Sphere-forming cell subsets with cancer stem cell properties in human musculoskeletal sarcomas

Musculoskeletal sarcomas are aggressive malignan- cies often characterized by an adverse prognosis despite the use of intense multiagent chemotherapy or molecular targeted therapy in combination to surgery and radiotherapy. Stem-like cells identi ed within solid tumors have been recently impli- cated in drug resistance, metastasis and local relapse. Here, we report the identi cation of putative cancer stem cells (CSCs) in sarcomas using a sphere culture system. These sarcospheres, able to grow in anchorage-independent and serum-starved conditions, express the pluripotent embryonic stem cell marker genes OCT3/4, Nanog and SOX2. Expression levels of these genes were greater in sarcospheres than in the parental tumor cultures. Importantly, the isolated tumor spheres transplanted into mice were tumorigenic and capable of recapitulating the human disease. Finally, we demonstrated that low (1%) O2 conditions, reproducing those found within the tumor micro- environment, signi cantly increase the number and the size of sarcospheres. The sphere formation assay is, therefore, a valu- able method for the isolation of putative CSCs from human sarcomas and its ef ciency is improved by controlling oxygen availability. This method provides a reliable preclinical model that can be used for future studies aimed at investigating crucial aspects of sarcoma biology, such as resistance to treat- ments and relapse.