Poster Presentation 29th Lorne Cancer Conference 2017

A RhoA-FRET biosensor mouse for intravital imaging in normal tissue homeostasis and disease contexts. (#242)

Max Nobis 1 , David Herrmann 1 , Sean C. Warren 1 , Shereen Kadir 2 , Wilfred Leung 1 , Monica Killen 1 , Astrid Magenau 1 , Morghan C. Lucas 1 , David Stevenson 2 , Nadine Reischmann 1 , Claire Vennin 1 , James R.W. Conway 1 , Alice Boulghourjian 1 , Anaiis Zaratzian 1 , Andrew M. Law 1 , David Gallego-Ortega 1 , Christopher J. Ormandy 1 , Stacey N. Walters 1 , Shane T. Grey 1 , Jacqueline Bailey 1 , Tatyana Chtanova 1 , Julian Quinn 1 , Paul A. Baldock 1 , Peter Croucher 1 , Juliane P. Schwarz 2 , Agata Mrowinska 2 , Lei Zhang 1 , Herbert Herzog 1 , Andrius Masedunskas 3 4 , Edna C. Hardeman 3 , Peter W. Gunning 4 , Gonzalo Del Monte-Nieto 5 6 , Richard P. Harvey 5 6 , Marina Pajic 1 , Ewan J. McGhee 2 , Anna-Karin E. Johnsson 7 , Owen J. Sansom 2 , Heidi C.E. Welch 7 , Jennifer P. Morton 2 , Douglas Strathdee 2 , Kurt I. Anderson 8 , Paul Timpson 1
  1. Garvan Institute of Medical Research, The Kinghorn Cancer Centre, St Vincent's Clinical School, Faculty of Medicine, Sydney, NSW, Australia
  2. Cancer Research UK Beatson Institute, Glasgow, Lanarkshire, UK
  3. Neuromuscular and Regenerative Medicine Unit, University of New South Wales, Sydney, NSW, Australia
  4. Oncology Research Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia
  5. Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia
  6. St. Vincent's Clinical School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, NSW, Australia
  7. Signalling Programme, Babraham Institute, Cambridge, Cambridgeshire, UK
  8. Francis Crick Institute, London, UK

Small GTPases such as RhoA enable cancer cells to migrate and metastasize. More specific, time-resolved monitoring of RhoA activity in tumours that have upregulated RhoA activity could therefore be done in an in vivo setting with the use of Förster resonance energy transfer (FRET) biosensors to monitor and thus potentially maximize the effect of therapeutic intervention.

Here we describe the generation and characterization of a RhoA-FRET biosensor mouse to examine RhoA activity in an in vivo setting in a variety of tissues as well as mouse models of mammary and pancreatic cancer. FLIM-FRET (fluorescence lifetime imaging microscopy) was performed in conjunction with a multiphoton set up in tissues and live mice using optical windows. RhoA activity was monitored live in mechanosensing of osteocytes in bones, during melanocyte and neutrophil migration, in crypt cells of the small intestine, in the mammary gland during gestation, as well as breast cancer and progressive stages of pancreatic ductal adenocarcinoma. Elevated levels of RhoA activity were observed in the polyoma-middle-T-antigen (PyMT) driven breast cancer model as well as at the invasive borders and liver metastasis of the KPC (KRasG12D/+ and p53R172H/+) driven pancreatic cancer model. Finally, longitudinal imaging of the indirect inhibition of RhoA activity live in vivo was achieved by employing optical windows implanted on top of developed tumours.

In conclusion the development and use of the RhoA-FRET biosensor mouse represents a strong resource in understanding tissue context specific signaling events during migration, mechanosensing and pharmacodynamics in vivo.