MuSpAn: a toolbox for multiscale spatial analysis

Abstract Advances in multiplex imaging and spatial omics have revolutionised spatial data generation in biology, revealing complex tissue organisation across multiple scales. However, methods for analysing these data have lagged behind, with fragmented, study-specific pipelines and limited guidance for tool selection. To address this, we introduce MuSpAn, a Multiscale Spatial Analysis package offering intuitive, flexible access to a wide range of mathematical tools - including spatial statistics, topological data analysis, geometry, and networks - within a unified framework. MuSpAn supports efficient data querying, is agnostic to imaging modality, and provides extensive documentation and community support. It enables users to create custom pipelines or conduct unbiased exploratory analyses. We demonstrate MuSpAn’s capacity to interrogate cross-compartmental cell interactions at multiple length scales in both normal and neoplastic tissue using mouse intestinal spatial transcriptomic datasets. Applied to a CMS4-like murine intestinal cancer model, MuSpAn identifies a continuum of fibroblastic functional phenotypes associated with discrete and coordinated fibroblast-immune interactions, highlighting its utility as a discovery tool across diverse biological contexts.