:NRASQ61K
kita–Gal4 × uas-‐HRAS
tp53−/−
PDAC p#1a–KRASG12V–GFP p#1a:Gal4–VP16 × uas–KRASG12V–GFP
T cell lymph/leuk rag2–myc rag2–lox–dsRED2–lox–EGFP–mMyc × hsp70–cre rag2–NOTCH1 rag2–myc × rag2–bcl2
B cell leuk B acQn–TEL–AML1 (ETV6–RUNX1)
Rhabdomyosarcoma rag2–KRASG12D
Neuroblastoma dβh:EGFP–MYCN dβh:EGFP and dβh:ALKF1174L
AML pu1–MYST3/NCOA2–EGFP
MPNST tp53−/−
Ewing’s hsp70 or β-‐acQn–EWSR1–FLI1 tp53−/−
Liver fabp10:LexPR; LexA:EGFP × cryB:mCherry; LexA:EGFP–krasG12V
fabp10:TA; TRE:xmrk; krt4:GFP
PNET zmyod–MYCN
CorQcotroph adenoma POMC–PTTG
TesQcular germ cell t. fugu flck–SV40 large T
Adapted from White et al 2013
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pNET and zebrafish
• Serendipity
• Z-myod: NMYC
• Low penetrance
• Strain not stable
Yang et al 2013
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Transgenesis in zebrafish
Prom. oncogene
linker
reporter
Conditional expression model +
reporter
Ins. prom STOP STOP reporter1 oncogene
linker
reporter2
Conditional expression model + reporter + inducible
Prom. oncogene Conditional expression model
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pNET model design
Ins. prom STOP STOP eBFP2 NMYC
P2A mCherry
Crys. pro eCFP
Ins. prom NMYC
P2A mCherry
Crys. pro eCFP
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Casper. Ubi:CreERT
+ TAM
pNET model (“Riddicks”)
X 20
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Targets of interest for KO
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Genome-‐ediBng strategies
Adapted from www.addgene.org
Clustered Regularly Interspaced Short Palindromic Repeats
Transcription activator-like effector nucleases
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CRISPR/Cas9
Building a positive control...
Chen et al 2013
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Genome-‐ediBng strategies
Adapted from www.pnabio.com
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Images from literature
Blackburn et al 2014
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Heterogeneity with xenograKs
Chapman et al 2014
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Chapman et al 2014
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XenograKs (zf embryos)
Chapman et al 2014.(Hurlstone lab)
Barriuso (Unpublished data. Hurlstone lab)
Adapted from Veinotte et al 2014
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Strategies
Nicoli et al 2007
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Strategies: new technique
BON cells injected in 2dpf embryo. Imaged at 24 hpi.
Barriuso (unpublished data)
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Angiogenesis assay
BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 24 hpi.
Barriuso (unpublished data) Nicoli 2007
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Angiogenesis assay
BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 24 hpi.
Two patterns of engraftment
SIV attraction Cuvier duct disruption
Barriuso (unpublished data)
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Angiogenesis assay
BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 72 hpi.
Barriuso (unpublished data)
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Angiogenesis assay
BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 72 hpi.
Barriuso (unpublished data)
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Future: TumourgraKs
PancreaQc cancer
Normal pancreas
2 dpf embryos
Marques et al 2009
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Hypothesis Co-clinical trials
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Xenografts
Cell lines Transgenic models
Tumourgrafts
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Working at a ZF facility
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Working at a ZF facility
RNA
DNA
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Aquarium
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Conclusions
• The number of transgenic models are increasing. • The versatility of the fish as model for cancer research makes it adaptable to complex scenarios as intratumoural heterogeneity studies. • KO models using the latest technologies (CRISPR/cas9) • Xenograft models. • The model is valid for well differentiated tumours. • The technologies applied are amenable for high throughput analysis.
• Next: tumourgrafts
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War against cancer
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Acknowledgements
Hurlstone lab Adam Hurlstone Rags Nagaraju Irene Barrinaga-‐Rementeria Laura Fernandez del Ama Anna Chapman Chris Dee Andy Badrock Helen Young Hannah Johnstone Mai AbdelaoumuQ
Wellbrock lab Claudia Wellbrock Imanol Arozarena
ChrisQe NET team Juan Valle Richard Hubner Maréad McNamara Angela Lamarca Lynn McCallum
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Muchas gracias