Intercellular heterogeneity is pivotal in carcinogenesis and tumor development and plays a central role in advanced metastatic disease, therapeutic resistance, and recurrence at the cellular, tissue and whole organism levels. Rapid advances in single-cell analysis technologies have enabled unprecedented insights into cellular machinery. Yet, the ultimate goal of directly correlating the cellular function with the transcriptional profile of a single cell for comprehensive interrogation of the dynamics of cellular variability remains elusive. We present a study on alterations in intercellular diversity in terms of functional and transcriptional phenotypes in the context of selective pressure based on integrated, multiplexed single-cell measurements of cellular respiration and gene transcription levels in the same single cells. Utilizing a Barrett's esophagus premalignant progression model, we investigated alterations in the intercellular diversity resulting from adaptation to the episodes of acute hypoxia that generated stringent selective pressure on a panel of human esophageal epithelial cell lines representing different stages (metaplasia and dysplasia) in the progression. We correlated alterations in cellular respiration with the expression levels of genes involved in the glycolysis and hypoxia response pathways in the same individual cells. Using dimensionality reduction approaches combined with multiparameter analysis we found evidence that population-level functional heterogeneity can be recapitulated and maintained by a small (∼10%) sub-population of cells that survive selective pressure. By combining functional and molecular profiling of the same individual cells we identified unique errant cellular functional phenotypes of individual cells that would remain hidden otherwise, but may harbor clues to phenotypic cancer progression dynamics. To our knowledge, this study is the first successful demonstration of an integrated, multiparameter approach to elucidate how heterogeneity at the transcriptional level manifests in the physiologic profile of individual cells at different stages of pre-malignant progression.

Citation Format: Laimonas Kelbauskas, Shashanka Ashili, Jia Zeng, Aida Mohammadreza, Kristen Lee, Dmitry Derkach, Benjamin Ueberroth, Weimin Gao, Thomas Paulson, Hong Wang, Yanqing Tian, Dean Smith, Brian Reid, Deirdre Meldrum. Single-cell analysis of functional heterogeneity dynamics in premalignant progression revealed by combined interrogation of functional and transcriptional phenotypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-027.