Diffuse midline glioma invasion and metastasis rely on cell-autonomous signaling

Abstract

Background: Diffuse midline gliomas (DMG) are pediatric tumors with negligible 2-year survival after diagnosis characterized by their ability to infiltrate the central nervous system. In the hope of controlling the local growth and slowing the disease, all patients receive radiotherapy. However, distant progression occurs frequently in DMG patients. Current clues as to what causes tumor infiltration circle mainly around the tumor microenvironment, but there are currently no known determinants to predict the degree of invasiveness.

Methods: In this study, we use patient-derived glioma stem cells (GSCs) to create patient-specific 3D avatars to model interindividual invasion and elucidate the cellular supporting mechanisms.

Results: We show that GSC models in 3D mirror the invasive behavior of the parental tumors, thus proving the ability of DMG to infiltrate as an autonomous characteristic of tumor cells. Furthermore, we distinguished 2 modes of migration, mesenchymal and ameboid-like, and associated the ameboid-like modality with GSCs derived from the most invasive tumors. Using transcriptomics of both organoids and primary tumors, we further characterized the invasive ameboid-like tumors as oligodendrocyte progenitor-like, with highly contractile cytoskeleton and reduced adhesion ability driven by crucial over-expression of bone morphogenetic pathway 7 (BMP7). Finally, we deciphered MEK, ERK, and Rho/ROCK kinases activated downstream of the BMP7 stimulation as actionable targets controlling tumor cell motility.

Conclusions: Our findings identify 2 new therapeutic avenues. First, patient-derived GSCs represent a predictive tool for patient stratification in order to adapt irradiation strategies. Second, autocrine and short-range BMP7-related signaling becomes a druggable target to prevent DMG spread and metastasis.

Keywords: DMG-H3K27M; GSC; invasion; metastasis; tumor organoids.

Graphical Abstract

Figure 1.

Variable tumor invasion extent correlates with survival and can be recapitulated by glioma stem cell (GSC) models in 3D. (A) Sagittal magnetic resonance images of 3 representative Diffuse midline gliomas (DMG) patients collected at diagnosis (top panels) and progression (bottom panels) displaying from left to right local, extensive locoregional (arrowheads indicate contiguous locoregional infiltration at relapse) and distant relapse (arrowheads indicate metastatic foci); initial pontine localization at diagnosis is pointed by arrowheads. (B) Kaplan–Meier curves of the overall survival (in months) of patients with local-to-locoregional (n = 53) and metastatic (n = 17) progression, according to the radiological assessment at first relapse; 2 patients were excluded as they died without relapse. P-value as by log-rank Mantel-Cox. (C) Overall survival in a cohort of 18 DMG patients with local-to-locoregional tumor evolution, according to the radiological invasion rate, assessed from 2 consecutive MRIs (P-value as by 2-tailed Pearson test). (D) Oncoplot displaying the mutational landscape for 7 recurrently mutated genes detected by whole-exome sequencing or targeted Sanger sequencing in tumor DNA in a cohort of 72 DMG collected at diagnosis (see methods and Supplementary Figure S6 for details on inclusion); white indicates data unavailable; LC, LR and MET indicate patients with subsequent local, locoregional or anytime-metastatic evolutions, respectively. (E) Representative phase-contrast images of invading gliomaspheres in Matrigel at 0-, 24-, and 40 hours post-embedding (scale bar = 600 μm). (F) Gliomasphere invasion ability was relative to the initial sphere area at 24 (light orange) and 40 hours (dark orange) after embedding. Results are ranked according to the average ± s.e.m. of at least 5 technical replicates per cell model. (G) Oncoplot of gliomaspheres displaying the mutational status obtained from whole-exon sequencing or targeted Sanger sequencing for 7 genes recurrently mutated in DMG. Metastatic progression is represented in red, locoregional progression in black, and local progression in light gray. (H) Comparison of relative gliomasphere area at 40 hours after embedding for models derived from patients with local/locoregional (n = 13) or distant metastatic progression (n = 9); average ± s.e.m is shown for both groups; P-value as by 2-tailed unpaired t-test. (I) ROC analysis of the invasion score measured at 40 hours after embedding in the panel of 22 models. The area under the curve is displayed as a measure of the performance of the assay to predict metastatic evolution (max = 1).

