Introduction: Glioblastoma stem cells (GSCs) are tumor-initiating cells and contribute to chemo-radiation resistance of glioblastoma multiforme (GBM) with poor prognosis. Cell-cell interactions in GBM niche regulate and maintain GSCs leading to tumor progression. Secretory molecules from different cell types in GBM niche play significant roles in intercellular communications. Therefore, we utilized phage screening technology to identify the secretory molecules regulating GSCs-microenvironment interactions and target their communications.
Methods: Phage display library consisting of 109 different combinations of 7 amino acid-long peptide sequences was screened for binding to GSCs in both in vitro and in vivo. 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 phage library into immunocompromised mice with intracranial primary GBM xenograft. Phage peptides that preferentially bound to the GSC population were recovered by dissociating the xenografted tumors and sorting for the CD133 surface marker. BLAST analysis was conducted to characterize the screened GSC-binding peptides. RT-qPCR, RNA-seq, and ChIP-seq were applied to validate the intercellular pathways. To elucidate the clinical significance of the findings, we used in silico analysis including protein interactions, tissue and histologic locations, and patient survivals.
Results: Angiotensin I converting enzyme (ACE), a key protein in renin-angiotensin system (RAS), was identified by BLAST analysis of the recovered peptides. RT-qPCR, RNA-seq, ChIP-seq, and in silico data showed that GSCs highly expressed renin and angiotensinogen, components of RAS, and differentiated cancer cells and endothelial cells expressed ACE. TCGA and histologic analysis were also consistent with these findings. Low expression of these RAS components correlated with better patient survivals.
Conclusions: This study suggests that inhibition of renin-angiotensin system in glioblastoma disrupts the intercellular communications among GSCs, differentiated cancer cells, and tumor endothelium and improves patient survival.
Patient Care: This research leads to an improved understanding of them mechanisms that drive proliferation of glioblastoma stem cells. This will allow for the development of therapeutic strategies to better treat glioblastoma.
Learning Objectives: 1) understand the cell-cell communications in glioblastoma renin-angiotensin model
2) understand the cancer stem cell hypothesis as it pertains to glioblastomas
3) understand phage display screening techniques and its application to explore novel targets in brain tumors.