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Single-cell analyses reveal YAP/TAZ as regulators of stemness and cell plasticity in glioblastoma

Nature Cancer volume 2, pages 174–188 (2021)Cite this article

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Abstract

Glioblastoma (GBM) is a devastating human malignancy. GBM stem-like cells (GSCs) drive tumor initiation and progression. Yet the molecular determinants defining GSCs in their native state in patients remain poorly understood. Here we used single-cell datasets and identified GSCs at the apex of the differentiation hierarchy of GBM. By reconstructing the GSCs’ regulatory network, we identified the YAP/TAZ coactivators as master regulators of this cell state, irrespectively of GBM subtypes. YAP/TAZ are required to install GSC properties in primary cells downstream of multiple oncogenic lesions and are required for tumor initiation and maintenance in vivo in different mouse and human GBM models. YAP/TAZ act as main roadblock of GSC differentiation, and their inhibition irreversibly locks differentiated GBM cells into a nontumorigenic state, preventing plasticity and regeneration of GSC-like cells. Thus, GSC identity is linked to a key molecular hub integrating genetics and microenvironmental inputs within the multifaceted biology of GBM.

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Fig. 1: A gene expression program identifying native GSCs.
Fig. 2: YAP/TAZ are master TRs of the GSC state.
Fig. 3: YAP/TAZ are required for oncogene-dependent transformation of primary normal neural cells.
Fig. 4: YAP/TAZ control GBM cell plasticity.
Fig. 5: YAP/TAZ are required to prevent GSC differentiation.
Fig. 6: YAP/TAZ are required for GBM initiation by preventing GSC differentiation.

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Data availability

All RNA-seq and microarray raw data generated for the present study, along with counts matrices and metadata for each sample, are publicly available in GEO under accession code GSE133471. The scRNA-seq data of primary glioblastoma samples from Darmanis et al. were downloaded as raw reads from GEO (GSE84465). The expression matrix and metadata of the Neftel dataset were downloaded from the Single Cell Portal of the Broad Institute (https://singlecell.broadinstitute.org/single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma#study-summary). Raw gene expression data (.CEL files) of the GBM TCGA cohort were downloaded from GEO (GSE83130). Raw gene expression (.CEL files) and clinical data of the REMBRANDT study were downloaded from GEO (GSE108474). BAM files of Ivy Atlas GBM samples were downloaded from the Anatomic Structures RNA-Seq repository of the Ivy Glioblastoma Atlas Project (http://glioblastoma.alleninstitute.org/rnaseq/bam.csv). Source data for Figs. 1e, 3b,e–f, 4c,f,g, 5a–f, 6b,f and Extended Data Figs. 1c, 2b,c, 3b, 4b, 6a–d, 7d, 8b, 9a,b and 10b,e,f have been provided with the paper. All other data supporting the findings of this study are available from the corresponding authors on reasonable request.

Code availability

All code used R 3.5.0 and publicly available packages cited in the paper. No custom functions were written for the analysis. STAR is available in GitHub (https://github.com/alexdobin/STAR). ARACNe-AP is available in GitHub (https://github.com/califano-lab/ARACNe-AP). HOMER is available at http://homer.ucsd.edu/homer/. MultiExperiment Viewer is available at http://mev.tm4.org.

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Acknowledgements

We thank I. Verma, J. Massagué and L. Naldini for plasmids and colleagues sharing their plasmids through Addgene (M.-C. Hung, L. Pedersen, C. Counter, C. Cepko, K. Hochedlinger and M. Meyerson). We thank D.J. Pan, D. Saur, J. Siveke and P. Bonaldo for gifts of mice, G. Basso for HuTu cells, G. Zuccolotto for the GFP/Luc-expressing lentiviral construct, V. Barbieri for in vivo experiments, V. Guzzardo for histology, S. Bresolin for microarrays, G. Leo for TAZ IHC analysis and M. Forcato for comments. M. Castellan was supported by a FIRC-AIRC fellowship for Italy. O.R. is supported by Fondazione Umberto Veronesi (Post-Doctoral Fellowship 2020). The research leading to these results has received funding from AIRC 5×1000 2018 ‘Metastasis as mechanodisease’ (ID, 22759) grant to S.P.; from AIRC IG Grant 2019 (ID, 23307) to S.P.; from the Italian Ministry of Education, University and Research under a MIUR-FARE grant to S.P. and a MIUR-PRIN Bando 2017 grant to S.P. (cod. 2017HWTP2K); from the European Research Council under the European Union’s Horizon 2020 research and innovation program (DENOVOSTEM grant agreement No 670126) to S.P.

