Density Gradients Separate Dormant and Treatment-Resistant Human Glioblastoma Cells

Glioblastoma (GBM) is among the most aggressive cancers. Despite aggressive radiotherapy and treatment with the alkylating agent temozolomide (TMZ), patients ultimately succumb to the disease. Although much interest has focused on highly tumorigenic GBM stem cells (GSCs), adaption of a concept from microbial research proposes that a minor population of dormant “persister” cells in cancer evade current therapies. To separate dormant and treatment-resistant tumor cells in human GBM tumorspheres, we have refined density gradient protocols previously used for separation of neurosphere-forming neural stem cells (NSCs). We find that a minor cell population in human GBM tumorsphere cultures and patient-derived tumor biopsies display increased cell density. These high-density GBM cells (HDGCs) display dormancy, variable expression of proposed GSC markers, and 10-100 fold higher levels of reprogramming gene expression compared to low-density GBM cells (LDGCs). Transcriptional profiling data confirmed the slow-cycling state of HDGCs. As a result, HDGCs show decreased tumorsphere formation capacity in vitro and reduced tumorigenicity in vivo. Using tumorspheres and xenografts, we demonstrated that HDGCs show increased treatment-resistance to ionizing radiation (IR) and temozolomide treatment compared to LDGCs. Similar to the NSC lineage, our data suggest that dormant HDGCs become increasingly sensitive to anti-proliferative therapies as they become activated and further differentiate. In conclusion, density gradients represents a marker-independent approach to separate dormant and treatment-resistant tumor cells in human GBMs and other solid cancers. 12 samples, no replicates, derived from 5 individual patients