Stem cells have been isolated by their ability to efflux
Hoechst 33342 dye and are referred to as the "side population" (SP). In this study, we used flow cytometry and
Hoechst 33342 dye efflux assay to isolate and characterize SP cells from six human
lung cancer cell lines (H460, H23,
HTB-58, A549, H441, and H2170). Nonobese diabetic/
severe combined immunodeficiency xenograft experiments showed that SP cells were enriched in
tumor-initiating capability compared with non-SP cells.
Matrigel invasion assay showed that SP cells also have higher potential for invasiveness. Further characterization of this SP phenotype revealed several stem cell properties. We found evidence for repopulating ability by SP to regenerate a population resembling the original population. SP displayed elevated expression of ABCG2 as well as other
ATP-binding cassette transporters and showed resistance to multiple chemotherapeutic drugs. Human
telomerase reverse transcriptase expression was higher in the SP, suggesting that this fraction may represent a reservoir with unlimited proliferative potential for generating
cancer cells.
mRNA levels of minichromosome maintenance (MCM) 7, a member of the MCM family of
proteins critical to the DNA replication complex, were lower in SP cells, suggesting that a majority of the SP fraction was in the G(0) quiescent state. Sixteen clinical
lung cancer samples also displayed a smaller but persistent SP population. These findings indicate that SP is an enriched source of lung tumor-initiating cells with stem cell properties and may be an important target for effective
therapy and a useful tool to investigate the tumorigenic process.