Subcellular progression of mesenchymal transition identified by two discrete synchronous cell lines derived from the same glioblastoma

Abstract

Glioblastomas (GBM) exhibit intratumoral heterogeneity of various oncogenic evolutional processes. We have successfully isolated and established two distinct cancer cell lines with different morphological and biological characteristics that were derived from the same tissue sample of a GBM. When we compared their genomic and transcriptomic characteristics, each cell line harbored distinct mutation clusters while sharing core driver mutations. Transcriptomic analysis revealed that one cell line was undergoing a mesenchymal transition process, unlike the other cell line. Furthermore, we could identify four tumor samples containing our cell line-like clusters from the publicly available single-cell RNA-seq data, and in a set of paired longitudinal GBM samples, we could confirm three pairs where the recurrent sample was enriched in the genes specific to our cell line undergoing mesenchymal transition. The present study provides direct evidence and a valuable source for investigating the ongoing process of subcellular mesenchymal transition in GBM, which has prognostic and therapeutic implications.

Introduction

Glioblastoma (GBM) is the most common primary malignant brain tumor and is one of the most aggressive cancers [1]. Despite the current best practice of maximal safe surgery, chemotherapy, and radiotherapy, the outcome of GBM is still devastating, in part due to intratumoral heterogeneity. Intratumoral heterogeneity implies spatially dispersed genetically diverse populations of cancer cells, distinguishing themselves in a variety of biological functions, including cell proliferation, invasion, immunity, and metastasis. Phylogenic clonal evolution giving rise to differential cellular hierarchies during oncogenesis usually leads to intratumoral heterogeneity in human GBMs. Therefore, intratumoral heterogeneity can contribute to treatment resistance or immune evasion [2].

Epithelial to mesenchymal transition, an EMT process, is a highly controversial term in GBM tumorigenesis. In gliomagenesis and its progression, a non-classical EMT-like or glial to mesenchymal transition is proposed to be linked to the mesenchymal nature of the neural cells during the neurodevelopmental process [3]. Another study reported the onco-plasticity of EMT-like or mesenchymal to epithelial transition (MET)-like conversion mediated by the epigenetic changes in the tumor microenvironment around glioma stem cells [4]. The well-known classification of glioma subtypes by gene expression profiles also uses several EMT-related genes (CD44, MERTK, TGFB1, NOTCH2 etc.), which are enriched in the mesenchymal subtypes [5, 6]. A recent study attempted to classify the GBMs using a series of EMT-associated genes to predict the differences in prognosis [7]. In addition, EMT has been widely described as a key factor that drives cellular heterogeneity influenced by both genetic and non-genetic determinants and even a small number of cells undergoing this process can cause evident intratumoral heterogeneity in cancers [8, 9]. However, the role of EMT and its clinical significance in GBM is still not well established due to the lack of direct evidence of its evolution in clinical samples and the rarity of cell lines that are derived from the same tissue to perform the verifying experiments.

Here, we isolated and established two distinct cell lines derived from the same GBM tissue sample. These cell lines have genetically developed into different cell lines by subculture. We compared and analyzed the genomic and biological signatures of the two cell lines and found that one of the cell lines exhibits mesenchymal transition characteristics, unlike the other. Furthermore, we reanalyzed publicly available single-cell RNA-seq data to identify analogous clusters to our case and found four similar GBM samples with occult clones of mesenchymal transition. We also found recurrent GBM samples showing evolution of mesenchymal transition clones characterized by upregulation of the EMT genes identical to the cell line which was not identified in the initial sample. These findings provide direct evidence to support the subcellular evolution of mesenchymal transition in GBM, which can be utilized in future investigations into intratumoral heterogeneity and the mesenchymal transition process.

