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p120-catenin-dependent collective brain infiltration by glioma cell networks

Nature Cell Biology volume 22, pages 97–107 (2020)Cite this article

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Abstract

Diffuse brain infiltration by glioma cells causes detrimental disease progression, but its multicellular coordination is poorly understood. We show here that glioma cells infiltrate the brain collectively as multicellular networks. Contacts between moving glioma cells are adaptive epithelial-like or filamentous junctions stabilized by N-cadherin, β-catenin and p120-catenin, which undergo kinetic turnover, transmit intercellular calcium transients and mediate directional persistence. Downregulation of p120-catenin compromises cell–cell interaction and communication, disrupts collective networks, and both the cadherin and RhoA binding domains of p120-catenin are required for network formation and migration. Deregulating p120-catenin further prevents diffuse glioma cell infiltration of the mouse brain with marginalized microlesions as the outcome. Transcriptomics analysis has identified p120-catenin as an upstream regulator of neurogenesis and cell cycle pathways and a predictor of poor clinical outcome in glioma patients. Collective glioma networks infiltrating the brain thus depend on adherens junctions dynamics, the targeting of which may offer an unanticipated strategy to halt glioma progression.

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Fig. 1: Multicellular glioma cell networks and perivascular invasion in mouse brain.
Fig. 2: Collective glioma cell invasion in 3D assays and organization of cell–cell junctions during glioma network invasion.
Fig. 3: p120-dependent cell–cell cooperation and migration persistence of collective invasion of glioma cells.
Fig. 4: Multicellular network restitution after p120 downregulation and rescue with p120 domain mutants.
Fig. 5: p120 is indispensable for glioma growth, network formation and diffuse brain infiltration.
Fig. 6: Neuronal related pathways regulated by p120-catenin in glioma cells and clinical relevance in glioma patients.

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Data availability

RNA–seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE73999. RNA expression data that support the findings of this study are available in the cBioportal (TCGA) and the GEO data repositories. The human glioblastoma analysis was first performed on the TCGA dataset, available as ‘Tumor Glioblastoma - TCGA - 540 - MAS 5.0 - u133a’2 in the R2 database (R2.amc.nl), and source data are available at https://www.cbioportal.org/. Analysis was performed on human glioblastomas data of the French dataset (gse16011), available as ‘Tumor Glioma - French - 284–540 - MAS 5.0 - u133p2’46 in the R2 database [R2.amc.nl].

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Acknowledgements

We thank E. Wagena, M. Vullings, K. Lenting, K. Verrijp, J. Mooren and B. Lemmers-Van de Weem for expert technical support in animal experiments, image analyses and immunohistochemistry. We further thank P. Derksen for providing constructs and M. Zegers for critical reading of the manuscript. This work was supported by The Netherlands Organization for Scientific Research (NWO-VICI 918.11.626), Pieken in the Delta Oost Nederland, the European Research Council (ERC-CoG DEEPINSIGHT, 617430), the National Institutes of Health (U54 CA210184-01), the Cancer Genomics Center, The Netherlands (to P.F.) and NWO-VENI (863.13.019 to C.E.J.D., SFB1080 and SFB834 to A.A.-P.).

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Authors and Affiliations

  1. Department of Cell Biology, Radboud University Medical Center, Nijmegen, The Netherlands

    Pavlo G. Gritsenko, Cindy E. J. Dieteren, Jan-Hendrik Venhuizen, Cornelia Veelken & Peter Friedl

  2. Department of Molecular Biology, Radboud University, Nijmegen, The Netherlands

    Nader Atlasy & Hendrik G. Stunnenberg

  3. Center for Molecular Medicine, University Medical Center, Utrecht, The Netherlands

    Nader Atlasy

  4. Protinhi Therapeutics, Nijmegen, The Netherlands

    Cindy E. J. Dieteren

  5. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Anna C. Navis

  6. Cognitive Neuroscience Department, Donders Institute, Radboud University Medical Center, Nijmegen, The Netherlands

    Dirk Schubert

  7. Institute of Cell Biology and Neuroscience and BMLS, Goethe University Frankfurt, Frankfurt, Germany

    Amparo Acker-Palmer

  8. Max Planck Institute for Brain Research, Frankfurt, Germany

    Amparo Acker-Palmer

  9. Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands

    Bart A. Westerman & Thomas Wurdinger

  10. Department of Biochemistry, Radboud University Medical Center, Nijmegen, The Netherlands

    William Leenders

  11. Department of Pathology, Amsterdam University Medical Centers/VUmc and Brain Tumor Center Amsterdam, Amsterdam, The Netherlands

