A quantitative analysis of the interplay of environment, neighborhood, and cell state in 3D spheroids

Abstract

1 Abstract 1 Cells react to their microenvironment by integrating external stimuli into phenotypic decisions 2 via an intracellular signaling network. Even cells with deregulated signaling can adapt to 3 their environment. To analyze the interplay of environment, neighborhood, and cell state 4 on phenotypic variability, we developed an experimental approach that enables multiplexed 5 mass cytometric imaging to analyze up to 240 pooled spheroid microtissues. This system 6 allowed us to quantify the contributions of environment, neighborhood, and intracellular 7 state to phenotypic variability in spheroid cells. A linear model explained on average more 8 than half of the variability of 34 markers across four cell lines and six growth conditions. We 9 found that the contributions of cell-intrinsic and environmental factors are hierarchically 10 interdependent. By overexpression of 51 signaling protein constructs in subsets of cells, we 11 identified proteins that have cell-intrinsic and extrinsic effects, exemplifying how cell states 12 depend on the cellular neighborhood in spheroid culture. Our study deconvolves factors 13 influencing cellular phenotype in a 3D tissue and provides a scalable experimental system, 14 analytical principles, and rich multiplexed imaging datasets for future studies.