Materials and methods

Cell lines and culture condition

An IRB-approved written informed consent was obtained from the patient to use the samples and to establish cell lines for research purposes (Seoul National University Hospital IRB No. H-0507-509-153 and H-1102-098-357). Tissues were freshly frozen in liquid nitrogen immediately after resection, and white blood cells (WBCs) were extracted from whole blood. Both tissue and WBC samples were then stored at − 80 °C until later use. The cell lines were established using the core area of the fresh tumor tissue just after resection. Solid tumor tissue was carefully minced with scissors and isolated into small mixtures by pipetting. Appropriate amounts of delicate tumor tissue fragments were seeded into 25 cm² cell culture flasks. The tumor cells were initially cultured in Opti-MEM medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) (O5). Cultures were maintained in RPMI 1640 medium containing 10% heat-inactivated FBS (R10). Initial passages were completed when abundant tumor cell growth was observed, and consecutive passages were done every 1 or 2 weeks. During passaging, adherent cells were recovered by pipetting by treatment with trypsin while growth was subconfluent. Differential trypsinization was used to obtain a pure tumor cell population when stromal cell growth was noted in the initial cultures. Cultures were maintained in humidified incubators at 37 °C with 5% CO₂ and 95% air. The established cell lines used in the present study are available at the Korea Cell Line Bank (https://cellbank.snu.ac.kr/). The cell lines were maintained in opti-DMEM medium supplemented with 20% fetal bovine serum (GIBCO).

Cell proliferation assay

Cell proliferation assays were performed with EZ-Cytox (Daeillab Service) on cells seeded at 1 × 10³ cells/well in 96-well plates and cultured for the designated times. The absorbance of the plates was measured using a microplate reader (Molecular Devices) at a wavelength of 450 nm.

Colony-forming assay

To grow colonies, cells were seeded in 6-well plates at 1000 cells/well density and incubated at 37 °C in an atmosphere of 5% CO₂ for 14 days. The cell colonies were fixed and stained with 0.05% crystal violet-methanol-acetic acid solution after 14 days. Stained colonies were scanned and score was calculated.

Western blot analysis

Western blot analysis was performed as previously described [10]. Antibodies against TGF-β (3711), Vimentin (5741), N-Cadherin (13,116), Claudin-1 (13,255), β-Catenin (8480), ZO-1 (8193), Snail (3879), Slug (9585), TCF/ZEB1 (3396), E-cadherin (3195), phosphorylated Smad2 at Ser465/467 (3108), Smad2 (5339), phosphorylated Smad3 at Ser423/425 (9520), Smad3 (9523), Smad4 (38,454) and ACTB (4967) were from Cell Signalling Technology; and HRP-conjugated IgGs (111-035-003 and 115-035-003) were from Jackson Immune Research. Immunoblots were visualized with a ChemiDoc XRS system (Bio-Rad). The density of the bands was measured using free image analyzer software (ImageJ V1.8x; National Institutes of Health, USA, http://rsb.info.nih.gov/ij/) [11].

Telomere repeat amplification protocol (TRAP) assay with ELISA

A TeloTAGGG Telomerase PCR ELISA PLUS kit (Roche) was used according to the manufacturer’s protocol to measure the telomerase activity. GBM tissues or cells were homogenized in ice-cold lysis buffer using an automill (Tokken). Briefly, after BCA protein quantification of the lysates, 10 µg proteins were incubated in a total volume of 50 µl reaction mixture at 25 °C for 30 min to allow the telomerase to add telomeric repeats to the end of the biotin-labeled primer. Then, PCR was conducted for 33 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s, followed by an additional extension time of 10 min at 72 °C and then holding the PCR tubes at 4 °C if not used immediately. The TA was measured at a reference wavelength of 450 and 690 nm. The relative TA (RTA) of each sample was calculated according to the instructions of the TeloTAGGG Telomerase PCR ELISA PLUS Kit.

C-circle assay

C-circle detection was executed as previously described [12]. Briefly, 30 ng DNA was combined with 7.5 U Φ29 DNA polymerase (NEB), 0.2 mg/ml BSA, 1 mM each dATP, dGTP and dTTP, 10 μl 2X Φ29 buffer, 0.1% (v/v) Tween 20, and incubated at 30 °C for 4 h or 8 h followed by 20 min at 70 °C. Amplification products were transferred on a Hybond N + nylon membrane (Bio-Rad) and processed using the TeloTAGGG Telomere Length Assay Kit (Roche). Chemiluminescent signals were visualized with a ChemiDoc XRS system (Bio-Rad), and the intensity of the spots was quantified with ImageQuant TL software (Bio-Rad).

In vivo xenograft tumor growth assay

To observe the in vivo proliferation of patient-derived primary GBM cells, a subcutaneous xenograft mouse model was constructed using 5-week-old female Balb/c nude mice. Primary GBM cells were cultured in Opti-MEM (LS31985070, Gibco) supplemented with 5% FBS (S 001-01, Welgene) and 1 × antibiotic–antimycotic (15240-062, Thermo Fisher Scientific) at 37 °C in a humidified incubator in the presence of 5% CO2. In total, 3 × 10⁶ cells were mixed with 100 µl of Opti-MEM and Matrigel mixture (Opti-MEM:Matrigel = 1:1) and subcutaneously injected into the mice. Tumor size was measured once a week using calipers, and tumor volume was calculated using the following formula: (length × width²)/2.