    Pieter Wesseling

  12. Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands

    Pieter Wesseling & Hendrik G. Stunnenberg

  13. The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Peter Friedl

  14. Cancer Genomics Center, Utrecht, The Netherlands

    Peter Friedl

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Contributions

P.G.G., N.A., C.E.J.D., W.L., P.W., A.A.-P., B.A.W., T.W., H.G.S. and P.F. designed the experiments and interpreted the data. P.G.G., N.A., C.E.J.D., A.C.N., J.-H.V., C.V., B.A.W. and D.S. performed experiments and analysed the data. P.G.G. and P.F. wrote the paper. All authors read and corrected the manuscript.

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Correspondence to Peter Friedl.

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Extended data

Extended Data Fig. 1 Identification of glioma cell networks and cohesive perivascular invasion zones in patient samples.

a-d, Morphological pattern and quantitative cell-cell junction analysis of glioma cell networks detected in 30 or 100 μm thick tissue slices. Samples included lower-grade astrocytoma with mutant IDH1 (LGA, grade II and III), glioblastoma with mutant (GBR132H) and wild-type IDH1 (GBwt). Detailed information in Supplementary Table 1. Images represent the peritumor region of the lesions. Identification of glioma cells in LGA and GBR132H via colocalization of IDH1R132H and nestin. In IDH1R132H negative glioblastoma, glioma cells are identified as nestin-positive but Myelin Basic Protein-(MBP) negative networks. Arrowheads in (a), filamentous protrusions between glioma cells. Similar results as in (a) were observed in brain samples from 8 (LGA) and 4 (GBwt) patients. Data represent 28–58 (b), 7–28 (c) or 14–34 (d) cells per sample from 6 (LGA), 1 (GBR132H) and 4 (GBwt) patients. e, Tumor-free human brain cortex and adjacent white matter region lacking IDH1R132H and interstitial nestin signal, but contain GFAP-positive astrocytes in the parenchyma. f, Representative large-field overview used for selecting region for high-resolution imaging. Nestin-positive but myelin basic protein-(MBP-) negative glioma cell network in GBwt sample intercalating with astrocyte (GFAP) and myelinated axonal (MBP) networks in tumor margin. g, Perivascular cohesive glioma cell layers aligning along blood vessel, identified by longitudinal or cross-sectioned lumen (V) in LGA sample. Inset, β-catenin-positive cell-cell junctions. h, Cell-cell proximity and number of directly neighboring cells during perivascular invasion (3D image analysis of LGA samples). Data represent 20 cells per sample from 2 LGA patients. i, Molecular topography of filamentous protrusions connecting glioma cells in patient-derived orthotopic E-468 xenograft. Identification of glioma cells using human-specific anti-nestin mAb. Arrowhead, nestin- and vimentin-positive filament, also containing microtubules. Similar results were observed in 3 mice. Scatter dot plots show the medians (red line) and values representing individual cells (b, c, h); in (d, h) values display relative fractions of connected glioma cells interacting with 0 up to 10 connected cells (boxes), median (red square), 25/75 percentiles (whiskers). Scale bars, 50 μm (a), 100 µm (e, f), 20 μm (g, i).

Source data

Extended Data Fig. 2 Subtype analysis and differential gene expression of E-98 and E-468 glioma cells.

a, Gene set enrichment analysis on non-targeting shRNA samples (from two independent RNA isolations per cell line) of E-98 (NT shRNA) and E-468 (NT shRNA) cell datasets using 3 glioblastoma subtype gene sets15. b, Hierarchical clustering and heatmap of mRNA expression of genes found to be deregulated after p120 knock-down (compare Extended Data Fig. 8). Data represent the gene expression in cells after lentiviral transduction with non-targeting shRNA (from two independent RNA isolations per cell line). c, Hierarchical clustering and heatmap of cadherin and protocadherin genes detected in at least one of the replicates. The Euclidean distance was used as metrics and the normalized expression level of genes used as input values and z-score values were used for visualizations.

