Introduction: Glioblastoma Multiforme (GBM) represents the most common and lethal primary brain tumor with conventional therapy offering only palliation. Radio- and chemo-resistance has been hypothesized to be due in part to the therapeutic resistance of GBM stem cells (GSCs), a subset of cells functionally defined by their ability to self-renew and propagate tumors. Development of effective GSC-specific therapeutics requires selective targeting of GSCs without disrupting normal stem cell maintenance. Therefore, we employed phage technology to identify novel proteins involved in regulating GSC maintenance.
Methods: A M13 bacteriophage peptide library consisting of 109 different combinations of 7 amino acid long peptide sequences was screened for binding to functionally validated GSCs in both in vitro and in vivo settings. In vitro screening was performed against GSCs isolated from human GBM tumors grown in culture following negative selection on non-GSCs. In vivo screening was performed by injecting the random phage library into immunocompromised mice with subcutaneous or intracranial xenografted primary GBM tumors cells. Phage peptides that preferentially bound to the GSC population were recovered by dissociating the xenografted tumors and sorting for the CD133 surface marker.
Results: In silico analysis of the recovered peptide sequences revealed several proteins that potentially play a key role in the cancer stem cell pathway. Among those proteins isolated was olig2, a known cancer stem cell specific marker. Evaluation of additional genes revealed proteins preferentially expressed in GSCs compared to their non-stem counterparts. Knockdown of these genes in GSCs using small hairpin RNA constructs showed phenotypic changes in cell growth. Expression of these proteins has also been shown to correlate with survival studies in GBM patients.
Conclusions: These data verify the use of phage display as a screening tool to isolate GSC-specific proteins that are potentially vital to their stem-like functions.
Patient Care: I have developed a novel screening strategy using phage display screening specifically against glioma stem cells using in vitro and in vivo models. Isolating protein targets that are vital to the specific functioning of glioma stem cells will allow the development of definitive therapeutics aimed specifically at targeting these pathways. This will allow isolation of the most crucial tumor propagating pathways while reducing unnecessary toxicity to adjacent cells.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) understand the cancer stem cell hypothesis as it pertains to gliomablastomas and 2) understand phage display screening techniques and how it can be used elucidate novel targets in glioma stem cells as well as other cell pathways, and 3) understand the methods used to validate proteins and their critical role in the functioning of glioma stem cells.
References: Bao S, Wu Q, McLendon RE, et al. (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 444: 756-60.
Liu JK, Teng Q, Garrity-Moses M, et al. A novel peptide defined through phage display for therapeutic protein and vector neuronal targeting. Neurobiol Dis. 2005; 19(3):407-18.