Introduction:
Glioblastoma multiforme (GBM) is the most malignant incarnation of glial tumour, with almost universal mortality. The treatment of GBM has improved only incrementally in the past several decades, with a current median survival of ~16 months. The difficulty of treating GBM stems from its diffuse, invasive growth pattern and placement within the brain, where large surgical margins are not possible. GBM also contains a subpopulation of cells, termed glioma stem cells (GSCs) that are highly resistant to chemoradiotherapy; so invasive cells beyond the tumour margins persist and cause recurrence and ultimately patient mortality. The lack of a definitive marker for the proposed GSC has hindered the ability to therapeutically target it. However, the GSC has similar properties to non-malignant, normal stem cells – high resistance to chemo/radiotherapy and a low mitotic index.
Methods:
We have exploited the reduced mitotic rate of these resistant cells to isolate them. Using a 7-day pulse-chase assay with the fluorescent dye Oregon Green we can identify and isolate by fluorescence imaging and FACS the subpopulation of cells with the lowest mitotic index and investigate them against a rapidly-dividing, non-label-retaining subpopulation.
Results:
It was found that cells that retain the highest concentration of Oregon Green are more resistant to chemotherapy (temozolomide), radiotherapy, and combination chemoradiotherapy. The quiescent cells are also larger, more complex, and more migratory than their proliferative counterparts and express greater levels of proteins associated with invasion.
Conclusion:
We have shown that quiescent cells are innately resistant to the current gold standard GBM treatments and display more malignant features in vitro than rapidly dividing cells. We have developed a platform for quick, easy, and reliable isolation of this quiescent fraction of cells to facilitate its interrogation. This method has the potential to identify future treatment targets, and could, in principle, be extended to other malignancies.