Figure 2.

Transcriptomic analysis reveals 2 types of diffuse midline glioma. (A) Representative brightfield and inset images of tumor organoids 10 days after embedding in Matrigel (scale bar = 600μm). (B) Unsupervised hierarchical clustering based on the expression of the 1000 most variable genes, displaying the existence of 2 distinct clusters of Diffuse midline gliomas (DMG) organoids, corresponding to moderately (5 C1 branches) or highly invasive (17 C2 branches) GSC-models; top bars represent the individual relative invasion score 40 hours post-embedding. (C). Relative invasion of glioma stem cells (GSCs) from C1 and C2 clusters measured at 40 hours; results represent average value for each model; P-value as by 2-tailed unpaired t-test. (D) Magnifications of the heatmap (indicated by * on the general heatmap) highlight the expression patterns of specific markers of oligodendrocyte progenitor cell specification and epithelial-to-mesenchymal transition. (E) Immunoblot on crude protein extracts of the 4 most invasive (red) and 4 least-invasive (blue) GSC models, displaying the detection of hOPC- and mesenchymal protein markers. (F) Gene set enrichment analysis analyses based on the comparison between the 2 molecular clusters of DMG organoids (top plots) or between available profiles of parental biopsies (n = 17) from individuals with non-metastatic versus metastatic progression (bottom), suggesting increased oligodendrocyte progenitor cell identity and reduced epithelial-to-mesenchymal transition in invasive 3D-GSC and metastatic samples. Normalized Enrichment Score and Benjamini-Hochberg adjusted P-value are shown on the plots; gene sets as by Hallmark and GO-related catalogues.

Figure 3.

Moderately and highly invasive diffuse midline gliomas cells have distinct actin cytoskeletons and adhesive properties. (A) Gene set enrichment analysis based on the comparison between the 2 molecular clusters of diffuse midline gliomas organoids (top plots) or between available profiles of parental biopsies (n = 17, bottom) from individuals with non-metastatic versus metastatic progression, showing reduced expression of focal adhesion components, extracellular matrix remodeling, and pro-angiogenic signals in invasive 3D-GSC and metastatic tumors. Normalized Enrichment Score and Benjamini–Hochberg adjusted P-values are shown on the plots; gene sets as by Hallmark and GO-related catalogs. (B) The indicated highly- and less invasive glioma stem cells (GSCs) were seeded on laminin-coated glass and then fixed and stained for vinculin (top panels) and F-actin (Phalloidine, bottom panels). Insets show higher magnification of boxed areas. Scale bar: 10 µm. (C) Quantification of moderately (in blue, n = 62 GSC-335, n = 37 GSC-375) and highly invasive GSC (in red, n = 50 GSC-290, n = 63 GSC-375) spread on glass substrate. (D) The indicated GSCs seeded on laminin-coated glass-bottom dishes were incubated for 30 minutes with Sir-Actin before being imaged for 45 minutes with a spinning disk microscope. Galleries of still pictures acquired every 5 minutes are shown. Arrowheads point to actin accumulation at the rear of migrating cells. Scale bar: 10 µm. (E) The indicated GSC models as in (D) were seeded on a laminin-coated glass-bottom dish before they were imaged every 10 minutes for 16 hours. Cell velocity was measured by manual tracking using the Chemotaxis tool of ImageJ; results represent 2 independent experiments with quantification of at least 25 cells per condition.

Figure 4.

