Hypoxia enhances tumor stemness by increasing the invasive and tumorigenic side population fraction

Although advances have been made in understanding the role of hypoxia in the stem cell niche, almost nothing is known about a potentially similar role of hypoxia in maintaining the tumor stem cell (TSC) niche. Here we show that a highly tumorigenic fraction of side population (SP) cells is localized in the hypoxic zones of solid tumors in vivo. We first identified a highly migratory, invasive, and tumorigenic fraction of posthypoxic side population cells (SPm_[hox] fraction) in a diverse group of solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, and small-cell lung carcinoma. To identify the SPm_(hox) fraction, we used an "injured conditioned medium" derived from bone marrow stromal cells treated with hypoxia and oxidative stress. We found that a highly tumorigenic SP fraction migrates to the injured conditioned medium in a Boyden chamber. We show that as few as 100 SPm_(hox) cells form rapidly growing tumors in vivo. In vitro exposure to hypoxia increases the SPm_(hox) fraction significantly. Quantitative real-time polymerase chain reaction and immunofluorescence studies showed that SPm_(hox) cells expressed Oct-4, a "stemness" gene having a potential role in TSC maintenance. In nude mice xenografts, SPm_(hox) cells were localized to the hypoxic zones, as demonstrated after quantum dot labeling. These results suggest that a highly tumorigenic SP fraction migrates to the area of hypoxia; this migration is similar to the migration of normal bone marrow SP fraction to the area of injury/hypoxia. Furthermore, the hypoxic microenvironment may serve as a niche for the highly tumorigenic fraction of SP cells.