Characterization of the Warburg Effect and Crabtree effect in Barrett's Esophagus Cell lines and Development of a Chip-Based Single-Cell Respirometry System

Abstract

This thesis consisted of two distinct parts: In the first part, Warburg and Crabtree effects on energy metabolism were characterized in cell lines derived from premalignant Barrett's esophagus patients; in the second part, I participated in developing a microscopy-based single-cell oxygen consumption rate measurement device for determining metabolic diversity in cell populations. Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in premalignant Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. We compared metabolic rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with the premalignant condition Barrett's esophagus (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from the dysplastic Barrett's esophagus have up to two-fold higher glycolysis compared to a cell line derived from non-dysplastic tissue; however, the dysplastic lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose, demonstrating an elevated Crabtree effect. Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment by increasing energy metabolic pathways typically associated with cancer cells. In the second part of my thesis, I assisted in the development and testing of a system to measure single-cell oxygen consumption rates (OCR). Compared to systems that perform similar measurements, our instrument was able to perform single-cell OCR measurements in gas-impermeable chambers on a population of CP-A with minimal toxicity. I also established methods for serially measuring cell cycle and OCR on single-cells cells in this system.