Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment

Abstract

Background Glioblastoma (GBM), a highly malignant brain tumor, invariably recurs after therapy. Quiescent GBM cells represent a potential source of tumor recurrence, but little is known about their molecular underpinnings. Methods Patient-derived GBM cells were engineered by CRISPR/Cas9-assisted knock-in of an inducible histone2B-GFP (iH2B-GFP) reporter to track cell division history. We utilized an in vitro 3D GBM organoid approach to isolate live quiescent GBM (qGBM) cells and their proliferative counterparts (pGBM) to compare stem cell properties and therapy resistance. Gene expression programs of qGBM and pGBM cells were analyzed by RNA-Seq and NanoString platforms. Findings H2B-GFP-retaining qGBM cells exhibited comparable self-renewal capacity but higher therapy resistance relative to pGBM. Quiescent GBM cells expressed distinct gene programs that affect cell cycle control, metabolic adaptation, and extracellular matrix (ECM) interactions. Transcriptome analysis also revealed a mesenchymal shift in qGBM cells of both proneural and mesenchymal GBM subtypes. Bioinformatic analyses and functional assays in GBM organoids established hypoxia and TGFβ signaling as potential niche factors that promote quiescence in GBM. Finally, network co-expression analysis of TCGA glioma patient data identified gene modules that are enriched for qGBM signatures and also associated with survival rate. Interpretation Our in vitro study in 3D GBM organoids supports the presence of a quiescent cell population that displays self-renewal capacity, high therapy resistance, and mesenchymal gene signatures. It also sheds light on how GBM cells may acquire and maintain quiescence through ECM organization and interaction with niche factors such as TGFβ and hypoxia. Our findings provide a starting point for developing strategies to tackle the quiescent population of GBM. Fund National Institutes of Health (NIH) and Deutsche Forschungsgemeinschaft (DFG).

Glioblastoma Stem Cells: Driving Resilience through Chaos

“…Single-cell sequencing studies have revealed high overlap between stem cell signatures and proliferative markers, suggesting that GSCs are more proliferative than was previously thought [24,25]. Quiescent GSCs may exist as a subpopulation that is maintained by signaling from the microenvironmental niche [112], giving rise to (or existing in parallel with) proliferative GSCs [23]. Although the relationship between proliferative and quiescent GSCs remains an open area of investigation, multiple studies have demonstrated that GSCs maintained in a quiescent state are more resistant to treatment [109,112,113].…”

“…Quiescent GSCs may exist as a subpopulation that is maintained by signaling from the microenvironmental niche [112], giving rise to (or existing in parallel with) proliferative GSCs [23]. Although the relationship between proliferative and quiescent GSCs remains an open area of investigation, multiple studies have demonstrated that GSCs maintained in a quiescent state are more resistant to treatment [109,112,113]. As previously noted, antiangiogenic agents such as bevacizumab have not been effective in clinical trials, despite demonstrating initial promise in preclinical models [110,114,115].…”

Mesenchymal Transformation: The Rosetta Stone of Glioblastoma Pathogenesis and Therapy Resistance

“…More recently, Tejero et al showed that a small group of quiescent cells in GBM cultures are more aggressive and therapy resistance. [116] By gene set enrichment analysis (GSEA), they identified that most of the PMT-associated genes were upregulated in therapy-resistant quiescent cells. [116] TMZ intrinsic and/or acquired resistance in GBM is not regulated by a single molecular event, rather by multiple proteins and TFs acting together.…”

Transcriptomic Profiling of 3D Glioblastoma Tumoroids for the Identification of Mechanisms Involved in Anticancer Drug Resistance

“…They found that in 3D glioblastoma multiforme PLA porous scaffolds compared to the 2D condition, genes in peroxisome proliferator-activated receptors and phosphatidylinositol 3'-kinase(PI3K)-AKT signaling pathways were up-regulated, while those in metabolism, or related to the extracellular matrix and transforming growth factor-beta pathways were down-regulated (12). Tejero, et al found that the 3D glioblastoma organoids promoted the appearance of a quiescent cell population that represented the self-renewal ability high therapy resistance, and mesenchymal gene signatures (13). Furthermore, Jia, et al found that the expression of genes associated with stemness, cell cycle, apoptosis, epithelia-mesenchymal transition, migration, invasion, and glioma malignancy, were upregulated in 3D glioma cell culture (14).…”