Poster Presentation 2019 Hunter Cell Biology Meeting

In vivo imaging of Rac1 targeting in metastatic breast cancer (#103)

Max Nobis 1 , Sean C Warren 1 , Kendelle J Murphy 1 , Alessia Floerchinger 1 , Andrew T McCulloch 1 , Janett Stoehr 1 , Pauline Melenec 1 , David Herrmann 1 , Anna-Karin E Johnsson 2 , Heidi CE Welch 2 , Owen J Sansom 3 , Jennifer P Morton 3 , Karen Blyth 3 , Kurt I Anderson 4 , 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. Signalling Programme, Babraham Institute, Cambridge, Cambridgeshire, UK
  3. Cancer Research UK Beatson Institute, Glasgow, Lanarkshire, UK
  4. Francis Crick Institute, London, UK

Small GTPases such as Rac1 enable cells to migrate during development as well as metastasize during cancer progression. More specific, time-resolved monitoring of key drivers of survival and metastasis in e.g. mammary cancer such as small GTPases could be done in an in vivo setting with the use of FRET-biosensor mice to track protein activity and the effect of therapeutic intervention.

Here, we describe the generation and characterization of a FRET-biosensor mouse to examine Rac1[1] activity in an in vivo setting in two mouse models of mammary cancer. Using time-correlated single photon counting (TCSPC) multiphoton microscopy allowed for the imaging of this signalling biosensor in tissues and live mice by the application of optical windows[2]. Elevated levels of Rac1 activity was observed in the polyoma-middle-T-antigen (PyMT) and Her2-driven breast cancer models. This activity was further upregulated at the invasive borders of these tumours. Two inhibitors of Rac1 activity were evaluated in 2D and 3D in vitro settings and NSC-23766 identified to successfully inhibit invasion of PyMT cells in a 3D context. Shear stress analysis revealed decreased invasion and survival of cells after treatment with NSC-23766. Finally, longitudinal imaging of the inhibition of Rac1 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 extra-cellular matrix and to the local vasculature. This allowed for the tailoring of targeted intervention in a spatiotemporal manner. Chronic treatment of a cohort of PyMT mice starting at the onset of tumour development until endpoint further revealed a significant reduction in metastatic burden in these animals.

In conclusion, the development and use of the FRET biosensor mice represents a strong resource in understanding tissue context specific signalling events during migration and drug target validation in vivo, identifying Rac1 as a strong therapeutic target in metastatic breast cancer.

  1. Johnsson, A.-K.E., Dai, Y., Nobis, M., et al. (2014) The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues. Cell Reports. 6, 1153–1164
  2. Ritsma, L., Steller, E.J.A., Ellenbroek, S.I.J., et al. (2013) Surgical implantation of an abdominal imaging window for intravital microscopy. Nature Protocols. 8, 583–594