FRET imaging of ERK activity in an orthotopic xenograft model of patient-derived pancreatic ductal adenocarcinoma using imaging window.

Intravital imaging of cancer xenograft models enables the observation of cells in their tissue microenvironment. Application of molecular activity probes to intravital imaging, however, often poses technical difficulties. Here we sought to visualize Extracellular signal-regulated kinase (ERK) activity in an orthotopic xenograft model of pancreatic cancer. Our custom-made pancreatic imaging window (PIW) enabled long-term tracking (> 2 weeks) of cancer xenografts showing heterogeneous growth potential. Our validation approach of Förster resonance energy transfer (FRET) imaging by a kinase-dead mutant ERK biosensor suggested the local pH may affect the ratiometric interpretation of FRET efficiency. Nevertheless, our FRET analysis allowed to detect pharmacological inhibition of ERK activity, which supports the validity of the experimental model to dissect heterogeneous drug responses of cancer cells in their microenvironment. Of note, we found that collagen signal was negatively correlated with local ERK activity heterogeneity, highlighting the benefit of our imaging system to observe the intricate pharmacodynamics modified by fibrosis. Our intravital live imaging platform paves the way for a novel pre-clinical model that links the molecular dynamics to the living tissue environment of pancreatic cancer.