Figure 1.

Cancer stem-like ESCC cells promote tumor initiation and metastasis. A tumorsphere culture system was used to isolate cancer stem-like ESCC cells in KYSE70, CE48T, and KYSE170 cells. The isolated KYSE70, CE48T, and KYSE170 cells were subjected to characterize the CSCs properties in vitro and in vivo. (a) The sphere-forming assay was used to evaluate self-renewal ability in ESCC cells. The cells were seeded into 10 cm ultra-low adhesion dishes and incubated in stem cell culture medium at a density of 1 × 10⁵ cells/dish. 7 days after seeding, tumorspheres were generated and the spheroid cells were then trypsinized and 1 × 10⁵ spheroid cells were used for the next passage. Three passages were processed and the percentage of tumor formation was counted. Scale bar: 100 µm. (b) and (c) The indicated mRNA and protein level was examined by qPCR and western blot in nonspheroid and spheroid ESCC cells. Quantified results of western blot indicated the relative protein expression level in spheroid cells compared with nonspheroid cells. (d) Tumorspheres were incubated in a serum-induced differentiation condition medium. Representative images of the differentiate spheroid cells were taken on day 0 (d0), day 1 (d1), day 4 (d4), and day 10 (d10) after incubation. The mRNA expression levels of indicated stemness genes and drug-resistant genes were examined in nonspheroid, spheroid, and differentiated KYSE70 cells by qPCR. (e) A series of nonspheroid and spheroid KYSE170-Luc cells were subcutaneously injected into immunodeficiency mice and monitor tumor growth weekly using an in vivo Imaging Systems (IVIS) system. (f) The tail vein injection model was used to investigate the metastatic ability in vivo. A total of1000 nonspheroid and spheroid KYSE170-Luc cells were injected into immunodeficiency mice using tail vein injections, the metastases were monitored weekly using an IVIS system. Four weeks after injection, the mice were sacrificed and the lungs were harvested and the metastases were examined by immunohistochemistry using human mitochondrial antibody. (Scale bar: 100 µm).

CSCs, cancer stem cells; ESCC, esophageal squamous cell carcinoma