Patients with treatment-refractory pancreatic cancer often succumb to systemic metastases1–3; however, the transcriptomic heterogeneity that underlies therapeutic recalcitrance remains understudied, particularly in a spatial context. Here we construct high-resolution maps of lineage states, clonal architecture and the tumour microenvironment (TME) using spatially resolved transcriptomics from 55 samples of primary tumour and metastases (liver, lung and peritoneum) collected from rapid autopsies of 13 people. We observe discernible transcriptomic shifts in cancer-cell lineage states as tumours transition from primary sites to organ-specific metastases, with the most pronounced intra-patient distinctions between liver and lung. Phylogenetic trees constructed from inferred copy number variations in primary and metastatic loci in each patient highlight diverse patient-specific evolutionary trajectories and clonal dissemination. We show that multiple tumour lineage states co-exist in each tissue, including concurrent metastatic foci in the same organ. Agnostic to tissue site, lineage states correlate with distinct TME features, such as the spatial proximity of TGFB1-expressing myofibroblastic cancer-associated fibroblasts (myCAFs) to aggressive ‘basal-like’ cancer cells, but not to cells in the ‘classical’ or ‘intermediate’ states. These findings were validated through orthogonal and cross-species analyses using mouse tissues and patient-derived organoids. Notably, basal-like cancer cells aligned with myCAFs correlate with plasma-cell exclusion from the tumour milieu, and neighbouring cell analyses suggest that CXCR4–CXCL12 signalling is the underlying basis for observed immune exclusion. Collectively, our findings underscore the profound transcriptomic heterogeneity and microenvironmental dynamics that characterize treatment-refractory pancreatic cancer.
