Comprehensive omics analyses profile genesets related with tumor heterogeneity of multifocal glioblastomas and reveal LIF/CCL2 as biomarkers for mesenchymal subtype

Abstract

Rationale: Around 10%-20% patients with glioblastoma (GBM) are diagnosed with more than one tumor lesions or multifocal GBM (mGBM). However, the understanding on genetic, DNA methylomic, and transcriptomic characteristics of mGBM is still limited. Methods: In this study, we collected nine tumor foci from three mGBM patients followed by whole genome sequencing, whole genome bisulfite sequencing, RNA sequencing, and immunohistochemistry. The data were further examined using public GBM databases and GBM cell line. Results: Analysis on genetic data confirmed common features of GBM, including gain of chr.7 and loss of chr.10, loss of critical tumor suppressors, high frequency of PDGFA and EGFR amplification. Through profiling DNA methylome of individual tumor foci, we found that promoter methylation status of genes involved in detection of chemical stimulus, immune response, and Hippo/YAP1 pathway was significantly changed in mGBM. Although both CNV and promoter methylation alteration were involved in heterogeneity of different tumor foci from same patients, more CNV events than promoter hypomethylation events were shared by different tumor foci, implying CNV were relatively earlier than promoter methylation alteration during evolution of different tumor foci from same mGBM. Moreover, different tumor foci from same mGBM assumed different molecular subtypes and mesenchymal subtype was prevalent in mGBM, which might explain the worse prognosis of mGBM than single GBM. Interestingly, we noticed that LIF and CCL2 was tightly correlated with mesenchymal subtype tumor focus in mGBM and predicted poor survival of GBM patients. Treatment with LIF and CCL2 produced mesenchymal-like transcriptome in GBM cells. Conclusions: Together, our work herein comprehensively profiled multi-omics features of mGBM and emphasized that components of extracellular microenvironment, such as LIF and CCL2, contributed to the evolution and prognosis of tumor foci in mGBM patients.

Keywords: CCL2; LIF; extracellular matrix; immune response; molecular subtype; multifocal GBM.

Figure 1

Analysis on genomes of tumor foci from the three mGBM patients. A) Representative MRI images of mGBM1 patient. Solid line circle in upper panels and dash line circle in lower panels indicate the tumor areas before and after surgical removal, respectively. B) Diagram of chromosomal copy number variation of tumor foci from the three mGBM patients. Red color and blue color represent chromosomal gain and loss, respectively. C) Distribution of cancer driver genes with mutations in tumor foci from the three mGBM patients. D) Evolution route of tumor foci from the three mGBM patients according to CNV and SNV. Red color font and green color font represent chromosomal gain and loss, respectively.

Figure 2

Analysis on genetic methylomes of tumor foci from the three mGBM patients. A) Heatmap diagram showing the comparison of methylated CpG with total CpG in different gene regions of tumor foci from the three mGBM patients. Red color and blue color represent hypermethylation and hypomethylation, respectively. B) Heatmap cluster of methylomes of tumor foci from the three mGBM patients. C) Venn diagram and scatter graph showing 19 GO genesets consistently enriched by genes with altered promoter CpG methylation in all three mGBM patients. D) FPKM value of YAP1 in tumor foci from the three mGBM patients. E) IHC of YAP1 in three tumor foci from mGBM1. H-Score (intensity × percentage) for each slide were labelled at the right of images. Scale bars: 100 μm.

Figure 3

Analysis on transcriptomes of tumor foci from mGBM1 and mGBM2. A) Heatmap diagram showing the cluster of gene expression in different tumor foci from mGBM1. B) Gene enrichment of significantly changed mRNA in mGBM1_C compared with the other three tumor foci in mGBM1. C) Gene enrichment of significantly changed mRNA in mGBM1_D compared with the other three tumor foci in mGBM1. D) Representative IHC images of indicated protein markers. Scale bars: 100 μm. E) Heatmap diagram showing the cluster of gene expression in different tumor foci from mGBM2. F) Gene enrichment of significantly changed mRNA in mGBM1_A compared with the other two tumor foci in mGBM2. G) Gene enrichment of significantly changed mRNA in mGBM1_A compared with the other two tumor foci in mGBM2.

Figure 4

Molecular subtype classification of tumor foci from the three mGBM patients. A) Venn diagram of counts of genes with promoter hypomethylation in individual tumor foci of three mGBM samples. B) Molecular subtype of individual tumor focus. C) Percentage of different molecular subtypes of GBM in mGBM and uGBM, respectively. D) The MRI images showing comparison of original tumor foci (mGBM1_B-D) with newly developed tumor foci (mGBM1_R1 and R2) after surgery and standard radio- and chemo-therapy. E) Schematic diagram of commonly altered genesets in our mGBM samples, mGBM vs. uGBM (TCGA_GBM), and mesenchymal vs. non-mesenchymal (TCGA_GBM).

Figure 5

Expression and clinical significance of LIF and CCL2 in GBM. A) Representative IHC images of indicated proteins on mGBM1_A-C. For p-STAT3 on mGBM1_C, there is a 2× enlargement (small rectangle to large rectangle) to show the nuclear localization of brown p-STAT3 signal. Scale bars: 100 μm. B) Representative Immunofluorescence images of indicated proteins on mGBM1_C. Dapi is used to show cell nuclei. Scale bars: 20 μm.

Figure 6

Clinical significance of LIF and CCL2 in GBM. A) mRNA expression of LIF and CCL2 in different molecular subtypes using TCGA_GBM database. B) Percentage of different molecular subtypes of GBM with LIF^High/CCL2^High and LIF^Low/CCL2^Low, respectively. C) Representative IHC images of indicated proteins on glioma tissue microarray. Scale bars: 100 μm. D) Kaplan-Meier survival analysis on protein levels of LIF or/and CCL2 in our glioma tissue microarray and mRNA levels of LIF or/and CCL2 in TCGA_GBM database. E) Measurement on protein levels of LIF and CCL2 in serum from 2 mGBM patients (mGBM1 and mGBM2) and additional 5 uGBM patients through ELISA.

Figure 7

The correlations among LIF/CCL2 co-expression, mGBM, and mesenchymal GBM. A) Enrichment of Verhaak_Glioblastoma_Mesenchymal geneset by different GBM phenotypes from TCGA_GBM database through GSEA. B) Enrichment of Hallmark_Epithelial_Mesenchymal_Transition geneset by different GBM phenotypes from TCGA_GBM database through GSEA. C) Cluster heatmap of transcriptomes of different GBM phenotypes from TCGA_GBM database through GSEA. D) Enrichment of Verhaak_Glioblastoma_Mesenchymal geneset by LN18 cells treated with LIF/CCL2 vs. vehicle. E) Enrichment of Hallmark_Epithelial_Mesenchymal_Transition geneset by LN18 cells treated with LIF/CCL2 vs. vehicle. F) Venn diagram of genes significantly upregulated in LN18 cells treated with LIF/CCL2 vs. vehicle and mesenchymal subtype vs. other subtypes from TCGA_GBM database. FC means Fold Change. G) Schematic diagram of tumor initiation and progression of mGBM.