Cancer cells display heterogeneous and dynamic states in glioblastoma, but how these malignant states arise and whether they follow a tractable cellular trajectory across tumours is poorly understood. Here, we generate a deep single cell and spatial multi-region atlas of glioblastoma that integrates transcriptomic, epigenomic and genomic analysis to comprehensively characterise their tumour heterogeneity. We describe spatially-patterned transitions of malignant cells from developmental-like towards glial injury response- and hypoxia-defined states during tumour expansion. This malignant cell trajectory dominates glioblastoma, manifesting across tumours and genetically distinct subclonal lineages that are finely spatially intermixed within tumours. Moreover, this trajectory unfolds across specialised myeloid signalling environments that mirror the spatial compartmentalization of malignant cells. Our findings define a stereotyped trajectory of cancer cells in glioblastoma and unify glioblastoma tumour heterogeneity into a tractable cellular and tissue framework.

Crohn’s disease often presents with fistulae, abnormal tunnels that connect the intestine to the skin or other organs. Despite their profound effect on morbidity, the molecular basis of fistula formation remains unclear, largely owing to the challenge of capturing intact fistula tracts and their inherent heterogeneity. Here we construct a subcellular-resolution spatial atlas of 68 intestinal fistulae spanning diverse anatomical locations. We describe fistula-associated epithelial, immune and stromal cell states, revealing abnormal zonation of growth factors and morphogens linked to establishment of tunnelling anatomy. We identify fistula-associated stromal (FAS) fibroblasts, which are assembled in concentric layers: a proliferative, lumen-adjacent zone beneath neutrophil and macrophage-rich granulation tissue, an active lesion core of FAS cells and a quiescent, pro-fibrotic outer zone. We examine the architecture of the extracellular matrix in the fistula tract and demonstrate that FAS populations associate with distinct collagen structures, exhibiting properties ranging from proliferation, migration and extracellular matrix remodelling to dense collagen deposition and fibrosis. We define niches supporting epithelialization of fistula tunnels and a FAS-like population that is detected at the base of ulcers in non-penetrating Crohn’s disease. Our study demonstrates that common molecular pathways and cellular niches underpin fistulae across intestinal locations, revealing the cellular protagonists of fistula establishment and persistence.