Parallelized multidimensional analytic framework applied to mammary epithelial cells uncovers regulatory principles in EMT

Abstract

A proper understanding of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. Here we combine state-of-the-art data acquisition platforms and bioinformatics tools to devise PAMAF, a workflow that simultaneously examines twelve omics modalities, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We apply PAMAF in an established in vitro model of TGFβ-induced epithelial to mesenchymal transition (EMT) to quantify >61,000 molecules from 12 omics and 10 timepoints over 12 days. Bioinformatics analysis of this EMT-ExMap resource allowed us to identify; –topological coupling between omics, –four distinct cell states during EMT, –omics-specific kinetic paths, –stage-specific multi-omics characteristics, –distinct regulatory classes of genes, –ligand–receptor mediated intercellular crosstalk by integrating scRNAseq and subcellular proteomics, and –combinatorial drug targets (e.g., Hedgehog signaling and CAMK-II) to inhibit EMT, which we validate using a 3D mammary duct-on-a-chip platform. Overall, this study provides a resource on TGFβ signaling and EMT.

Reconstruction of single cell lineage trajectories and identification of diversity in fates during the epithelial-to-mesenchymal transition

“…Such a scope, however, might not capture the complete EMT landscape. First, although we’ve identified early divergences in EMT trajectories, actual phenotypic changes may become apparent later, potentially attributable to differences between RNA expression and protein levels due to RNA-protein discordance 63 . In addition, variabilities in gene expression can arise from intrinsic noise 64 , extracellular environmental changes 65,66 , and differing epigenetic states 67 .…”

DESP: Demixing Cell State Profiles from Dynamic Bulk Molecular Measurements

“…In that study, cells of the human mammary epithelial cell-line MCF10A were treated with TGF-Beta to induce epithelial-to-mesenchymal transition (EMT) and samples were aliquoted at ten timepoints for multi-omics profiling. Details on the experiment and the downloadable datasets can be accessed at (Paul et al, 2023) 13 .…”

“…Single-cell RNA-Seq quantified the expression of 9,785 genes in 1,913 cells at eight consecutive timepoints, with roughly 200 cells per timepoint. Details on the experiment and the downloadable data can be found at (Paul et al, 2023) 13 . Low-quality genes detected in less than 5% of all cells were discarded (17 genes).…”

“…EMT is a complex biological process by which epithelial cells lose their adhesion and gradually differentiate into mobile mesenchymal cells and is of particular importance to the biomedical community due to its integral role in wound healing, tissue development, and cancer progression 12 . We analyzed bulk quantitative proteomics and scRNA-Seq data generated at eight consecutive timepoints representing MCF10A mammary epithelial cells undergoing EMT 13 , and we show that DESP is able to identify the corresponding cellular proteomes and recover expected patterns of canonical EMT markers. Additionally, DESP goes further by identifying the proteomic profile of transient cell states key to the differentiation process that were not detected at the transcriptomics level and whose signal is significantly diluted in the bulk proteomics due to the confounding presence of other cell states in the mixture.…”

“…We applied DESP to a multi-omics time-course dataset investigating the molecular changes of pre-cancerous cells undergoing TGF-Beta-induced epithelial-to-mesenchymal transition (EMT) in-vitro 12,13 . EMT is a complex biological process by which epithelial cells lose their adhesion and gradually differentiate into mobile mesenchymal cells and is of particular importance to the biomedical community due to its integral role in wound healing, tissue development, and cancer progression 12 .…”