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Author notes

  1. These authors jointly supervised this work: M. Cordenonsi, S. Piccolo.

Authors and Affiliations

  1. Department of Molecular Medicine, University of Padua, Padua, Italy

    Martina Castellan, Alberto Guarnieri, Atsushi Fujimura, Francesca Zanconato, Giusy Battilana, Tito Panciera, Hanna Lucie Sladitschek, Paolo Contessotto, Anna Citron, Michelangelo Cordenonsi & Stefano Piccolo

  2. Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy

    Andrea Grilli, Oriana Romano & Silvio Bicciato

  3. Department of Medicine - Surgical Pathology and Cytopathology Unit, University of Padua, Padua, Italy

    Matteo Fassan

  4. Department of Woman and Children Health, University of Padua, Padua, Italy

    Elena Porcù

  5. Department of Surgery, Oncology and Gastroenterology, University of Padua, Padua, Italy

    Antonio Rosato

  6. Veneto Institute of Oncology IOV-IRCCS, Padua, Italy

    Antonio Rosato

  7. IFOM, the FIRC Institute of Molecular Oncology, Milan, Italy

    Stefano Piccolo

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  1. Martina Castellan
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Contributions

M. Castellan performed most of the in vitro and in vivo experiments and contributed to writing. A.F. carried out the initial experiments of this study. A. Guarnieri and G.B. carried out experiments with HuTu cells. T.P. optimized mouse astrocyte isolation and infection. F.Z. contributed to manuscript preparation. F.Z., H.L.S., P.C. and A.C. optimized technical procedures critical for experiments in vivo and in vitro; M.F. performed histology and histopathological evaluations. E.P. and A.R. performed brain tumor experiments. O.R., A. Grilli and S.B. performed bioinformatic analyses. S.P. and M. Cordenonsi conceived the initial hypothesis and experimental design, organized the work and wrote the manuscript.

Corresponding authors

Correspondence to Michelangelo Cordenonsi or Stefano Piccolo.

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Extended data

Extended Data Fig. 1 Identification of the gene expression program of GSCs.

a, Single-cell differentiation trajectory of GBM cells reconstructed by Monocle2 using single-cell RNA-seq data of the indicated cell populations from primary GBM samples of the Darmanis dataset. b, Gene set enrichment analysis (GSEA) for association between the cell populations at the start and at the end of the pseudotime trajectory of the neoplastic cells of the Darmanis datasets (as depicted in Fig. 1c), and gene sets denoting the identity of specific cell types. Gene lists denoting early neural progenitor cells (RG: Radial Glia; oRG: outer Radial Glia; vRG: ventricular Radial Glia) or neural stem cells (NSC) are indicated in red; those identifying neurons, astrocytes or committed neuronal progenitors (OPC: Oligodendrocyte Progenitor Cells; INP: Intermediate Neuronal Progenitors) are, respectively, in blue, purple and blue-green colors; gene lists enriched in the putative GSC and DGC populations are highlighted in orange and in light blue, respectively. Signatures are available in Supplementary Table 1. GSEA calculated FDR adjusting for multiple comparisons; details of p-value and FDR calculation are described in the GSEA website (http://software.broadinstitute.org/gsea/index.jsp). Related to Fig. 1c. c, Log2 expression levels of the indicated oRG (top graphs), NSC and GSC (middle graphs) and INP markers (bottom graphs) in the subpopulations of neoplastic cells of the Darmanis dataset that are at the start (GSC, n = 221 cells) and at the end (DGC, n = 221 cells) of the pseudotime trajectory depicted in Fig. 1c. Data are presented as mean + s.d. p-values were determined by unpaired two-tailed t test. d, RNA velocities (arrows) of neoplastic cells of the Darmanis dataset projected in the space of the first two principal components. Red and blue dots are the cells that are at the start (GSC) and at the end (DGC) of the pseudotime trajectory depicted in Fig. 1c.

