Award Title: High content FLIM, FRET, and localisation imaging to characterise inter-cellular heterogeneity in PI3K signalling to glycolysis in breast cancer and improve treatment strategies
Funder: Cancer Research UK

Abstract: Background  Breast cancer is diagnosed in over 50,000 women in the UK every year and almost a quarter will die from the disease. Improved treatments are needed, but the development of curative therapies remains challenging. Heterogeneity between cells in levels of target inhibition, subsequent modulation of downstream target function, and variation in the dependence of key cell functions on the target all contribute to incomplete tumour cell elimination and therapy failure. Phosphoinositide-3 kinase (PI3K) signalling is activated in breast cancer due to mutations in either PIK3CA or PTEN; furthermore, several phase II & III clinical trials are ongoing with PI3K inhibitors in breast cancer. Live cell imaging with single cell resolution combined with the implementation of fluorescent ‘biosensors’ enables the direct observation of intercellular heterogeneity and its linkage to cell fate. These data will inform the development of more effective strategies to target PI3K signalling in cancer patients.    Aims  1. Development of an automated multiplexed FLIM and homoFRET platform linked to complex analytic methods for long term time-lapse studies of heterogeneity in PI3K signalling and cell fate  2. Understanding of how intercellular heterogeneity in response is linked to the emergence of resistance and improving strategies to reduce variance in therapeutic responses to PI3K targeting in breast cancer    Methods  Heterogeneity in connectivity strengths between different nodes of cell signalling/regulation can undermine therapy efficacy. We will establish and use multiplexed high-throughput biosensor imaging to characterise intercellular variation at regulatory nodes of PI3K activity, Akt, glucose metabolism, and energy stress in breast cancer cell lines and organoids in response to PI3K&AKT inhibitors. Long term imaging using a unique high-content analysis (HCA) platform providing fluorescence lifetime imaging (FLIM) of heteroFRET, fluorescence anisotropy imaging of homoFRET, and intensity-base localisation analysis will enable heterogeneity in the activity of key molecules or pathways and the strength of their connectivity to be linked to cell fate in response to therapy. Finally, we will test strategies to limit intercellular variability and thereby improve the uniformity of therapeutic responses. The first strategy involves targeting PI3K-glycolysis signalling at multiple points to limit relief of negative feedback mechanisms. The second strategy involves limiting the activity of epigenetic regulators to reduce variability in cell state.    How the results of this research will be used  The multiplexed, single cell time-course analysis will enable an improved understanding of therapy response in heterogeneous cancer cell populations, and thereby the development of more effective combination therapies.

Lay Abstract: Background  Breast cancer is diagnosed in over 50,000 women in the UK every year and almost a quarter will die from the disease. Improved treatments are needed, but the development of curative therapies remains challenging. Heterogeneity between cells in levels of target inhibition, subsequent modulation of downstream target function, and variation in the dependence of key cell functions on the target all contribute to incomplete tumour cell elimination and therapy failure. Phosphoinositide-3 kinase (PI3K) signalling is activated in breast cancer due to mutations in either PIK3CA or PTEN; furthermore, several phase II & III clinical trials are ongoing with PI3K inhibitors in breast cancer. Live cell imaging with single cell resolution combined with the implementation of fluorescent ‘biosensors’ enables the direct observation of intercellular heterogeneity and its linkage to cell fate. These data will inform the development of more effective strategies to target PI3K signalling in cancer patients.    Aims  1. Development of an automated multiplexed FLIM and homoFRET platform linked to complex analytic methods for long term time-lapse studies of heterogeneity in PI3K signalling and cell fate  2. Understanding of how intercellular heterogeneity in response is linked to the emergence of resistance and improving strategies to reduce variance in therapeutic responses to PI3K targeting in breast cancer    Methods  Heterogeneity in connectivity strengths between different nodes of cell signalling/regulation can undermine therapy efficacy. We will establish and use multiplexed high-throughput biosensor imaging to characterise intercellular variation at regulatory nodes of PI3K activity, Akt, glucose metabolism, and energy stress in breast cancer cell lines and organoids in response to PI3K&AKT inhibitors. Long term imaging using a unique high-content analysis (HCA) platform providing fluorescence lifetime imaging (FLIM) of heteroFRET, fluorescence anisotropy imaging of homoFRET, and intensity-base localisation analysis will enable heterogeneity in the activity of key molecules or pathways and the strength of their connectivity to be linked to cell fate in response to therapy. Finally, we will test strategies to limit intercellular variability and thereby improve the uniformity of therapeutic responses. The first strategy involves targeting PI3K-glycolysis signalling at multiple points to limit relief of negative feedback mechanisms. The second strategy involves limiting the activity of epigenetic regulators to reduce variability in cell state.    How the results of this research will be used  The multiplexed, single cell time-course analysis will enable an improved understanding of therapy response in heterogeneous cancer cell populations, and thereby the development of more effective combination therapies.

Research Activity Codes: 4.2 EVALUATION OF MARKERS AND TECHNOLOGIES;5.1 PHARMACEUTICALS
Health Categories: CANCER AND NEOPLASMS