Extended Data Fig. 3 Variability of cell-cell interactions and functional coupling of glioma cells during migration.

a, Quantification of next-neighbor proximity, migration speed and cell-cell interactions in E-98 glioma cell invasion in reconstituted basement membrane (rBM) assay after 48 h of invasion culture. Data represent 272 cells (left panel), 8 spheroids (middle panel) or 73 cells (right panel) from two representative from 3 independent experiments. Scatter dot plots show the medians (red line) and values representing individual cells (left plot) or spheroids (middle plot); in the right graph values display relative fractions of connected glioma cells interacting with 0 up to 10 connected cells (boxes), median (red square), 25/75 percentiles (whiskers). b, Cell pattern of E-98 glioma invasion (48 h) from multicellular spheroid (Sph) into 3D astrocyte scaffolds. Arrowheads indicate filamentous cell contacts. Similar results were observed in 3 independent experiments. c, Functional cell-cell coupling in E-98 and E-468 cells measured as intracellular calcium transients and inhibition of multicellular calcium transients in E-98 cells in the presence of carbenoxolone (CBX, 50 µM). Cell identification for ratiometric time-series (left panels) and derived calcium transients in neighboring cells (right panel). Dots, cells assessed for calcium transients displayed in the graphs. Sph, spheroid. Similar results were observed in 3 independent experiments. d, Near-instantaneous and sequential calcium transients in E-98 and E-468 cell clusters and networks. Example micrograph highlighting neighboring cells participating in calcium wave (left panel) and fields of connected cells recorded over 2 min (right image). e, Calcium waves and propagation speed (in brackets). The propagation speed in E-98 is consistent with the velocity of calcium waves between astrocytes (approx. 6 μm/s)33. Similar results were observed in 3 independent experiments. f, The number of cells in synchronized clusters of E-98 and E-468 cells. Data represent 22 (E-98), 11 (E-98, CBX) and 18 (E-468) cell clusters from 3 independent experiments. Scatter dot plot shows the medians (red line) and values representing individual cell clusters (dots). P values were obtained using two-tailed Mann-Whitney test. Scale bars, 50 µm.

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Extended Data Fig. 4 Expression and subcellular distribution of p120 and other cell-cell adhesion molecules in human glioma cells.

a, Surface expression of receptors implicated in cell-cell interactions in E-98 cells (FACS data). Similar results were observed in 2 independent experiments. b, Increased expression of p120-catenin in human gliomas of different grade. Data are derived from French dataset (MAS5.0-u133p2) comprising grade I-IV gliomas (n = 284 patients)46. Expression reflects log level. Box plots display the median (black line), 25/75 percentiles (boxes) and maximum/minimum (whiskers) from 8 (normal brain), 8 (grade I), 13 (grade II), 16 (grade III) and 159 (grade IV) glioma patients. c, p120 (brown label in large panels) and IDH1R132H (insets) immunohistochemistry in glioblastomas and low grade astrocytoma (LGA) samples. P120 positivity in both tumor core and diffuse brain infiltration region. IDH1R132H positivity in grade III glioma sample was used to confirm glioma cell origin. Similar results were observed in 2 independent immunohistochemistry staining of samples from 2 (LGA), 1 (GBR132H) and 2 (GBwt) patients. Scale bars, 500 µm (overviews) and 20 µm (details).

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Extended Data Fig. 5 Molecular targeting of glioma cell-cell interaction and collective invasion in vitro.