Telomere length fragmentation assay

Telomere lengths were determined by Southern blot using a TeloTAGGG Telomere Length Assay Kit (Roche) according to the manufacturer’s protocol. Briefly, 1 µg of each DNA sample was digested with RsaI and HinfI for overnight at 37 °C, electrophoresed on a 0.8% agarose gel at 50 V for 4 h, and transferred to a nylon membrane by Southern blotting. The membrane was blocked and hybridized overnight to a digoxigenin (DIG)-labeled probe specific for telomeric repeats. Then, it was incubated with anti-DIG-alkaline phosphatase (1:1000 dilution) for 30 min and processed using the substrate in the TeloTAGGG Telomere Length Assay kit (Roche). After chemiluminescence signals were visualized with a ChemiDoc XRS system (Bio-Rad), telomere length was calculated with Telo Tool version 1.3.

Whole exome sequencing and data analysis

Extracted DNAs from the samples were checked for quality control with Bioanalyzer. The sequencing library was prepared by random fragmentation of the DNA or cDNA followed by 5′ and 3′ adapter ligation. Library preparation was done using the SureSelectXT library prep kit. Whole exome sequencing (WES) was performed with the Illumina platform at Macrogen, Korea, and generated BCL binary was converted into raw FASTQ files utilizing the Illumina bcl2fastq package. Next FASTQ files were mapped to the reference genome (hg19) with BWA (0.7.12), and PCR duplicates were marked with Picard (1.130) [13]. Base recalibration and SNP and INDEL calling was done using GATK (v3.4.0) [14] We processed five different samples (blood, tumor, parent cell, subclone #5, and subclone #11) to obtain the WES data from the same patient. To track genomic alterations across the samples, the generated bam and vcf files were then analyzed with SuperFreq [15]. Typically, the SuperFreq program is designed for clonal analysis with samples related by timepoints. It tracks the SNP/INDEL and CNV from the samples and calls out clones or clusters according to the mutation changes. Since our samples do not contain any temporal information, we took advantage of the SuperFreq to track mutational differences and sample-specific mutations for both SNP/INDEL and CNV of our five samples. Mutation variations were clustered according to their variant allele frequency throughout the samples.

RNA-sequencing and data analysis

Extracted RNAs from the samples were checked for quality control with Bioanalyzer. Library preparation was conducted using a Lexogen Quantseq 3’ mRNAseq library prep kit. Single-end mRNA sequencing was performed with Illumina Nextseq500 at ebiogen, Korea. Generated FASTQ files were mapped to the reference genome (hg19) with Bowtie2. Normalization of the count was done with EdgeR and batch effects were removed with the help of the R package limma (doi: https://doi.org/10.1093/nar/gks042 and https://doi.org/10.1093/nar/gkv007) [16, 17]. We then compared the general RNA-seq expression profile of the genes across the samples with Pearson correlation analysis using R. Differential gene expression (DEG) analysis between subclone #11 and #5 were done using the ExDEGA software provided by ebiogen Korea. Significant upregulated genes in subclone #11 compared to subclone #5 was selected with the filtering criteria of P value < 0.05 and Log2 fold change 11/5 ≥ 2. Then functional clustering of the upregulated genes in subclone #11 was done using DAVID v6.8 [18]. For the RNA-seq of GBM longitudinal samples, a total of 55 pairs (initial and recurrent) of tumor samples RNA was extracted and the library was prepared with a SureSelectV6 mRNA seq library preparation kit. RNA-sequencing was performed with Illumina Hiseq2500 at Macrogen, Korea. FASTQ files were processed and normalization of the count was done as described above. Then the differences between gene expression were calculated by subtracting the normalized count of each initial sample from its paired recurrent sample.

Single cell RNA-seq analysis

We downloaded GBM single-cell RNA-seq data from four recently published studies (GSE117891, GSE131928, GSE125587, phs001287), which included a total of 71 samples from 65 patients [19,20,

Availability of data and materials

Data and materials for the study are included in the manuscript and supplementary information.