Cell Lineage-Based Stratification for Glioblastoma

Abstract

Glioblastoma, the predominant adult malignant brain tumor, has been computationally classified into molecular subtypes whose functional relevance remains to be comprehensively established. Tumors from genetically engineered glioblastoma mouse models initiated by identical driver mutations in distinct cells of origin portray unique transcriptional profiles reflective of their respective lineage. Here, we identify corresponding transcriptional profiles in human glioblastoma and describe patient-derived xenografts with species-conserved subtype-discriminating functional properties. The oligodendrocyte lineage-associated glioblastoma subtype requires functional ERBB3 and harbors unique therapeutic sensitivities. These results highlight the importance of cell lineage in glioblastoma independent of driver mutations and provide a methodology for functional glioblastoma classification for future clinical investigations.

Keywords: Erbb3; GBM; PDX; cell of origin; glioblastoma; molecular classification; molecular subtype; mouse model; neural stem cell; oligodendrocyte lineage cell.

Figure 1.. Nf1, Trp53, and Pten inactivation in adult NSCs versus OLCs generates distinct GBM subtypes.

(A) Nestin-CreER^T2;Rosa-lsl-Yfp and NG2-CreER™;Rosa-lsl-tdTom transgenes were used to drive reporter expression in SVZ NSCs and OLCs, respectively after tamoxifen induction at 1 month of age and analysis 1 week later. YFP expression was revealed by anti-GFP staining while TOMATO expression was endogenous. Scale bars, 1000 μM. (B) Representative images of Type 1 and 2 GBM analyzed by hematoxylin and eosin (H&E) staining. Regions marked by the white box (left, scale bars, 1000 μM) are magnified (right, scale bars, 50 μM). (C) Volcano plot illustrating DEGs (green dots) in Type 1 and 2 GBM (n=7 each for Type 1 and Type 2 GBM; adjusted p<0.1; 2-fold change). (D) Western blot analysis in Type 1 (n=7) and Type 2 (n=7) primary GBM tissue samples. (E) Representative immunofluorescence images in Type 1 and 2 tumors. Scale bars, 50 μM. In A-B and E, results are representative of n≥3 biological replicates. See also Figure S1 and Table S1.

Figure 2.. Type 1 and Type 2 GBM cells exhibit conserved molecular and histological phenotypes as primary cultures and following intracranial transplantation.

(A) Western blot analysis in Type 1 (n=5) and Type 2 (n=6) mouse GBM primary cultures. (B) Representative H&E staining for tumors generated by intracranial transplantation of Type 1 (n=3) and 2 (n=3) cultures into nude mice. Scale bars: 1000 μM (left); 100 μM (right). (C) Representative western blot analysis in transplanted Type 1 and 2 tumors. (D-E) Growth factor dose response for EGF (D) and NRG1 (E) (concentrations from 0–40 ng/ml) of Type 1 and Type 2 cells measured by ATP assays (CellTiter-Glo®; n=3 replicates). Data are presented as mean ± standard deviation (S.D.). See also Figure S2.

Figure 3.. Type I & Type II classification of human GBM cases.

(A) Work flow showing stepwise identification of human Type I and Type II GBM core samples. Transcriptional data from 498 IDH1 wild-type TCGA GBM were analyzed (z-score threshold of 0.5) to identify Type I and Type II candidates. GSVA using human homologues of mouse Type 1 versus Type 2 GBM DEG was applied. (B) Hierarchical clustering analysis dendrogram of human and mouse GBM profiles using mouse Type 1 and 2 GBM DEG. (C) Multi-dimensional scaling (MDS) plot using global methylation status of TCGA Type I (n=7) and Type II (n=9) GBM core samples on HM450K platform with 1000 most variable probes. (D) Gene expression heatmap of Type I and Type II GBM signature genes, TIsig and TIIsig, in TCGA core (n=52), extended (n=87), and Rembrandt (n=28) Type I, as well as TCGA core (n=38), extended (n=54) and Rembrandt (n=26) Type II samples. (E) Progression free survival of available data from Type I (n=139), Type II (n=92) and remaining non-Type I&II (n=267) TCGA GBM patients (Type I vs. Type II, p=0.011, likelihood ratio test). See also Figure S3 and Tables S2 and S3.

Figure 4.. Human Type I and II GBM signature genes and mutations.

(A) mRNA expression (shown as log fold change) of representative lineage markers that are differentially expressed in human Type I vs. Type II GBM in TCGA core samples (U133 microarray data), mouse Type 1 vs. Type 2 GBM (microarray data), and NSCs vs. OLCs (Mizrak et al., 2019). mNSC, mouse Neural Stem Cell; mOPC, mouse Oligodendrocyte progenitor cell. (B) Toppcluster analysis (https://toppcluster.cchmc.org/) showing genes and biological processes associated with TIsig and TIIsig. (C) Gene amplification in TCGA Type I and Type II GBM (***p<0.001, Fisher’s exact test for two proportions). (D) Frequency of GBM driver mutations in Type I and Type II GBM (*p<0.05, Fisher’s exact test for two proportions). (E) Frequency of ERBB3 gene amplification in the TCGA dataset. See also Figure S4 and Table S4.

Figure 5.. Type I and II GBM identification in patient-derived cultures and xenografts.