a-d, Transient downregulation of candidate proteins involved in cell-cell junction stability of E-98 cells. (a) Relative protein levels detected by Western blot (total proteins) or flow cytometry (surface receptors) after transient downregulation using SMARTpool siRNA. Similar results were observed in 2 independent siRNA transfections. Unmodified scan of the Western-blot is provided in Source data. (b) Numbers in the table express the protein ratio between p120 and non-targeting (NT) siRNA conditions. (c) Morphology and (d) quantification of single-cell fraction during radial migration of E-98 glioma cells from spheroids on rBM-coated surface after single- or triple interference with cell-cell junction proteins. Similar results were observed in 3 independent siRNA transfections. Data in (d) represent 9–15 spheroids per condition from 3 independent siRNA transfections. Scatter dot plots show individual spheroids and medians (red line). e-l, Quality control and functional consequences of stable downregulation of p120 in glioma cells. e-g, Extent of downregulation of p120 protein (e, f) and p120 mRNA (f) and persistence of vimentin expression in E-468 cells (g). Similar results were observed in 2 (e, f) or 3 (g) independent shRNA lentiviral transductions. Unmodified scan of the Western-blot (e) is provided in Source data. Immunostaining of human vimentin was used for the detection of glioma cells in the mouse brain. h-j, Effect of four independent shRNA sequences on p120-catenin protein levels detected by immunofluorescence (h), the number of cell-contacts between neighboring cells (i) and cell density as measure for growth (j). Data represent 313–2353 (h), 196–228 cells (i) and 669–3153 (j) cells per shRNA sequence per cell line from 3 independent shRNA lentiviral transductions. Values in (h) show the means. Values in (j) represent the means of cell number per area (650×486 μm). Box plots in (i, j) display the median (black line), 25/75 percentiles (boxes) and maximum/minimum (whiskers). P values, two-tailed Mann-Whitney test. Based on efficacy of p120 downregulation and representativity, sequence TRCN0000122988 was used for further in vitro and in vivo experiments. k, Cells in S/G2/M phase detected by geminin staining >30 (E-98) or 8 days (E-468) after transduction with p120 shRNA. Data represent 238–475 cells in 5–6 areas (650×486 μm) per condition in one representative of 3 independent shRNA transductions. P values, two-tailed Mann-Whitney test. In contrast to E-98 cells which grew well in liquid culture before p120 downregulation, E-468 cells were derived directly from mouse brains and failed to grow in vitro. l, Reduced growth of E-98 cells in neurobasal spheroids after p120 downregulation. Data represent 18 (NT shRNA) and 53 (p120 shRNA) spheroids from one representative of 3 independent shRNA transductions. P values, two-tailed Mann-Whitney test. m, n, Myosin light-chain phosphorylation (ROCK target) after p120 downregulation in E-468 cells in the absence or presence of ROCK inhibitor Y-27632 (10 µM) detected by cell-based immunofluorescence. Data represent 84–171 cells from one representative of 3 independent shRNA transductions. P values, two-tailed Mann-Whitney test. Scatter dot plots show the medians (red line) and values representing individual spheroids (d, l) or areas covered by cells per field (650 × 486 μm) in 96 well plates (k, n). Scale bars, 100 µm (c, g, k, m), 200 µm (l).

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Extended Data Fig. 6 Myosin light-chain 2 phosphorylation and β-catenin expression in E-468 cells and radial invasion from spheroids after p120 downregulation and reexpression of p120 mutants.

a-d, Myosin light-chain 2 (Ser 19) phosphorylation (a, b) and β-catenin expression (c, d) during E-468 cell culture on rBM after transduction with non-targeting shRNA, human p120 shRNA, or human p120 shRNA with additional transduction with mouse p120-eGFP constructs (indicated in Fig. 4c) encoding wild-type p120-catenin, p120 with K401M substitution and p120 with 622–628 deletion. Similar results as in (a, c) were observed in 3 independent shRNA lentiviral transductions. Mean intensity of phospho-Ser19-MLC2 (b) or β-catenin (d) immunofluorescence in p120 shRNA cells after re-expression of p120-catenin constructs. Data in (b, d) represent the fluorescence intensity per imaging field (650×486 μm) covered by multiple cells from 20–34 (b) or 26–33 (d) fields per condition from 3 independent lentiviral transductions. Scatter dot plots in (b, d) show the normalized fluorescence intensity per area (650 × 486 μm) and medians (red line). P values, one-way ANOVA Dunnett’s multiple comparison test. ***, P < 0.0001. e, Overviews of migration and intercellular organization of E-468 cells invading from multicellular spheroids along 3D rBM interface after 3-day culture (described in Fig. 2a). Similar results were observed in 2 independent shRNA transfections. E-468 cells were transduced with non-targeting or human p120 shRNA and additionally mouse p120-eGFP constructs encoding wild-type p120-catenin, p120 with K401M substitution and p120 with 622–628 deletion. Arrowheads indicate filamentous cell-cell contacts. Scale bars, 20 µm (a, c); 200 µm (e).

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Extended Data Fig. 7 Inhibition of multicellular networks and diffuse infiltration into mouse brain after p120 downregulation.

a, Impaired tumor growth, loss of network formation and diffuse brain infiltration of E-98 cells after p120 downregulation. Overviews (upper panels) and zoom images (lower panels) from 200 μm thick brain slices. Arrowheads, filaments in multicellular networks of human vimentin-positive E-98 cells diffusely infiltrating the brain. Data represent 9 (NT shRNA) or 10 (p120 shRNA) mice from 2 independent experimental series (a1) and 49 cells (NT shRNA) or 89 (p120 shRNA) cells from two mice per condition (a2). P values, two-tailed Mann-Whitney test. b, Persistent deficiency of p120 signal in vivo in p120 shRNA knock-down in E-98 cells 4 weeks after intracranial tumor implantation. Similar results were observed in two mice per condition. c, Sustained glioma growth deficit in vivo after p120 downregulation. Frequency of geminin-positive glioma cells after the indicated time of post-implantation in mouse brain. Data represent 42–201 (E-98) and 108–574 (E-478) cells from 4–5 (E-98) or 5–21 (E-468) imaging fields from two mice per condition. P values, two-tailed Mann-Whitney test. Scatter dot plots show the medians (red line) and values representing individual mice (a1), individual cells (a2) or imaged areas in brain slices (c). Scale bars, 2 mm (a, upper panels), 50 μm (a, lower panels; b, c).

