Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive, metastatic and chemoresistant malignancy that is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualised transient manipulation or "priming" of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signalling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in both mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care gemcitabine/Abraxane chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues and stromal stiffness to chemosensitisation, while fluid flow-induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a subset of patients with a poor prognosis and that are likely to respond to FAK priming prior to chemotherapy.