Bone morphogenetic pathway 7 (BMP7) expression stimulates invasion in diffuse midline glioma. (A) Scatter plot highlighting differentially expressed genes (adj-P < 0.01) between the 2 invasive clusters and those presenting elevated Pearson correlation (r > 0.55) with continuous-trait invasive ability; overlap between the 2 is presented as in the color legend. (B) Correlation between BMP7 RNAseq expression (transcripts per million reads on x-axis) and relative invasion score measured at 40 hours in GSCs; r and P-value as by 2-tailed Pearson correlation. (C) Two-dimensional representations of the OC-like versus AC-like (x-axis) and OPC-like (y-axis) scores for H3-K27M diffuse midline gliomas cells colored by the different tumor metaprograms (left), or colored according to BMP7 normalized expression levels as indicated by the color scale (right). This indicates that BMP7 expression is restricted to the OPC-like metaprogram and to a lesser extent the AC-like program. (D) Violin and dotplot representing the normalized and scaled expression levels of BMP7 across the different tumor cell metaprograms, showing OPC-like-1 as the prominent cell population expressing BMP7 (E, G) Live cell microphotographs displaying the invasion of cells after transduction of 2 non-targeting control shRNAs (Renilla, LIN28) and 2 BMP7-specific shRNAs in highly invasive GSC-290 and GSC-293 models; scale bars = 800 μm. (F, H) Quantification of the relative size of gliomaspheres relative to the initial size at embedding over 48 hours; gliomaspheres of 20 000 cells expressing control (Renilla-, Lin28-) or BMP7-shRNA; bars represent the average ± s.e.m.; P-value as by 2-way ANOVA multiple comparison test. (I) Schematic strategy and (J) results of transwell migration assay performed on highly invasive GSC-290 and moderately invasive GSC-375 patient-derived models in the presence of recombinant BMP7 (100 ng/mL added in the top reservoir) and/or Alk/BMPR inhibitor LDN-193189 (650 nM). Migration is displayed as increasing cell confluence in the bottom compartment over time relative to the initial top confluence. Representative results from 1 out of 2 independent experiments; results represent the average ± s.e.m. of at least 5 technical replicates per condition; p-value as by 2-way ANOVA multiple comparison test.

Figure 5.

Bone morphogenetic pathway 7 (BMP7) regulates a mesenchymal-to-ameboid phenotypic switch. (A) Heatmaps representing the ranked expression of BMP7 (left) by the 22 3D-GSC models compared to the normalized enrichment in the expression of epithelial-to-mesenchymal transition signature as by single sample gene set enrichment analysis (see methods); scatter plot showing inverse correlation between these 2 traits is represented in the right panel; r and P as by 2-tailed Pearson correlation. (B) Moderately invasive GSC-375 cells were seeded on laminin-coated glass coverslips and treated (or not) with 100 ng/mL recombinant BMP7 for 24 hours, as indicated, before being fixed and stained for vinculin. Insets show higher magnification of boxed areas. Scale bar: 10 µm. (C) Quantification of the length of cells’ long axis cells as in (B). Data are expressed as maximal length in µm of individual cells from 2 independent experiments. (D) Cells as in (B) were seeded on a laminin-coated glass-bottom dish before being imaged every 10 minutes for 16 hours. Cell velocity was measured by manually tracking 60–100 cells per condition over 16 hours using the Chemotaxis tool of ImageJ. (E) shBMP7-expressing highly invasive GSC-290 cells were seeded on laminin-coated glass coverslips and treated (or not) with 100 ng/mL recombinant BMP7 for 24 hours, as indicated, before being fixed and stained for vinculin. Insets show higher magnification of boxed areas. Scale bar: 10 µm. (F), Quantification of the length of cells’ longest axis in cells as in (E). Data are expressed as the average of maximal length in µm of individual cells from 2 independent experiments.

Figure 6.

Bone morphogenetic pathway 7 (BMP7) activates a kinase-mediated signaling promoting GSC invasion. (A) Immunoblots performed on GSC_290 (left) and GSC-275 cells (right), after 2 hours of incubation either in medium without any growth factor (starved) or in complete GSC medium without BMP7, or in complete medium with 100 ng/mL recombinant BMP7 for 0, 15, 30, 60, and 120 minutes before harvesting the cells; P = phosphorylated, T = total protein. (B) schematic representation of the signaling cascade activated by secreted BMP7. Interaction of BMP7 with cognate Alk receptors induces the sequential phosphorylation and activation of canonical SMADs, the PI3K/AKT axis, or MAP-kinases MEK and ERK to promote the activity of Rho-GTPase; MAPK activations associated with pro-invasive phenotypic switch and increased cell motility mediated by the actin-mediated propulsion. (C) Representative video-microscopy images 40 hours post-embedding of GSC-290 gliospheres treated with different amounts of LDN-193189, E0126, FR180204, and Y-27632 at embedding; scale bars = 800 μm. (D) Quantification of relative invasion of the different conditions in representative experiments. GSC medium with DMSO at corresponding concentration was used as a control.