(A) Western blot analysis in a panel of 17 patient-derived GBM cells designated as Type I and II candidates. (B) Heat map showing primary cell enrichment scores for DEG that distinguished core Type I & II GBM (TIDEGScore and TIIDEGScore) in 14 PDXs. (C) Representative images of H&E and IHC staining for EGFR and ERBB3 of Type I and Type II GBM PDX brain sections. Scale bars, 50 μM. (D) Immunofluorescence staining for Type I markers, SOX9 and SLC1A3, and Type II markers, SOX10 and FA2H, on GBM PDX brain sections. Scale bars, 50 μM. In C-D, results are representative of n≥3 biological replicates. See also Figure S5 and Table S5.

Figure 6.. Differential response to Tucatinib and Dasatinib by Type 2/II GBM.

(A-B) Dose response curves of primary mouse Type 1 & 2 (A) and human Type I & II (B) GBM cells to Tucatinib. Cells were treated with Tucatinib and analyzed 96 h after exposure using ATP assay (CellTiter-Glo®; n=3 replicates). Data are presented as mean ± S.D. (C-D) Western blot analyses for GBM cells from human Type I (C) and human Type II (D) treated with Tucatinib. Primary cells were cultured as monolayer and growth factor starved for 24 h, then treated with Tucatinib at 0.5 μM, 1 μM and 2 μM for 1 h, followed by stimulation with EGF+NRG1 (10 ng/ml each) together. Cells were harvested for analysis 30 min post-stimulation. (E-F) Dose response growth curves of mouse Type 1 & 2 cells (E) and human Type I & II cells (F) to Dasatinib. Cells were treated with Dasatinib and analyzed 96 h after exposure using ATP assay (CellTiter-Glo®; n=3 replicates). Data are presented as mean ± S.D. (G-H) Western blot analyses for GBM cells from human Type I (G) and human Type II (H) treated with Dasatinib. Primary cells were cultured as monolayer and growth factor starved for 24 h, then treated with Dasatinib for 1 h, followed by stimulation with EGF or NRG1 (10 ng/mL) individually. Cells were harvested for analysis 30 min post-stimulation. In C-D and G-H, results are representative of n≥3 biological replicates. See also Figure S6 and Table S6.

Figure 7.. Dasatinib attenuates Type 2 & II GBM growth in vivo.

(A) Experimental scheme for subcutaneously (SQ) transplanted mouse Type 1 and Type 2 GBM cells. (B) Type 1 GBM (#3605) SQ tumors harvested from vehicle- and Dasatinib-treated mice. (C-D) Tumor volume (C, p=0.12, student’s t-test) and tumor weight (D, p=0.17, student’s t-test) of Type 1 GBM (#3605) SQ tumors from vehicle-treated (n=7) and Dasatinib-treated (n=12) mice. Data are presented as mean ± SD. (E) Type 2 GBM (#3112) SQ tumors harvested from vehicle- and Dasatinib-treated mice. (F-G) Tumor volume (F, **p=0.0035, student’s t-test) and tumor weight (G, **p=0.0011, student’s t-test) of Type 2 GBM (#3112) SQ tumors from vehicle-treated (n=8) and Dasatinib-treated (n=12) mice. (H) Representative images of H&E and anti-Ki67-stained sections of Type 1 GBM (#3605) and Type 2 GBM (#3112) from vehicle- and Dasatinib-treated mice. Scale bars, 50 μM. (I) Quantification of Ki67⁺ cell densities in Type 1 (#3605) and Type 2 (#3112) GBM SQ tumors after vehicle (n=4 Type 1, n=3 Type 2) or Dasatinib (n=4 Type 1, n=4 Type 2) treatment. Data are presented as mean ± S.E.M. ;N.S., not significant; *p=0.0369, student’s t-test. (J) Experimental scheme for intracranially transplanted human Type I GBM cells (#1156-Luc, 5×10⁵ cells/mouse). Tumor bearing mice were gavaged with vehicle or Dasatinib, once per day, 5 days per week, starting at 2 weeks post-transplantation. Luminescence imaging monitored tumor growth until week 13 and MRI at week 16. (K) Plot of luminescence intensity of transplanted Type I GBM (#1156-luc) mice treated with vehicle (n=4) or Dasatinib (n=4). (L) Representative luminescence images of mice bearing Type I GBM (#1156-luc) at week 0 and week 13 following vehicle and Dasatinib treatment. (M) Experimental scheme for intracranially transplanted human Type II GBM cells (#160403-Luc, 5×10⁵ cells/mouse). Tumor growth was monitored with luminescence imaging weekly and MRI at week 6. (N) Plot of Type II GBM (#160403-luc) cells luminescence intensity over time for vehicle- and Dasatinib-treated mice. Luminescence intensity fold change was normalized to week 0 of individual mouse and presented as average ± S.D. (n=4 at each time point). *p=0.030, student’s t-test. (O) Representative luminescence images of intracranially transplanted Type II GBM (#160403-luc) mice at week 0 and week 5 following vehicle or Dasatinib treatment. (P) Kaplan-Meier survival curve of intracranially transplanted Type II GBM (#160403-luc) mice treated with vehicle (n=4) or Dasatinib (n=4). ***p=0.0041, Log-rank (Mantel-Cox) test. (Q) H&E and anti-Ki67 analysis of Type II tumors with vehicle and Dasatinib treatment. Scale bar, 50 μM. (R) Quantification of Ki67⁺ cell densities in Type II GBM (#160403-luc) intracranial tumors after vehicle (n=4) and Dasatinib (n=4) treatment. Data are presented as mean ± S.E.M. *p=0.0484, student’s t-test. See also Figure S7 and Table S7.