Autofluorescence of NADH is a new biomarker for sorting and characterizing cancer stem cells in human glioma

BACKGROUND: The existing cell surface markers used for sorting glioma stem cells (GSCs) have obvious limitations, such as vulnerability to the enzymatic digestion and time-consuming labeling procedure. Reduced nicotinamide adenine dinucleotide (NADH) as a cellular metabolite with property of autofluorescence has the potential to be used as a new biomarker for sorting GSCs. METHODS: A method for sorting GSCs was established according to the properties of the autofluorescence of NADH. Then, the NADH(high) and NADH(low) subpopulations were sorted. The stem-like properties of the subpopulations were evaluated by qRT-PCR, western blot analyses, limiting dilution assay, cell viability assay, bioluminescence imaging, and immunofluorescence analysis in vitro and in vivo. The relationship between CD133(+)/CD15(+) cells and NADH(high) subpopulation was also assessed. RESULTS: NADH(high) cells expressed higher stem-related genes, formed more tumor spheres, and harbored stronger pluripotency in vitro and higher tumorigenicity in vivo, compared to NADH(low) subpopulation. NADH(high) glioma cells had the similar stemness with CD133(+) or CD15(+) GSCs, but the three subpopulations less overlaid each other. Also, NADH(high) glioma cells were more invasive and more resistant to chemotherapeutic drug temozolomide (TMZ) than NADH(low) cells. In addition, the autofluorescence of NADH might be an appropriate marker to sort cancer stem cells (CSCs) in other cancer types, such as breast and colon cancer. CONCLUSION: Our findings demonstrate that intracellular autofluorescence of NADH is a non-labeling, sensitive maker for isolating GSCs, even for other CSCs.