Source data

Extended Data Fig. 2 Validation of the G-STEM signature.

a-b, Violin plots showing the expression of the G-STEM signature (right panels in (b)) on the cells at the start (Low; red dots in the left panels in (b)) of the pseudotime trajectories (a) of patient-specific cohorts of the Darmanis dataset, vs. the neoplastic cells that are on the opposite ends of the same trajectories (High; blue dots in the left panels in (b)). The p-values were determined by two-tailed Mann-Whitney test. c, Violin plots showing the expression of the G-STEM signature (right panel) on the cells at the start (Low; red dots in the middle panel) of the pseudotime trajectory (left panel) of the sole neoplastic cells of the Darmanis dataset, vs. the cells that are on the opposite ends of the same trajectory (High; blue dots in the middle panel). The p-values were determined by two-tailed Mann-Whitney test.

Source data

Extended Data Fig. 3 Characterization of the G-STEM signature.

a, Graphs depicting the most significant GO terms emerging from the Gene Ontology analyses of the genes composing the G-STEM and the DGC signatures. The full lists of significant GO terms of both signatures are in Supplementary Table 3. b, Log2 expression levels of the indicated components of the G-STEM signature in the subpopulations of neoplastic cells of the Darmanis dataset that are at the start (GSC, n = 221 cells) and at the end (DGC, n = 221 cells) of the pseudotime trajectory depicted in Fig. 1c. Data are presented as mean + s.d. p-values were determined by unpaired two-tailed t test.

Source data

Extended Data Fig. 4 Validation of the G-STEM signature in large datasets of GBM patients.

a, Gene set enrichment analysis (GSEA) for association between the cell population at the start of the pseudotime trajectory of the neoplastic cells of the Neftel datasets (as depicted in Fig. 1e) vs. all the other neoplastic cells and gene sets denoting the identity of specific cell types. Abbreviations and color codes are as in Extended Data Fig. 1b. Signatures are available in Supplementary Table 1. GSEA calculated FDR adjusting for multiple comparisons; details of p-value and FDR calculation are described in the GSEA website (http://software.broadinstitute.org/gsea/index.jsp). Related to Fig. 1e. b, Violin plots showing the expression of the G-STEM signature (bottom panels) on the cells at the start of the pseudotime trajectory (GSC; red dots in the top panels) of small tumor cohorts of the Neftel dataset, pre-sorted according to the Proneural, Classical or Mesenchymal classification of GBMs, vs. all the other neoplastic cells of the same cohorts (NON GSC; light blue dots in the top panels) of the same dataset. The p-values were determined by two-tailed Mann-Whitney test. (c) Kaplan–Meier analysis representing the probability of survival in n = 541 GBM patients from the TCGA dataset (left panel), n = 210 GBM patients from the REMBRANDT dataset (middle panel), and n = 390 GBM patients carrying wild-type IDH1 from the TCGA dataset (right panel), stratified according to high or low GSC-signature. The p-value of the Log-rank (Mantel-Cox) test reflects the significance of the association between GSC-signature ‘low’ and longer survival. G-STEM expression is prognostic for the vast majority of GBM, that is IDH1-wild type tumors (93%, of those annotated in the TGCA dataset; n = 390 out of 419 IDH1-annotated samples).

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Extended Data Fig. 5 A computational procedure to identify candidate TRs controlling the gene expression program of GSCs.

a, Overview of the experimental flow for inference of the master Transcriptional Regulators (TRs) of the GSC state using the Rhabdomant pipeline on the Darmanis sc-RNA-seq dataset of primary GBM samples. See Methods for details. b, List of candidate master Transcriptional Regulators (TRs) emerging from the analysis of the Darmanis dataset of scRNA-seq dataset with the Rhabdomant pipeline, ordered on the base of their normalized enrichment signal (NES). The Rhabdomant pipeline calculated FDR adjusting for multiple comparisons; see Methods for details about p-value and FDR calculation. The lists of candidate master TRs of the GSC and of the DGC state are highlighted in orange and in light blue, respectively. The most significant candidate master TRs of the GSC state are indicated in red.

