Altered O-linked glycosylation in benign and malignant meningiomas

Human malignant meningioma cell lines and culture

IOMM-Lee, a human meningioma cell line (WHO grade III), was purchased from the American Type Culture Collection (Manassas, VA, USA). HKBMM, a human meningioma cell line (WHO grade III), was kindly provided by Associate Professor Norie ARAKI (Kumamoto University, Kumamoto, Japan). Both cell lines were cultured in complete medium containing Dulbecco’s modified Eagle’s medium (DMEM, cat. no. 12100-046; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% penicillin-streptomycin (cat. no. 15140-122; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 10% fetal bovine serum (FBS, cat. no. 10270-098; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained in a humidified incubator with 5% CO₂ at 37 °C.

Human benign meningioma cells isolation and culture

Benign meningioma tissues were obtained from two patients who underwent surgical resection at Suranaree University of Technology Hospital, Thailand and were kept in a frozen medium containing 90% FBS, and 10% DMSO. Written informed consent was obtained from each subject. The Suranaree University of Technology Ethics Committee approved the study protocol (registration number: EC-64-154). The two benign meningioma tissues were designated as SUT-MG12 and SUT-MG14. SUT-MG12 and SUT-MG14 were derived from a 70- and 63-year-old female patient, respectively. The histopathological examination of the two benign meningioma tissues was determined and confirmed to be meningothelial meningioma, WHO grade I. The frozen benign meningioma tissues were rinsed in PBS and resuspended in a complete medium for primary cell establishment. The tissues were minced into 2–3 mm³ pieces. Tissue fragments were transferred into a 15 mL tube for the washing step. The fragments were resuspended in pre-warmed complete medium with collagenase solution at 1 mg/mL (cat no. 17100-017; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated for 45 min at 37 °C with gentle stirring every 5 min. The digested tissues were sieved through 100 and 70 µm nylon filters (cat nos. 93070 and 93100; SPL Life Sciences, Pochon, South Korea). Cell suspensions were centrifuged and washed with complete medium. The cells were then resuspended in DMEM with 1% penicillin-streptomycin, 20% fetal bovine serum, and 2 mM L-glutamine (cat. no. 25030-081; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained in a humidified incubator with 5% CO₂ at 37 °C. Primary benign meningioma cells with 90% confluence were split by trypsinization. The medium was changed twice a week. The passage number of the primary cell culture was between 3 and 10, after applying subsequent experiments.

Immunocytochemistry and lectin staining

Cells were seeded at 3–5 × 10⁵ cells into a 24-well plate. After seeding for 48 h, cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.2% Triton X-100 in PBST for 10 min. Endogenous hydrogen peroxide activity was blocked with 0.3% of hydrogen peroxide for 30 min. Then, nonspecific binding was blocked with 3% bovine serum albumin (BSA) for 30 min. Sixteen biotinylated lectins (Vector Laboratories, Inc., Newark, CA, USA) or primary antibodies were probed at 25 °C overnight, on a shaker including (cat.no. BK-1000 and BK-3000; Vector Laboratories, Inc., Newark, CA, USA), vimentin (dilution 1:100, cat. no. SC-6260; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), MUC1 (dilution at 1:150 cat. no. SC-7313; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and SSTR2A (dilution at 1:100, cat. no. Ab134152; Abcam, Cambridge, UK) (Table 1). Then, ABC-Peroxidase Solution (cat. no. PK-4000; Vector Laboratories, Inc., Newark, CA, USA) was used for 1 h at room temperature as the secondary antibody to determine the lectin signal. Meanwhile, horseradish peroxide-conjugated secondary antibody (1:100), (GE Healthcare, Buckinghamshire, UK) was used for 1 h to determine the protein expression of vimentin, MUC-1, and SSTR2A. Visualization was accomplished using a SignalStain® DAB substrate kit (cat. No. 8059; Cell Signaling Technology, Inc., Danvers, MA, USA). Images were visualized using an inverted microscope with an original magnification of ×200.

Gene expression analysis by GEO database

The gene expression data (GEO series GSE16581) of the meningiomas were retrieved from the Gene Expression Omnibus (GEO) database ((https://www.ncbi.nlm.nih.gov) accessed on 20 June 2022). The GEO series GSE16581 comprises expression data from 43 WHO grade I, 19 WHO grade II and 6 WHO grade III meningiomas. All expression data were log2 transformed.

Gene expression analysis by quantitative PCR

Cells with 90% confluence in a 60 mm culture dish were harvested by TRIzol reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA was extracted according to the manufacturer’s protocol. The cDNA synthesis using the SensiFAST cDNA synthesizer kit was performed. The primer sets for all the glycosyltransferases are listed in Table 2. The mRNA expression levels of all the glycosyltransferases were analyzed in both primary benign meningioma cells and malignant meningioma cell lines using quantitative PCR (qPCR) as previously described (Talabnin, Talabnin & Wongkham, 2020). The gene amplification condition was performed as previously described (Talabnin, Talabnin & Wongkham, 2020). β-Actin was used as the internal control to normalize the expression of the target genes. Relative mRNA expression was determined using the 2Ct method (Livak & Schmittgen, 2001).

Statistical analysis

The independent t-test was used to determine the differential expression of glycosyltransferase genes between the primary benign meningiomas and the malignant meningiomas. All analyses used GraphPad Prism software (version 8.0; GraphPad Software, Inc., La Jolla, CA, USA). A P < 0.05 indicated a statistically significant difference.

