Esophageal cancer tumorspheres involve cancer stem-like populations with elevated aldehyde dehydrogenase enzymatic activity.

Cancer stem cells (CSCs) form spheres in vitro in serum-free suspension culture. Sphere formation is particularly useful to enrich the potential CSC subpopulations as a functional approach. Few reports are currently available on tumorspheres in esophageal cancer (EC). The present study focused on evaluating the cancer stem-like properties and analyzing the difference between spheroid and adherent cells of the Eca109 human EC cell line. Immunofluorescence and immunoblotting analysis revealed that EC tumorspheres expressed the stem cell markers Nanog and Oct4 more highly, but showed a decreased expression of the differentiation marker CK5/6. The spheroids were chemoresistant to cisplatin compared to the adherent cells (32.5 vs. 135.8 µM in IC50). Side population cells increased in tumorspheres compared to adherent cells (0.7 vs. 5.6%). A marked upregulation of drug-resistant genes (ABCG2 and MDR1) was observed in sphere-forming cells. We compared the profiles of adherent and spheroid cells by microarrays and obtained one representative differentially expressed gene, aldehyde dehydrogenase (ALDH). We also verified that the cancer stem-like cells of EC contained a high ALDH enzymatic activity. ALDH-positive cells were enriched by 11- to 12-fold in spheroids, compared to adherent cells (2.5 vs. 28.6%). Immunofluorescence and immunoblotting analysis also revealed a higher expression of ALDH in EC tumorspheres. In conclusion, our study verified that sphere-forming culturing can be utilized to demonstrate the putative esophageal CSCs, and identified a potential esophageal CSC surface marker, ALDH.