Extended Data Fig. 6 YAP/TAZ are required for GSC maintenance in vivo.

a-c, Effects of YAP/TAZ knockout on the growth of established subcuteaneous GBM-like lesions. Transformed cells were obtained by dissociation of gliomaspheres obtained from HER2CA- (a), shNF1/shp53- (b) or KRasG12V/shp53- (c) transformed R26CAGCreERT2; Yapfl/fl; Tazfl/fl newborn mouse astroglial cells (as in Fig. 3), and then injected in NOD-SCID mice. When subcutaneous tumors reached approximately 0.5 cm of diameter, mice were either fed with Tamoxifen food to induce YAP/TAZ knockout (YAP/TAZ KO), or maintained under normal diet (YAP/TAZ wt). Graphs are growth curves of YAP/TAZ wt (KRasG12V/shp53-, n = 4 mice; HER2CA, n = 6 mice; shNF1/shp53, n = 5 mice) and YAP/TAZ KO (KRasG12V/shp53-, n = 4 mice; HER2CA, n = 4; shNF1/shp53, n = 8 mice) tumors (average volume ± s.e.m.). d,e, Effects of YAP/TAZ knockout in tumors derived from KRasG12V/shp53 gliomaspheres, following the experimental setup described in a-c. d, Dot plot for tumor weight at sacrifice (YAP/TAZ wt, n = 8; YAP/TAZ KO, n = 6). Mean ± s.e.m. of the distribution are also shown. p-value was calculated by unpaired two-tail t-test. e, Representative H&E stainings. Scale bar, 2.5 mm. N, necrotic area; *, Matrigel residue. f, Tabular results showing the number of NOD/SCID mice displaying subcutaneous tumor formation after injection of cells dissociated either from gliomaspheres derived from HER2CA-transformed primary newborn astroglial cells (Primary tumors), or from HER2CA-gliomaspheres derived from one of the Primary tumors (Secondary tumors).

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Extended Data Fig. 7 Ex-vivo reprogramming of normal neural cells into GSC-like cells.

a, GFAP and SOX2 stainings (scale bars, 50 μm) of the mouse SVZ, representative of n = 3 mice. Nuclei were counterstained with DAPI. b,c, GFAP, NESTIN and SOX2 stainings (scale bars, 50 μm) in mouse newborn astroglial cells, representative of two independent experiments. d, Gliomaspheres emerging from newborn astroglial cell cultures transformed by the indicated oncogenes (P0 spheres) were dissociated to single cells and replated at clonal density for gliomasphere formation (P1 to P10 spheres). Results are representative of three experiments with n = 3 replicates each. Data are presented as scatter dot plots and bar graphs showing mean with s.d. e, Left panel: H&E staining of a lesion obtained after intracranial transplantation of shNf1/shp53-transformed astroglial cells. N, necrotic area. Scale bar, 2.5 mm. Middle panel: High magnification of the same tumor, showing large polynucleated cells (arrowheads). Right panel: TAZ IHC on the same tumor. Scale bars, 100 μm. Experiments were independently repeated on n = 10 mice, with similar results. f, H&E staining of subcutaneous tumors obtained by injecting cells dissociated from gliomaspheres carrying the indicated oncogenic lesions, representative of: KRasG12V/shp53, n = 4 tumors; HER2CA, n = 6 tumors; shNf1/shp53, n = 5 tumors. N, necrotic areas. Scale bars, 250 μm. g, Number of mice displaying tumor formation after injection of cells dissociated from KRasG12V/shp53-gliomaspheres at the indicated cell dilutions. h, Top, Schematic representation of the serial transplantation assay performed with HER2CA-transformed cells (see Methods for details). Bottom, H&E staining (scale bars, 2.5 mm) of tumors obtained after each round of transplantation, representative of n = 4 primary tumors, n = 8 secondary tumors and n = 4 tertiary tumors, respectively. Numbers of mice developing tumors per numbers of transplanted mice are indicated in each picture. i, GSEA curves of the G-STEM and the DGC signatures in KRasG12V/shp53-tumors compared to the astroglial cells from which they derive. Signatures are available in Supplementary Table 7.

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Extended Data Fig. 8 Oncogenic insults activate YAP/TAZ in transformed primary astroglial cells.

a, Bright-field and fluorescent pictures (representative of n = 5 independent samples each) of newborn astroglial cells transduced with lentiviral vectors encoding for the YAP/TAZ reporter 8xGTIIC-RFP-DD52, and with lentiviral vectors encoding for the indicated oncogenes or, as negative control, with empty vector, as in Fig. 3b. Images were taken 4 days after inducing oncogenic reprogramming by incubating cells in NSC medium. Scale bars, 50 μm. b, Compendium of Fig. 3c. Efficiency of Yap and Taz downregulation in R26CAG-CreERT2; Yapfl/fl; Tazfl/fl mouse newborn astroglial cells treated with either vehicle (Control) or 4OH-TAM (YAP/TAZ KO), as measured by qRT-PCR (mean + s.d. of all independent samples of three experiments). p-values are calculated by two-way ANOVA with Sidak’s multiple comparisons.

