ALDEFLUOR-positive cell population from GI-101A cells displays properties of CSCs in both cell culture and animal models. (A-F) Representative FACS sorting of ALDEFLUOR-positive cells from GI-101A cell line. The ALDEFLUOR sorting on GI-101A cells was electronically gated firstly to exclude dead cells (R1), doublets (R2) and aggregates (R3). Incubation of cells with ALDEFLUOR substrate in the absence of DEAB induces a shift in BAAA fluorescence, defining the ALDEFLUOR-positive population, which represents 6.43 ± 0.98% of the total live single cells in GI-101A comparing 0.35 ± 0.98% of the total live single cells in the presence of DEAB. The purity of sorted cells was checked finally. (G-L) The ALDEFLUOR-positive cells have higher tumorigenic potential and latency in athymic nude mice. Tumor-forming occurrence was observed at different time points (G-J). (K) Representative tumor development in nude mouse at the ALDEFLUOR-positive cells’ injection site (50,000 cells injected). Smaller tumor was detected at the ALDEFLUOR-negative cells’ injection site (50,000 cells injected). (L) Tumor growth was monitored weekly by measuring the tumor volume of five mice in each group. The curves were plotted for the numbers of cells injected (50,000 cells and 5,000 cells) and for each population (ALDEFLUOR-positive, ALDEFLUOR-negative). Tumor growth kinetics correlated with the latency and size of tumor formation and the number of ALDEFLUOR-positive cells. No tumor was detected when 500 cells were injected, whereas ALDEFLUOR-positive cells produced tumors that grew at a rate that directly correlated with the number of cells injected. (M) Statistical evaluation of the mammospheres formation efficiency of GI-101A ALDEFLEOR-positive and ALDEFLEOR-negative cells.