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- Creators: School of Life Sciences
Description
Single cell heterogeneity plays an important role in the onset and progression of a variety of disease pathologies. One of the most notable examples of the impact of heterogeneity in the complexity of a disease is cancer. Traditionally, molecular analyses on cancer-related samples have been performed on bulk populations of cells, with the resultant data only representative of an average of the population, thereby concealing potentially relevant information about individual cells. Performing these studies at the single cell level is proposed to address this issue. However, current methods for the isolation and analysis of single cells often require specialized and expensive equipment that may be prohibitive to labs wishing to perform such analyses. Herein, a method for the isolation and gene expression analysis of single cells is described that (1) relies only on readily available, inexpensive materials, (2) is compatible with phase and fluorescent microscopy, and (3) allows for the ability to track specific cells throughout all measurements. This method utilizes random seeding of single cells on 72-well Terasaki plates (also called microtest plates) that have 20 µl, optically clear flat-bottomed wells in order to circumvent the need for specific hardware for cell isolation. Suspensions of the Barrett’s esophagus epithelial cell line CP-D stably expressing turboGFP and a related, GFP-negative BE cell line, CP-A, were prepared, seeded at a concentration of approximately 1-2 cells/well and incubated overnight. Wells containing single cells were visually identified using phase-contrast and fluorescent microscopy. Single cells were then lysed directly in the well, total RNA was isolated, and RT-qPCR was performed. RT-qPCR results reflected the ability to distinguish between turboGFP-expressing and non-expressing cells that matched previous identification by microscopy. These results indicate that this is a convenient and cost-effective method for studying gene expression in single cells.
ContributorsZiegler, Colleen Patricia (Author) / Chao, Joseph (Thesis director) / Tran, Thai (Committee member) / Yaron, Jordan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2013-05
Description
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