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Extended Data Fig. 9 YAP/TAZ are required for GSC maintenance in vitro.

a, Control experiment of Fig. 5a–e. Gliomaspheres derived from HER2CA-transformed Yapfl/fl; Tazfl/fl newborn astroglial cells, not expressing CREERT2, were treated with either ethanol (Vehicle) or 4OH-TAM (TAM). Panels are representative images (left; scale bar, 100 µm) and quantifications (right; mean ± s.d. of two independent experiments, each performed with two replicates) of the number of gliomaspheres/cm2 in vehicle versus 4OH-TAM-treated samples. p-values were determined by two-way ANOVA with Sidak’s multiple comparisons test. In the absence of CREERT2 expression, treatment with 4OH-TAM is inconsequential for gliomasphere formation, indicating that gliomasphere disaggregation shown in Fig. 4a–e is specifically caused by YAP/TAZ deletion. b, P2 gliomaspheres derived from R26CAG-CreERT2; Yapfl/fl; Tazfl/fl newborn astroglial cells transformed with the indicated oncogenes were dissociated to single cells and replated at clonal density for P3 gliomasphere formation in presence of ethanol (YAP/TAZ wt), or of 4OH-TAM to induce YAP/TAZ knockout (YAP/TAZ KO). Data are presented as scatter dot plots (n = 3 replicates each) and bar graphs showing mean with s.d. The p-values were calculated by unpaired two-tailed t-test.

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Extended Data Fig. 10 YAP/TAZ are required for GBM initiation in vivo.

a-c, Immunocompromised mice were injected intracranially with KRasG12V/shp53-transformed Yapfl/fl;Tazfl/fl cells, also transduced with dual luciferase-GFP expression vectors. Control animals (n = 6) were injected with cells transduced with Ad-GFP, whereas YAP/TAZ KO animals (n = 5) were injected with cells transduced with Ad-Cre. a, Representative images of brain bioluminescence. b, Bioluminescence quantification shown as scatter dot plots and bar graphs showing mean with s.d; p-value was calculated by unpaired two-tailed t-test. c, Representative H&E stainings. Scale bars, 2.5 mm in left panels and 250 μm in the magnification shown on the right. Arrowheads highlight the presence of large, polynucleated cells. d-f, Immunocompromised mice were injected intracranially with HuTu13 cells transduced with dual luciferase-GFP expression vectors, and transfected with siCo (Control; n = 5) or siYAP/TAZ (YAP/TAZ depleted; n = 5). d, Representative images of brain bioluminescence. e, Bioluminescence quantification shown as scatter dot plots and bar graphs showing mean with s.d.; unpaired two-tailed t-test p-values are shown. f, Representative H&E stainings. Scale bars, 2.5 mm in left panels and 250 μm in the magnification shown on the right. ‘N’ indicates necrosis. g-i, CT2A cells were transduced with dual luciferase-GFP expression vectors and injected intracranially in syngeneic mice. Control animals (n = 5) were injected with cells expressing anti-GFP shRNA, whereas YAP/TAZ-depleted animals (n = 5) were injected with cells expressing doxycycline-inducible YAP and TAZ shRNAs. g, Representative brain bioluminescences at one day and 14 days after injection. h, Bioluminescence quantification at three different time points shown as scatter dot plots and bar graphs showing mean with s.d.; unpaired two-tailed t-test p-values are shown. i, Representative H&E stainings. Scale bars, 2.5 mm in left panels and 250 μm in the magnification shown on the right. N, necrotic areas. j, GFP and TUJ1 stainings in sections from YAP/TAZ-wt and YAP/TAZ-KO subcutaneous shNF1/shp53-induced tumors (representative of n = 3 independent samples each). Scale bars, 50 µm.

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Supplementary information

Reporting Summary

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Castellan, M., Guarnieri, A., Fujimura, A. et al. Single-cell analyses reveal YAP/TAZ as regulators of stemness and cell plasticity in glioblastoma. Nat Cancer 2, 174–188 (2021). https://doi.org/10.1038/s43018-020-00150-z

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