Cellpolaris: A novel tool to quantify cell polarity reliably at scale in fixed and living cells

Abstract Cell polarity, the asymmetric distribution of cellular constituents, is critical for many cellular functions, including cell migration, cell-cell communication, and cell fate regulation, and is often deregulated in disease. Due to technical challenges during image analysis, studies analyzing cell polarity thus far rely on laborious manual visual inspection of a handful of cells, limiting statistical power, the strength of conclusions, and overall scientific progress. Despite the importance of cell polarity, tools capable of quantifying cell polarity accurately with high sensitivity and high-throughput in different cell types are lacking. Consequently, the role of cell polarity in hematopoietic stem and progenitor cells remains poorly understood, and little is known about how cell polarity affects hematologic disease progression. To address these issues and enable new scientific insights through high-quality cell polarity quantifications, we developed Cell Polarity at Scale (CellPolariS), a user-friendly, open-source image analysis tool. CellPolariS quantifies cell polarity with high sensitivity and throughput regardless of cell shape and cell type. Our systematic and quantitative analysis of CellPolariS shows that CellPolariS is more sensitive, precise, and robust when compared to classical manual and semi-automated analysis, while classic polarity quantification methods often don't work and/or produce biased results when cells change size, shape, and/or fluorescence distribution. CellPolariS' novel and innovative algorithm overcomes these issues, and our extensive validation and benchmarking show that CellPolariS can detect the previously reported CDC42 and Tubulin polarity loss in aged hematopoietic stem and progenitor cells with increased sensitivity. We also demonstrate scalability by quantifying for the first time how CDC42 polarity changes during differentiation, by measuring over a hundred thousand hematopoietic stem and progenitor cells across 13 different cell types, increasing the throughput by 2-3 orders of magnitude compared to other methods. Using CellPolariS massively increased throughput and sensitivity, we show that CDC42 polarity changes dynamically with the cell cycle in hematopoietic stem and progenitor cells. Furthermore, using CellPolariS on many hours-long live cell imaging experiments of hematopoietic cells, we discover that contrary to current assumptions, cell polarity is a highly dynamic process and that cells can reversibly a polar to an apolar state within minutes. In summary, CellPolariS is the very first fully automated, standardized tool capable of quantifying cell polarity reliably in any cell type, irrespective of cell shape, size, and fluorescence. CellPolariS provides novel and exciting new ways to study normal and mutant hematopoietic cells' behavior in various contexts and has already revealed never-before-seen insights into fundamental processes and mechanisms of blood stem cell and progenitor regulation. Importantly, CellPolariS is fully generalizable and can also be used to study non-hematopoietic cells.