Figure 3: Top-down and bottom-up approaches to discover multicellular structures from multiplexed imaging.

(a) Section of tonsil tissue stained with hematoxylin and eosin (H&E). Area 1 is a blood vessel-associated area, area 2 is a lymphoid structure, and area 3 is not defined by readily identifiable structural elements. (b) Example of graph from top-down analysis: e.g. fractions of cells determined by multiplexed imaging in regions pre-selected as areas of interest marked 1, 2, and 3 in panel a. (c) Bottom-up analysis enables identification of multicellular structures that are not obvious in H&E-stained images (area 3 from panel a) while also capturing known structures (areas 1 and 2 from panel a). Tissue representations colored by cell type identity (left panel) and cell neighborhood association (middle panel) as identified using bottom-up clustering of single-cell marker expression and cell co-localization profiles are shown. The right panel shows the network of neighborhood interaction rules (i.e. a hierarchical representation of recurrent co-localization of neighborhoods) that defines the structural composition of the tissue.