There is no CSC marker based hierarchy in CSC marker expressing tumors.

(A) Schematic diagram shows the construction of serial single-cell derived sublines (SCDSLs). CSC+ and CSC− cells from the original tumor cell lines were selected to generate SCDSLs and arbitrarily classified as generation 1 SCDSLs (including G1+ and G1−). CSC+ and CSC− cells from G1 SCDSLs were selected to generate G2 SCDSLs (including G1+G2+, G1−G2+, G1+G2− and G1−G2−). We repeated the procedure until G3 SCDSLs were obtained. (B–E) The percentage of CSC marker positive cells in SCDSLs from KG-1, MCF-7, SHG44, Caco-2 and A375 was analyzed by flow cytometry when the cell quantity reached approximately 1×106. Box plots show the percentage of CSC marker positive cells in single CSC+-derived (B) and CSC−-derived (C) sublines from different generations, with the whiskers representing the minimum and maximum values, the central lines representing the median value, and the boxes representing the 25th and 75th percentile. Histograms show the percentage of CSC marker positive cells in single CSC+-derived (D) and CSC−-derived (E) sublines from different generations. Data represent mean ± SEM from 100 samples, each from one independent serial SCDSL construction.