Phage display discovery of novel molecular targets in glioblastoma-initiating cells.
Glioblastoma is the most common primary intrinsic brain tumor and remains incurable despite maximal therapy. Glioblastomas display cellular hierarchies with self-renewing glioma-initiating cells (GICs) at the apex. To discover new GIC targets, we used in vivo delivery of phage display technology to screen for molecules selectively binding GICs that may be amenable for targeting. Phage display leverages large, diverse peptide libraries to identify interactions with molecules in their native conformation. We delivered a bacteriophage peptide library intravenously to a glioblastoma xenograft in vivo then derived GICs. Phage peptides bound to GICs were analyzed for their corresponding proteins and ranked based on prognostic value, identifying VAV3, a Rho guanine exchange factor involved tumor invasion, and CD97 (cluster of differentiation marker 97), an adhesion G-protein-coupled-receptor upstream of Rho, as potentially enriched in GICs. We confirmed that both VAV3 and CD97 were preferentially expressed by tumor cells expressing GIC markers. VAV3 expression correlated with increased activity of its downstream mediator, Rac1 (ras-related C3 botulinum toxin substrate 1), in GICs. Furthermore, targeting VAV3 by ribonucleic acid interference decreased GIC growth, migration, invasion and in vivo tumorigenesis. As CD97 is a cell surface protein, CD97 selection enriched for sphere formation, a surrogate of self-renewal. In silico analysis demonstrated VAV3 and CD97 are highly expressed in tumors and inform poor survival and tumor grade, and more common with epidermal growth factor receptor mutations. Finally, a VAV3 peptide sequence identified on phage display specifically internalized into GICs. These results show a novel screening method for identifying oncogenic pathways preferentially activated within the tumor hierarchy, offering a new strategy for developing glioblastoma therapies.
1] Department of Neurosurgery, Cleveland Clinic, Cleveland, OH, USA [2] Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Liu
1] Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA [2] Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA.
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Lubelski
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Schonberg
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Wu
Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
J S
JS
Hale
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Flavahan
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Mulkearns-Hubert
Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Man
1] Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA [2] Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA.
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Hjelmeland
1] Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA [2] Department of Radiation Oncology, Cleveland Clinic, Cleveland, OH, USA.
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Yu
1] Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA [2] Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.
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Lathia
1] Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA [2] Department of Molecular Medicine, Cleveland Clinic, Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, USA.
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D000818:Animals; D001932:Brain Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000502:physiology; D045744:Cell Line, Tumor; D061505:Cell Surface Display Techniques; D005909:Glioblastoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D064593:Heterografts; D006801:Humans; D051379:Mice; D058990:Molecular Targeted Therapy; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D015398:Signal Transduction
D010455:Peptides