Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemo-resistant malignancy and is characterized by a dense, desmoplastic stroma that increases the biomechanical force load on cancer cells and is thought to both promote and restrict disease advancement. Using the p53 mutant KPC mouse model of PDAC, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signalling. We used the fluorescent ubiquitination-based cell cycle indicator (FUCCI) cell cycle reporter for in vivo imaging to monitor dynamic changes in FAK activity, stromal biomechanics and gemcitabine/Abraxane-induced effects. Critically, micropatterned pillar plates and stiffness-tuneable matrices were used to pinpoint the contribution of mechanical environmental cues to chemo-sensitization, 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. In a prevision medicine approach, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy. This dataset has led to the establishment of a Phase II clinical trial of first-line FAK inhibition in combination with Gemcitabine/Abraxane in PDAC Patients (NCT05355298).