Generation of Fluorescent Esophageal Adenocarcinoma Cells for Lineage Tracing Within Composite 3D Culture Models
Poster Number
14
Faculty Mentor
Matthew Stern, Ph.D.; sternm@winthrop.edu
College
College of Arts and Sciences
Department
Biology
Faculty Mentor
Matthew Stern, Ph.D.
Abstract
While relatively rare, the aggressive nature and poor prognosis associated with esophageal carcinoma make it a particularly dangerous form of cancer. One approach to discovering esophageal cancer treatment options includes the development of composite 3D culture models. Fluorescent labeling allows each cell type within a 3D culture model to be distinguished from others, facilitating independent study via lineage tracing. Our goal was to generate fluorescent esophageal cancer cells. Of primary interest was the Homo sapiens esophageal adenocarcinoma cell line OE19 that was transfected with a plasmid carrying the mCherry (red) fluorescence marker and neomycin resistance gene. We hypothesized that if geneticin antibiotic (G418) was introduced to transfected OE19 cultures, the percentage of mCherry expressing cells would be enhanced due to G418 selection against cells lacking the plasmid. To test our hypothesis, 400 mg/mL of G418 was applied to multiple OE19 passages during routine cell maintenance. Fluorescence-activated cell sorting (FACS) was then used to sort each cell based on mCherry expression. In addition, OE19 cultures under G418 selection were stained with Hoechst dye (cyan), to visualize nuclei as reference for mCherry appearance during analysis via fluorescent microscopy. The data obtained from FACS analysis indicated that 5-7% of OE19 sample cultures expressed the mCherry label. This was supported by fluorescent microscopy. Low cell viability following cell sorting did not allow for continued culture of sorted cells. Future directions include increasing the selection pressure on OE19 cells by raising the concentration of G418 and using modified FACS procedures to improve post-sorting viability.
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Grant Support
South Carolina INBRE (NIH-NIGMS P20GM103499)
Type of Presentation
Poster presentation
Start Date
16-4-2021 11:30 AM
Generation of Fluorescent Esophageal Adenocarcinoma Cells for Lineage Tracing Within Composite 3D Culture Models
While relatively rare, the aggressive nature and poor prognosis associated with esophageal carcinoma make it a particularly dangerous form of cancer. One approach to discovering esophageal cancer treatment options includes the development of composite 3D culture models. Fluorescent labeling allows each cell type within a 3D culture model to be distinguished from others, facilitating independent study via lineage tracing. Our goal was to generate fluorescent esophageal cancer cells. Of primary interest was the Homo sapiens esophageal adenocarcinoma cell line OE19 that was transfected with a plasmid carrying the mCherry (red) fluorescence marker and neomycin resistance gene. We hypothesized that if geneticin antibiotic (G418) was introduced to transfected OE19 cultures, the percentage of mCherry expressing cells would be enhanced due to G418 selection against cells lacking the plasmid. To test our hypothesis, 400 mg/mL of G418 was applied to multiple OE19 passages during routine cell maintenance. Fluorescence-activated cell sorting (FACS) was then used to sort each cell based on mCherry expression. In addition, OE19 cultures under G418 selection were stained with Hoechst dye (cyan), to visualize nuclei as reference for mCherry appearance during analysis via fluorescent microscopy. The data obtained from FACS analysis indicated that 5-7% of OE19 sample cultures expressed the mCherry label. This was supported by fluorescent microscopy. Low cell viability following cell sorting did not allow for continued culture of sorted cells. Future directions include increasing the selection pressure on OE19 cells by raising the concentration of G418 and using modified FACS procedures to improve post-sorting viability.
