Phage display discovery of novel molecular targets in glioblastoma-initiating cells

Abstract

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-proteincoupled-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.

GFAPδ/GFAPα ratio directs astrocytoma gene expression towards a more malignant profile

“…Our classification of these genes as high-malignant or low-malignant is based on their correlation to astrocytoma malignancy and is supported by previous studies. Increased expression of VAV3 in high compared to low grade astrocytoma [ 34 , 35 ], increased NOS2 expression in tumours of higher compared to lower malignancy and in brain tumours compared to healthy tissue [ 36 – 39 ], and increased SPC24 expression in other tumour types compared to healthy tissue [ 40 ] all support their tumorigenic function. This is emphasized by the negative prognostic effect of VAV3 and NOS2 expression in grade IV astrocytoma and for SPC24 in liver tumours [ 40 ].…”

Tumor-targeting peptides from combinatorial libraries

“…Expression level of VAV3 in recurrent glioblastoma is higher than that of primary glioblastoma [61]. Elevated VAV3 expression correlates with higher grade tumor and poor prognosis.…”

“…Targeting VAV3 by ribonucleic acid interference decreased GIC growth, migration, invasion and in vivo tumorigenesis. A VAV3-targeting peptide SSQPFWS was identified by the Rich group through in vivo biopanning of the Ph.D.-7 library in mice implanted subcutaneously with patient-derived glioblastoma xenografts [61]. This peptide specifically internalized into GICs.…”

Isolation and characterisation of antibodies against human and canine EphA2_ targets for comparative studies in brain cancer

“…However, GBM is incurable and patients need to undertake other treatments to prolong the survival and relieve the GBM symptoms. Identifying the GBM biomarkers are significantly crucial for early diagnosis, prognosis and treatment management, and has been extensively reported [339][340][341].…”

“…Phage display technology can provide the tools for the isolation of antibodies that bind specifically to the cell and organ-specific biomarkers in their native conformation [341,342]. The generated antibodies can form the basis of therapeutic and diagnostic reagents.…”