Characterization of Cancer Stem Cells in Esophageal Adenocarcinoma

Introduction: Despite modern treatment of esophageal cancer, the prognosis remains poor. Recurrences still can occur in a short period of time. Cancer stem cells might contribute to this phenomenon. Cancer stem cells are chemoradiation resistant and are thus able to generate a new tumor. Therefore, it is important to find potential markers to identify these cells in order to improve therapy. In esophageal cancer, cancer stem cell markers remain unidentified. Also, the regulation of these cancer stem cells needs to be elucidated. miRNAs are important in tumorigenesis and are involved in the regulation of cancer stem cells. However, the role of miRNAs in the regulation of esophageal cancer stem cells remains unclear. The goal of my project is to identify potential cancer stem cell markers in esophageal adenocarcinoma, e.g. CD24, CD44, CD44v6, CD26 and CD133, and to find out whether miRNAs are involved in regulating these cancer stem cells. Material and Methods: Cells were stained with fluorescence-conjugated antibody against the previous mentioned markers and sorted into two populations, one marker-positive and one marker-negative population. A sphere formation assay with 10.000 cells was performed for both sorted populations of every marker. A comparison has been made in the sphere forming ability between both populations after 11-13 days. Furthermore, a miRNA PCR array is done to identify the most down-regulated miRNA in the population of the most significant marker. To understand whether this miRNA can regulate the potential cancer stem cell population, flow cytometry is performed with the most down-regulated miRNA modificated cells to determine the effect on the potential cancer stem cell population. A sphere formation assay is performed to further confirm the results. Results: Cells that stained positively for CD44v6 (p=0.0003) and CD24 (p=0.01) showed significant increase in sphere enrichment in BE3 whereas only CD44v6 (p=0.0009) showed a significant increase in OE33. miR-205 (0.25 fold) appeared to be the most down-regulated miRNA in the CD44v6+ population. CD44v6 expression was significant lower (p=0.005) in the miR-205 overexppressed cells and significantly higher (p=0.02) in miR-205 repressed cells compared to the control. Moreover, miR-205 suppressed cells showed more sphere formation compared to the control. Conclusion: CD44v6 is a putative marker to identify a cancer stem cell-like population in esophageal adenocarcinoma. Furthermore, miR-205 is down-regulated in esophageal adenocarcinoma which results in an increase of CD44v6 cancer stem-cell like population and sphere formation. Therefore, miR-205 is likely a key player in the regulation of esophageal adenocarcinoma cancer stem cells.