Decoding multicellular niche formation in the tumour microenvironment from nonspatial single-cell expression data

Accurate inference of granular cell states that co-occur within the tumour microenvironment (TME) is central to defining pro- and anti-tumour environments. Here, we describe how ecosystems of cell populations can be robustly inferred from nonspatial single-cell RNA-seq (scRNA-seq) atlases. Leveraging a unique discovery-validation setup across eight scRNA-seq datasets profiling pancreatic ductal adenocarcinoma (PDAC), we show highly consistent co-occurrence of fine-grained cell states across patients and characterize the positive predictive value of such analyses. Building on this, we develop a novel probabilistic model to quantify multi-cellular ecosystems directly from such atlas-scale scRNA-seq datasets. By mapping these ecosystems to spatial transcriptomics data, we demonstrate that such ecosystems represent bona fide spatial variation within individual tumours despite being learned from nonspatial data. Importantly, through mapping these ecosystems across two large clinical cohorts, we show they are more predictive of therapy response than any individual cell state and are associated with specific tumour somatic mutations. Together, this work lays the foundation for inferring reproducible multicellular ecosystems directly from large nonspatial scRNA-seq atlases.