New horizons for animal models in neuroendocrine tumours.

Using the zebrafish as model.

Jorge Barriuso Marie-Curie Fellow Hurlstone lab University of Manchester

Do  we  need  animal  models?  

Oncogenes Tumour suppressor genes

celullarstroma Immune

system Vasculature ECM

Do  we  need  animal  models?  

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.

pNET  model  

Hanahan 1985

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

pNET  and  zebrafish  

•  Serendipity

•  Z-myod: NMYC

•  Low penetrance

•  Strain not stable

Yang et al 2013

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

pNET  model  design  

Ins.  prom   STOP   STOP  eBFP2   NMYC  

P2A   mCherry  

Crys.  pro   eCFP  

Ins.  prom   NMYC  

P2A   mCherry  

Crys.  pro   eCFP  

Casper. Ubi:CreERT

+ TAM

pNET  model  (“Riddicks”)  

X 20

Targets  of  interest  for  KO    

Genome-‐ediBng  strategies  

Adapted from www.addgene.org

Clustered Regularly Interspaced Short Palindromic Repeats

Transcription activator-like effector nucleases

CRISPR/Cas9  

Building a positive control...

Chen et al 2013

Genome-‐ediBng  strategies  

Adapted from www.pnabio.com

Images  from  literature  

Blackburn et al 2014

Heterogeneity  with  xenograKs  

Chapman et al 2014

Chapman et al 2014

XenograKs  (zf  embryos)  

Chapman et al 2014.(Hurlstone lab)

Barriuso (Unpublished data. Hurlstone lab)

Adapted from Veinotte et al 2014

Strategies  

Nicoli et al 2007

Strategies:  new  technique  

BON cells injected in 2dpf embryo. Imaged at 24 hpi.

Barriuso (unpublished data)

Angiogenesis  assay    

BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 24 hpi.

Barriuso (unpublished data) Nicoli 2007

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)

Angiogenesis  assay    

BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 72 hpi.

Barriuso (unpublished data)

Angiogenesis  assay    

BON cells injected in 2dpf fli-1:eGFP embryo. Imaged at 72 hpi.

Barriuso (unpublished data)

Future:  TumourgraKs  

PancreaQc  cancer  

Normal  pancreas  

2  dpf  embryos  

Marques et al 2009

Hypothesis Co-clinical trials

+

Xenografts

Cell lines Transgenic models

Tumourgrafts

Working  at  a  ZF  facility    

Working  at  a  ZF  facility    

RNA

DNA

Aquarium

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

War against cancer

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  

Muchas gracias