Live cell imaging and analysis reveal cell phenotypic transition dynamics inherently missing in snapshot data

Abstract

22Recent advances in single-cell techniques catalyze an emerging field of studying how cells 23 convert from one phenotype to another, in a step-by-step process. Two grand technical 24 challenges, however, impede further development of the field. Fixed cell-based approaches can 25 provide genome-wide snapshots of cell status but have fundamental limits on revealing temporal 26 information, and fluorescence-based live cell imaging approaches provide temporal information 27 but are technically challenging for multiplex long-term imaging. We first developed a live-cell 28 imaging platform that tracks cellular status change through combining endogenous fluorescent 29 labeling that minimizes perturbation to cell physiology, and/or live cell imaging of high-30 dimensional cell morphological and texture features. With our platform and an A549 VIM-RFP 31 EMT reporter line, live cell trajectories reveal parallel paths of epithelial-to-mesenchymal 32 transition missing from snapshot data due to cell-cell heterogeneity. Our results emphasize the 33 necessity of extracting dynamical information of phenotypic transitions from multiplex live cell 34 imaging. 35 36 MAIN TEXT 37 42physiological and pathological conditions. A well-studied example is the epithelial-to-43 mesenchymal transition (EMT), central to many fundamental biological processes including 44 embryonic development and tissue regeneration, wound healing, and disease-like states such as 45 fibrosis and tumor invasiveness (1). One additional example is artificially reprogramming 46 differentiated cells, such as fibroblasts, into induced pluripotent stem cells and other differentiated 47 cell types such as neurons and cardiomyocytes (2). CPT is ubiquitous in biology, and a 48 mechanistic understanding of how a CPT proceeds, emerges as a focused research area with an 49 ultimate goal of achieving effective control of the phenotype of a cell. 50 The CPT studies can also be placed in another large context of studying dynamical processes of 51 escaping from a metastable state or relaxing to a newly-established stationary state (3). Such 52 problem has been a focused and still active research topic in physics and chemistry for more than 53 a century. Formulating CPT as such a problem allows one to apply techniques such as modern 54 transition path sampling and transition path theory (4), and control theories of dynamical systems, 55 for mechanistic understanding of the transition bottlenecks, and manipulating the transitions such 56 as accelerating and directing reprogramming and differentiation processes, and slowing 57 down/preventing undesirable pathological processes. In return CPT studies can provide 58 unprecedented multi-dimensional information that is difficult to obtain for a molecular system, 59 and thus ideal for testing and advancing theoretical studies of non-equilibrium transition 60 processes. A framework of quantitative description of CPT processes is necessary to catalyze 61 such epochal convergence of traditionally separated f...

Comparative Study of Transcriptomics-Based Scoring Metrics for the Epithelial-Hybrid-Mesenchymal Spectrum

“…EMT involves a multitude of changes at both molecular and morphological levels. Various attempts to characterize the spectrum of EMT at molecular and/or morphological levels have been made recently, enabled by latest developments in multiplex imaging, single-cell RNAseq and inducible systems (Mandal et al, 2016;Pastushenko et al, 2018;Stylianou et al, 2018;Cook and Vanderhyden, 2019;Devaraj and Bose, 2019;Karacosta et al, 2019;Wang W. et al, 2019;Watanabe et al, 2019;Lam et al, 2020). These approaches have highlighted the dynamical nature of EMT in driving cancer progression in patients (Jolly and Celia-Terrassa, 2019), and the heterogeneity in EMT status in cell lines and patient samples (Panchy et al, 2020;Shen et al, 2020).…”

Comparative study of transcriptomics-based scoring metrics for the epithelial-hybrid-mesenchymal spectrum

“…EMT involves a multitude of changes at both molecular and morphological levels. Various attempts to characterize the spectrum of EMT at molecular and/or morphological levels have been made recently, enabled by latest developments in multiplex imaging, single-cell RNA-seq and inducible systems (Mandal et al, 2016;Pastushenko et al, 2018;Stylianou et al, 2018;Cook and Vanderhyden, 2019;Devaraj and Bose, 2019;Karacosta et al, 2019;Wang et al, 2019b;Watanabe et al, 2019). These approaches have highlighted the dynamical nature of EMT in driving cancer progression in patients (Jolly and Celia-Terrassa, 2019).…”

Multi-Stability and Consequent Phenotypic Plasticity in AMPK-Akt Double Negative Feedback Loop in Cancer Cells

“…Besides these specific examples, EMP and CSCs are archetypal examples of phenotypic switching reported in many carcinomas [ 42 , 43 ] and non-epithelial tumors [ 44 , 45 , 46 , 47 ]. Recent progress in collecting high-throughput spatiotemporal data and mapping the regulatory networks underlying these axes of plasticity has led to developing complex mechanism-based and population-based mathematical models to decode dynamical traits of phenotypic switching such as dose-dependence, reversibility, hysteresis and transition rates among the cell states [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ].…”