Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-ß as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Proteomic Analysis Implicates Vimentin in Glioblastoma Cell Migration.

We previously showed lithium chloride (LiCl) and other inhibitors of glycogen synthase kinase-3 (GSK-3) including 6-bromo-indirubin-3-oxime (BIO), can block glioblastoma (GBM) cell migration. To investigate the mechanisms involved we used two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify proteins altered after treatment of U251 GBM cells with 20 mM LiCl. Downregulation of the intermediate filament protein vimentin was the most significant change identified. Analysis of patient tumor samples revealed that vimentin is expressed abundantly in GBM, and is prognostic especially in lower grade tumors. Additionally, siRNA-mediated vimentin knockdown impaired GBM migration. Western blotting showed that treatment with LiCl or small molecule GSK-3 inhibitors led to the rapid downregulation of detergent soluble vimentin levels across a panel of GBM-derived cells. Fluorescence reactivation after photobleaching (FRAP) microscopy studies showed a significant reduction in the ability of the vimentin cytoskeleton to recover from photo-bleaching in the presence of LiCl or BIO. Biochemical studies revealed that GSK-3 and vimentin directly interact, and analysis of vimentin revealed a GSK-3 consensus phosphorylation site. We conclude that anti-migratory compounds with the ability to inhibit GSK-3 have effects on vimentin cytoskeletal dynamics, which may play a role in their anti-invasive activity.

The functional synergism of microRNA clustering provides therapeutically relevant epigenetic interference in glioblastoma.

MicroRNA deregulation is a consistent feature of glioblastoma, yet the biological effect of each single gene is generally modest, and therapeutically negligible. Here we describe a module of microRNAs, constituted by miR-124, miR-128 and miR-137, which are co-expressed during neuronal differentiation and simultaneously lost in gliomagenesis. Each one of these miRs targets several transcriptional regulators, including the oncogenic chromatin repressors EZH2, BMI1 and LSD1, which are functionally interdependent and involved in glioblastoma recurrence after therapeutic chemoradiation. Synchronizing the expression of these three microRNAs in a gene therapy approach displays significant anticancer synergism, abrogates this epigenetic-mediated, multi-protein tumor survival mechanism and results in a 5-fold increase in survival when combined with chemotherapy in murine glioblastoma models. These transgenic microRNA clusters display intercellular propagation in vivo, via extracellular vesicles, extending their biological effect throughout the whole tumor. Our results support the rationale and feasibility of combinatorial microRNA strategies for anticancer therapies.

MiR-19b non-canonical binding is directed by HuR and confers chemosensitivity through regulation of P-glycoprotein in breast cancer.

MicroRNAs and RNA-binding proteins exert regulation on >60% of coding genes, yet interplay between them is little studied. Canonical microRNA binding occurs by base-pairing of microRNA 3'-ends to complementary "seed regions" in mRNA 3'UTRs, resulting in translational repression. Similarly, regulatory RNA-binding proteins bind to mRNAs, modifying stability or translation. We investigated post-transcriptional regulation acting on the xenobiotic pump ABCB1/P-glycoprotein, which is implicated in cancer therapy resistance. We characterised the ABCB1 UTRs in primary breast cancer cells and identified UTR sequences that responded to miR-19b despite lacking a canonical binding site. Sequences did, however, contain consensus sites for the RNA-binding protein HuR. We demonstrated that a tripartite complex of HuR, miR-19b and UTR directs repression of ABCB1/P-glycoprotein expression, with HuR essential for non-canonical miR-19b binding thereby controlling chemosensitivity of breast cancer cells. This exemplifies a new cooperative model between RNA-binding proteins and microRNAs to expand the repertoire of mRNAs that can be regulated. This study suggests a novel therapeutic target to impair P-glycoprotein mediated drug efflux, and also indicates that current microRNA binding predictions that rely on seed regions alone may be too conservative.

Disruption of the ß1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner.

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPß1L (ß1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting ß1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of ß1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPß1L in enabling immortality in TERT promoter mutant glioblastoma.

Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles.

Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1-dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.