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Extended Data Fig. 8 Gene expression regulation in glioma cells in response to downregulation of p120 and clinical relevance of p120-catenin in glioma patients.

a. Proportional Venn diagrams of total number of down- (n = 1,148 and n = 1,242) or upregulated (n = 785 and n = 403) genes in E-98 and E-468 cells respectively after p120 downregulation (p values were obtained using hypergeometric test). b, Ranked list heatmap of all relevant GO term biological functions resulting from gene ontology analysis of deregulated genes in each p120 knock-down cell line. Absolute Log2 transformed Benjamini-Hochberg FDR values per each GO term were used as input in which the upregulated terms were converted to a positive and the downregulated terms to a negative number in the color scale scoring. The annotation was carried out using the DAVID server and GO database and the FDR < = 10–3 was determined as cutoff for selecting the relevance of the GO terms. To determine the significance of deregulation, log2-fold change of  > 1.5 for upregulation and < −1.5 for downregulation and two-sided test p value < 0.05 and FDR < 0.05 were used as cut offs. The gene ontology analysis was performed based on data obtained from two independent RNA isolations per condition (n = 2). c, Gene enrichment analysis of the p120 signature in human glioma. Expression levels of p120 correlate to migration/ECM gene signatures. Ranked expression panels of p120 RNA expression as determined by MAS5.0 normalized u133a or u133p2 RNA expression arrays (Verhaak, TCGA 540 tumors, ID2000-01-01)45 and low- to high-grade glioma (French 284 samples including normal controls, GEO ID: gse16011)46, correlated to z-values of a migration signature;66 n = 540 and n = 284 patients, respectively (http://www.broadinstitute.org/gsea/msigdb/cards/WU_CELL_MIGRATION.html), and an ECM signature (Segal Stanford cancer modules, http://ai.stanford.edu/~erans/cancer/modules/module_47.html). d. Kaplan Meier survival curves of p120 mRNA expression within glioblastoma subtypes. Trends show decreased survival in the p120high subgroup, but do not reach statistical significance due to low case numbers (proneural: n = 24; mesenchymal: n = 27; classical: n = 17). P-values, two-tailed Mann Whitney test (raw, non-Bonferroni-corrected). e, Expression of p120 mRNA in the three subtypes of glioblastoma, as defined by Verhaak et al45 (TCGA dataset, MAS5.0 - u133a, 540 patients of which n = 68 subtype classified patients are shown). The time to progressive disease is indicated as a heatmap value for each patient (red indicates immediate progressive disease; blue indicates delayed progressive disease). This shows that p120 is highly expressed in the classical and mesenchymal subgroups which have a rapid progressive disease, compared to the proneural subtype. P-value, ANOVA-test (one-way analysis of variance). f, Types of collective glioma cell invasion in response to different microanatomy of brain subregions. Perivascular cohesive strands move through aligned confined space forming epithelial-like AJs whereas multicellular networks with neuronal-like filaments support intercellular connections during interstitial invasion of brain stroma. Black arrows, kinetics of cell-cell interactions. Blue arrows, direction of migration.

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Supplementary Tables

Supplementary Table 1. Patient samples used for 3D reconstruction of glioma networks and perivascular invasion. Supplementary Table 2. GO biological function terms of up- and downregulated genes for E-98 and E-468 cell lines. The significance of the GO terms were calculated using one-tail Fisher exact probability test implemented in the DAVID online database. The GO terms are ordered based on Benjamini FDR values. The gene ontology analysis was performed based on data obtained from two independent RNA isolations per condition (n = 2).

Supplementary Video 1

3D reconstruction of glioma cell network from low-grade astrocytoma (LGA) human sample (40 µm z-stack, 2 µm step size). Nine representative glioma cells positive for IDH1 R132H mutation and nestin were selected as examples for an approach to network quantification. Arrowheads indicate filament connections between the cells (coloured lines), as quantified for Extended Data Fig. 1. Similar results were observed in the brain samples from eight low-grade IDH1R132H gliomas (Supplementary Table 1).

