Following incubation, the individual oxygen consumption rate of each cell is determined by reversibly creating a gas-impermeable chamber of 140?pL via hermetically sealing the bottom array containing single cells with a top array containing a ratiometric optical oxygen sensor covalently attached to the bottom and the walls of the microwells (Fig. manifests in the physiologic profile of individual cells in the context of disease progression. Rapid advances in the field of analysis technologies for molecular profiling at the single cell level have resulted in unprecedented insights into and discoveries about the cellular machinery and its functional relevance in normal and disease says1,2,3,4,5,6. However, the ultimate goal of relating cellular function with the molecular phenotype and genotype in the same cell remains elusive. Functional heterogeneity exists even in isogenic cell populations and is pivotal in key processes including development, homeostasis, disease etiology, and response to pharmacological brokers7,8,9,10,11. Dissecting the distinct functions intercellular variability plays in disease says, especially as a prerequisite PF-05231023 of evolution during the development of cancer12,13,14, holds the promise of novel treatment strategies and efficient drug targets15. While several groundbreaking technologies for genotyping, gene transcription, protein expression level, and metabolic profiling at the single cell level exist16,17,18,19,20,21,22,23, each of PF-05231023 them provides only one type of molecular information thus limiting the ability to link differences at the genome or transcriptome level and their phenotypic manifestation in individual cells. Several new techniques for simultaneous characterization of genomic, transcriptomic, and epigenomic molecular profiles of individual cells have recently been reported1,5,24,25, yet they represent tools for end-point analysis and do not offer the ability to directly correlate functional parameters of the same cell with its biomolecular profile. We present an integrated approach for combined profiling JMS of functional and molecular phenotypes of the same individual cells while enabling unbiased, functional readout-based analysis and selection of single cells for downstream analysis. We created a novel integrated platform and approach that combines a technology for respiration rate determination of single cells with a method for harvesting the same cells26,27, followed by gene expression level analysis on the same individual cells. To demonstrate the biological power of the approach, we studied how the selective environment of multiple cycles of acute hypoxia affects physiological and transcriptional heterogeneity in pathologic progression represented by premalignant progression of Barretts esophagus (BE). BE is usually a metaplastic precursor lesion of the esophagus that increases the risk of developing esophageal adenocarcinoma (EAC)28. As in many other solid cancers, progressing BE is usually associated with genomic instability and heterogeneity that evolve in EAC29,30. Hypoxia, incurred by the hyperproliferative phenotype of cancer cells that outpaces neovascularization in tumors, is usually common in many types of solid cancers and is known to play a central role in carcinogenesis, progression, and metastasis31,32,33,34,35,36. In BE, episodes of chronic acid reflux cause the epithelial cells to be exposed to periods of bile, hypoxia, and low pH. It is hypothesized that this interplay between these changes in esophageal environment and immune system response plays a central role in progressing from BE to EAC via selection for the fittest PF-05231023 clones that can expand after other competing clones have been eradicated. We found differential cellular heterogeneity dynamics in the premalignant PF-05231023 progression stages in response to acute hypoxia episodes. While we did not detect significant alterations in the cell respiration phenotype among different progression stages, overall gene expression heterogeneity decreased in metaplasia, the early stage of progression, as a result of hypoxia. In contrast, high-grade dysplasia, the late progression stage, showed an increase in gene expression level variability, suggesting an increased ability of the cell populace in the late progression stage to adapt and survive under stress. We demonstrate the ability of our approach to identify single cells with aberrant phenotypes by combining physiologic and gene expression profiles. Results As a demonstration of the power of our technology (Fig. 1), we performed a study of the integrated dynamics of cellular oxygen consumption and gene transcription heterogeneity in the same single cells in the context of selective pressure conferred by repeated acute hypoxia episodes. The main goal of the study was to gain a detailed insight into the.
