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In vitro measurements of cellular respiration have proven to be key biomarkers for the early onset of tumor formation in certain pathological mechanisms.1 The examination of isolated single cells has shown promise in predicting the onset of cancerous growth much earlier than current methods allow.2 Specifically, measurements of the oxygen consumption rates of precancerous cells have elucidated outliers which predict the early onset of esophageal cancer.2 Single cell profiling can fit in to current pathology studies and can serve as a step along the way, much like PCR or gel assays, in detecting biomarkers earlier than current clinical methods.3 Measurement of these single cell metabolic rates is currently limited to 25 cells per experiment. It is the aim of this project to increase throughput from 25 cells to 225 cells per experiment via the implementation of new hardware and software which fit with current methods to allow the same experimental structure. Successful implementation of such methods will allow for more rapid and efficient data collection, facilitating quantitative results and nine times the yield from the same experimental manpower and funding. This document focuses on the implementation ultra high density (UHD) hardware consisting of a pneumatic molar design, angular adjustment features and a mechanical Z-stage. These components have produced the most encouraging results thus far and are the key changes in transitioning to higher throughput experiments.
ContributorsUeberroth, Benjamin Edward (Author) / Kelbauskas, Laimonas (Thesis director) / Ashili, Shashanka (Committee member) / Myers, Jakrey (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2013-05
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Esophageal adenocarcinoma (EAC) is one of the most lethal and fastest growing cancers in the United States. Its onset is commonly triggered by metaplastic transformation of normal squamous esophageal epithelial cells to Barrett's esophagus (BE) cells in response to acid reflux. BE patients are believed to progress through non-dysplastic metaplasia and increasing grades of dysplasia prior to EAC. Conventional cancer diagnostic tools rely on bulk-cell analyses that are incapable of identifying intratumoral heterogeneity or rare driver cells that play important roles in cancer progression. An improved single-cell method of cancer diagnosis would overcome this challenge by detecting cancer initiating cells before they progress into untreatable stages. In this study, using EAC as a model, we attempted to identify a more effective method of cancer diagnosis. We quantified the single- and bulk-cell mRNA expression of genes that have been proposed to be instrumental in the progression of EAC through BE. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis was performed on human primary cells to measure the mRNA expression levels of BE- and EAC-associated genes. Our results showed high levels of heterogeneity of CDX2 and TFF3 at the single-cell resolution in human BE and EAC samples. Additionally, while expression of VEGF is generally low at the bulk-cell level, our results showed that a few, rare cells had significantly higher VEGF expression levels than the majority of cells in the EAC sample. In conclusion, we have affirmed that EAC cancer cells, as well as BE cells, show high levels of heterogeneity. Based on the VEGF gene expression pattern, single-cell analysis could potentially be more effective for identifying rare, but essential cells for cancer progression, which could then be targeted for treatment. Future studies will focus on analyzing human samples from thousands of normal and cancer subjects to validate the use of single-cell profiling in cancer.
ContributorsHaeuser, Kelsey Lynn (Author) / Tran, Thai (Thesis director) / Kelbauskas, Laimonas (Committee member) / Gao, Weimin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-12