Supplementary Video 2

3D reconstruction of E-98 xenograft in the peritumoral region of mouse brain tissue reflecting the invasion zone (100 µm z-stack, 2 µm step interval). Identification of glioma cells via human nestin staining. Arrowheads show filaments connecting glioma cells in a network-like manner. Related to Fig. 1a. Similar results were observed in three mice intracranially implanted with E-98 cells.

Supplementary Video 3

3D reconstruction of E-468 xenograft in the peritumoral region of mouse brain tissue reflecting the cell network in the invasion zone (100 µm z-stack, 2 µm step interval). Related to Fig. 1a. Identification of glioma cells via human nestin staining. Similar results were observed in three mice intracranially implanted with E-468 cells.

Supplementary Video 4

High-resolution 3D rotation of filamentous contacts between E-468 glioma cells in mouse brain tissue (45 µm z-stack, 2 µm step). Connecting filaments are positive for nestin (red; human-specific antibody), vimentin (green) and tubulin (blue). Cell nuclei, cyan (DAPI). Related to Fig. 1a and Extended Data Fig. 1i. Similar results were observed in three mice intracranially implanted with E-468 cells.

Supplementary Video 5

Invasion of E-468 cells (LifeAct/eGFP) in 3D astrocyte scaffolds (left) and detail showing actin enrichment and transient stability of filamentous junctions. Migration tracks (red lines) are overlaid for three representative cells. Related to Fig. 2i-l. Similar results were observed in three independent experiments.

Supplementary Video 6

Example for intercellular calcium wave propagation in different multicellular fields of E-98 spheroid invasion area visualized as Fura-2 340/380 nm ratio. Left: raw data. Right: pseudocoloured ratio with masked cell areas. Related to Extended Data Fig. 3c-f. Similar results were observed in three independent experiments.

Supplementary Video 7

Example for intercellular calcium wave propagation in different multicellular fields of E-468 spheroid invasion area visualized as Fura-2 340/380 nm ratio. Left: raw data. Right: pseudocoloured ratio with masked cell areas. Related to Extended Data Fig. 3c-f. Similar results were observed in three independent experiments.

Supplementary Video 8

Inhibition of intercellular calcium wave propagation in E-98 spheroid invasion area by CBX. Left: raw Fura-2 340/380 nm ratio images. Right: pseudocoloured ratio with masked cell areas. Related to Extended Data Fig. 3c,f. Similar results were observed in three independent experiments.

Supplementary Video 9

Inhibition of intercellular calcium wave propagation in E-98 and E-468 spheroid invasion area after p120-catenin downregulation. Related to Fig. 3b. Similar results were observed in two independent experiments.

Supplementary Video 10

Invasion of E-98 and E-468 (LifeAct/eGFP) in 3D astrocyte scaffolds (15 µm z-stack, 5 µm step interval). Three different z-sections are depth-encoded by colour from red (lower) to green (middle) and blue (upper). After p120-catenin downregulation, cells lose cell–cell junctions and switch to a rounded morphology with poor directional persistence and ability to migrate away from the origin. Related to Fig. 3d. Similar results were observed in three independent experiments.

Supplementary Video 11

3D whole-brain reconstruction by serial vibratome sectioning to quantify diffuse brain infiltration in E-468 glioma xenografts expressing NT or p120-catenin targeting shRNA. The brain fraction infiltrated by glioma cells was obtained by dividing the pixel sum occupied by human vimentin-positive events (false-colour green/yellow, glioma cells) divided by the total brain area from all 200 μm-thick slices. Related to Fig. 5b,c. Similar results were observed in eight (NT shRNA) and eight (p120 shRNA) mice from two independent implantation series.

Supplementary Video 12

3D confocal microscopy of E-468 xenografts in mouse brain tissue to identify the cell network in the invasion zone in human vimentin-positive control lesions expressing NT shRNA and reduced network formation in residual microlesions after downregulation of p120-catenin (100 µm z-stack, 3 µm step). Related to Fig. 5d,f. Similar results were observed in eight (NT shRNA) and eight (p120 shRNA) mice from two independent implantation series.

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Gritsenko, P.G., Atlasy, N., Dieteren, C.E.J. et al. p120-catenin-dependent collective brain infiltration by glioma cell networks. Nat Cell Biol 22, 97–107 (2020). https://doi.org/10.1038/s41556-019-0443-x

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