ClumPyCells resolves spatial aggregation in complex tissues overcoming size biases

Abstract The spatial arrangement of cells within a tissue microenvironment shapes their interactions and cell states, which are essential for tissue development, homeostasis, and disease. Spatial -omics technologies can precisely map the location of each cell within complex tissue structures, while also profiling their protein content and transcriptional diversity. Various approaches have been developed to analyze spatial patterns of cell aggregation, repulsion, or random distribution within tissues. However, differences in cell morphology within a tissue can introduce significant bias. Cell size in particular is not accounted for and introduces challenges when quantifying the aggregation of cells or their molecular features. To overcome such limitations, we present ClumPyCells: a statistical framework that measures cell and marker aggregation within tissue while correcting for size morphology. ClumPyCells enables interpretation of cell aggregation, bypassing interfering cell types or tissue regions unrelated to the desired spatial correlation. We demonstrate the capabilities of ClumPyCells across several tumor types, including melanoma and colorectal cancer, and spatial -omics technologies such as spatial transcriptomics and proteomics, while benchmarking how cell-size differences contribute to misinterpretations. By correcting for disruptive cell types within a region of interest, ClumPyCells will determine new tissue patterns and structures without morphological interference.