Flash Talk & Poster Presentation 32nd Lorne Cancer 2020

In vivo imaging of the Akt-FRET biosensor mouse (#107)

Max Nobis 1 , James RW Conway 2 , Sean C Warren 1 , Young Kyung Lee 1 , Andrew T McCulloch 1 , Astrid Magenau 1 , Xanthe Metcalf 1 , Janett Stoehr 1 , Lea Abdulkhalek 1 , Kendelle J Murphy 1 , Brooke A Pereira 1 , Pauline Melenec 1 , David Herrmann 1 , Sharissa L Latham 1 , David R Croucher 1 , Helen Lenthall 1 , Elissa K Deenick 1 , Tri G Phan 1 , Stacey N Walters 1 , Shane T Grey 1 , Yan-Chuan Shi 1 , Lei Zhang 1 , Herbert Herzog 1 , Antonella Papa 3 , Owen J Sansom 4 , Jennifer P Morton 4 , Jody J Haigh 5 , 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. Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
  3. Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Mebourne, VIC, Australia
  4. Cancer Research UK Beatson Institute, Glasgow, Lanarkshire, UK
  5. Research Institute in Oncology and Hematology, Department of Pharmacology and Therapeutics, University of Manitoba, Manitoba, Canada

Akt (protein kinase B) is a key regulator in a variety of cellular processes such as glucose metabolism, cell survival, apoptosis, transcription, proliferation and cell migration. Its activity is aberrantly upregulated in a plethora of cancers, metabolic and immune disorders, including but not limited to breast, pancreatic and prostate cancer. More specific, time-resolved monitoring of key drivers of metabolism and proliferation in tissue specific contexts can be achieved 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 Akt [1] activity in an in vivo setting in a variety of tissues and cancers. Time-correlated single photon counting (TCSPC) multiphoton microscopy allowed for the imaging of this signalling biosensor in live mice by the application of optical windows [2]. Elevated levels of Akt activity were observed in the pancreatic cancer models, including PTEN loss driven PDAC as reported previously [3] and Akt activity mapped over the course of disease progression. Whole body PTENG129E/+ mutation or loss (PTENfloxed/+) mice were also crossed to the Akt-FRET biosensor mouse and Akt activity measured in several cancers such as lymphomas, adrenal, mammary and prostate cancer. Cell lines established from these tumours retained the AKT-FRET reporter expression and inhibition of Akt was mapped in vitro over time in 2D and 3D contexts. Akt activity was further successfully quantified in the brain via neuropeptide Y receptor-Cre (NPY-Cre) mediated expression, pancreatic beta islets (RIP-Cre) and the native pancreas (Pdx1-Cre). Metabolic challenge in mice bearing optical windows over white fat or brown fat was moreover successfully imaged live in vivo in Akt-FRET mice following ip administration of glucose or insulin.

In conclusion, the described Akt-FRET biosensor mouse can be applied to a wide range of metabolic, immune and cancer settings and used successfully in characterizing disease etiology and monitoring treatment outcome.

  1. Komatsu N, et al. (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–56
  2. Ritsma L, et al. (2013) Surgical implantation of an abdominal imaging window for intravital microscopy. Nature Protocols. 8, 583–594
  3. Conway JRW, et al. (2018) Intravital Imaging to Monitor Therapeutic Response in Moving Hypoxic Regions Resistant to PI3K Pathway Targeting in Pancreatic Cancer. Cell Rep. 23, 3312–3326