Poster Presentation 30th Lorne Cancer Conference 2018

Intravital window imaging of RhoA-, Rac1- and Akt-FRET biosensor mice in normal tissue homeostasis, disease contexts and treatment. (#232)

Max Nobis 1 , David Herrmann 1 , Sean C Warren 1 , James RW Conway 1 , Pauline Melenec 1 , Janett Stoehr 1 , David Stevenson 2 , Marina Pajic 1 , Ewan J McGhee 2 , Jody J Haigh 3 , Anna-Karin E Johnsson 4 , Heidi CE Welch 4 , Michael S Samuels 5 , Owen J Sansom 2 , Jennifer P Morton 2 , Douglas Strathdee 2 , Karen Blyth 2 , Kurt I Anderson 6 , 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. Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia
  4. Signalling Programme, Babraham Institute, Cambridge, Cambridgeshire, UK
  5. Centre for Cancer Biology, SA Pathology and University of South Australia School of Medicine, University of Adelaide, Adelaide, South Australia, Australia
  6. Francis Crick Institute, London, UK

Small GTPases such as Rac1 and RhoA enable cells to migrate during development as well as metastasize during cancer progression. Furthermore, in pancreatic cancer the PI3K pathway is aberrantly regulated in 21% of cases. More specific, time-resolved monitoring of key drivers of survival and metastasis in e.g. pancreatic cancer such as Akt and small GTPases could therefore be done in an in vivo setting with the use of genetic FRET-biosensors to track their activity and the effect of therapeutic intervention.

Here, we describe the generation and characterization of these FRET-biosensor mice to examine RhoA1, Rac12 and Akt kinase activity in an in vivo setting in a variety of cell types in homeostasis as well as in mouse models of cancer. Using multiphoton microscopy allowed for the imaging of these signaling biosensors in tissues and live mice by the application of optical windows3. Elevated levels of Rac1 and 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/+) pancreatic cancer model. Finally, longitudinal imaging of the indirect inhibition of Rac1 and RhoA activity live in vivo was achieved by employing optical windows implanted on top of developed tumours. The therapeutic response was further correlated live to the local extra-cellular matrix and to the local vasculature. This will allow tailoring of targeted intervention in a spatiotemporal manner.

In conclusion, the development and use of the FRET biosensor mice represents a strong resource in understanding tissue context specific signaling events during migration and drug target validation in vivo.

  1. Nobis, M., Herrmann, D., Warren, S.C., Kadir, S., Leung, W., Killen, M., Magenau, A., Stevenson, D., Lucas, M.C., Reischmann, N., et al. (2017). A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts. Cell Rep. 21, 274–288.
  2. Johnsson, A.-K.E., Dai, Y., Nobis, M., Baker, M.J., McGhee, E.J., Walker, S., Schwarz, J.P., Kadir, S., Morton, J.P., Myant, K.B., et al. (2014). The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Rep. 6, 1153–1164.
  3. Ritsma, L., Steller, E.J.A., Ellenbroek, S.I.J., Kranenburg, O., Borel Rinkes, I.H.M., and van Rheenen, J. (2013). Surgical implantation of an abdominal imaging window for intravital microscopy. Nat. Protoc. 8, 583–594.