Establishment of the primary benign meningioma cells

A well-characterized benign meningioma cell line is not available due to the senescence of nonmalignant cells occurring in vitro. Thus, two primary benign meningioma cells (WHO grade I)—SUT-MG12 and SUT-MG14—were isolated from two meningothelial meningioma tissues and performed primary culture technique. The morphological analysis demonstrated that SUT-MG12 and SUT-MG14 are spindle-shaped cells with large nuclei (Fig. 1). The primary culture showed that SUT-MG12 and SUT-MG14 could proliferate and multiply after more than 10 passages, but the proliferation rate declined after passage no. 10, and the cells were enlarged and underwent senescence. Thus, the cells in passage nos. 3–10 were used for subsequent experiments. Using immunocytochemical analysis, protein expressions of vimentin (Vim) and somatostatin receptor 2A (SSTR2A) were observed in these two primary benign meningioma cells (WHO grade I) including the well-established meningioma cell lines—HKBMM and IOMM-Lee (WHO grade III). However, the protein expression of mucin 1 (MUC1) was nominal (Fig. 1). The data from the primary culture, morphological analysis, and immunocytochemistry demonstrated the benign meningiomas characteristic of SUT-MG12 and SUT-MG14.

Alteration of glycan expression in meningiomas

Altered expression of glycans have been associated with tumor development and progression (Thomas, Rathinavel & Radhakrishnan, 2021). For screening of the potential glycan structures between the benign and malignant meningiomas, a specific lectin technique is a good candidate. Using lectin-cytochemistry, the glycan profiles of primary benign meningioma cell lines—SUT-MG12 and SUT-MG14—were compared with the malignant meningioma cell lines—HKBMM and IOMM-Lee. Sixteen lectins with different glycan specificities (Table 1) were investigated. ConA (for mannose), RCA (for GalNAc), WGA, and GSLII (for GlcNAc) were stained strongly positive in both primary benign and malignant meningioma cell lines. At the same time, UEAI (for fucose), SBA (for GalNAc), DSL, LEL, and STL (for GlcNAc) were only slightly stained (positive). In contrast, DBA, VVL (for GalNAc), PNA, ECL, and Jacalin (for Gal) were strongly stained (positive) in primary benign meningioma cell lines compared with negative or slightly positive stained in malignant meningioma cell lines (Figs. 2–4). Furthermore, the alteration of sialylation in meningiomas was determined using MAL II (alpha 2,3 sialylation) and SNA (alpha 2,6 sialylation). Strong positive staining of SNA was observed in primary benign meningioma cell lines (Fig. 4, Table 1). These findings demonstrated the differential expression of glycans in the primary benign and malignant meningiomas, the high expression of GalNAc, Gal glycans, and alpha 2,6 sialylation was observed in primary benign meningioma cell lines—SUT-MG12 and SUT-MG14 (WHO grade I)—compared with malignant meningioma cell lines—HKBMM and IOMM-Lee (WHO grade III).

Altered expression of mucin-type O-linked glycosyltransferases in meningiomas

To explore whether the specific changes of the O-linked glycosylation in benign and malignant meningiomas are due to the aberrant expression of the mucin-type O-linked glycosyltransferase. The expression of the mucin-type O-linked glycosyltransferase genes—including C1GALT1, COSMC, C2GNT1, C2GNT2, and B3GNT6—were investigated through the GEO database (GSE16581) and all underwent qPCR. In the GEO database, the respective mRNA expressions of C1GALT1, COSMC, C2GNT1, C2GNT2, and B3GNT6 was highly expressed in Grade I, Grade II, and Grade III meningiomas (Fig. 5A). Meanwhile, COSMC was only significantly increased in Grade II and III vs Grade I meningiomas (P < 0.05). We then further investigated the mucin-type O-linked glycosyltransferase gene using qPCR in primary benign—SUT-MG12 and SUT-MG14—and malignant—HKBMM and IOMM-Lee—meningioma cell lines. The gene expression experiments demonstrated that mRNA expression of C1GALT1 and COSMC were highly expressed in both the primary benign and malignant meningioma cell lines. In contrast, C2GNT2 was highly expressed in malignant meningioma cell lines compared to primary benign meningioma cells (Fig. 5B).

Additionally, investigations of membrane-bound mucin genes (MUC1, MUC3, MUC4, and MUC13), associated with altered O-linked glycosylation in various cancers, were investigated through the GEO database (GSE16581) and underwent qPCR. In the GEO database, the respective mRNA expressions of MUC1, MUC3, MUC4, and MUC13 was detected in Grade I, Grade II, and Grade III meningiomas. Still, only MUC1 expression was significantly increased in grade II and III meningiomas (Fig. 6A). In contrast, MUC1 and MUC4 were highly expressed in primary benign—SUT-MG12 and SUT-MG14—meningioma cell lines (Fig. 6B).

Altered sialyltransferases expression in meningiomas

Changes in sialylated glycans have been linked to facilitate malignant cell transformation, immune evasion, and metastatic spread (Hugonnet et al., 2021). Additionally, high expression of alpha 2,6 sialylation was observed in primary benign meningioma cell compared to malignant meningioma cell lines (Fig. 4). To draw a full picture of the alteration of O-linked glycosylation in benign and malignant meningiomas, the expression of “capping enzymes”—sialyltransferases were demonstrated. For the extension of the mucin-type O-linked glycosylation, the expression of six sialyltransferases (STs)—ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL6, ST6GAL1, and ST6GALNAc1—were investigated using the GEO database (GSE16581) and underwent qPCR. In the GEO database, mRNA expression of ST3GAL4 was significantly decreased in grade II and III meningiomas (P < 0.05), while the others (ST3GAL1, ST3GAL3, ST3GAL6, ST6GAL1, and ST6GALNAc1) were not (Fig. 7A). In the meningioma cell lines, mRNA expression of ST6GAL1 was highly expressed in the primary benign meningiomas—SUT-MG12 and SUT-MG14—while the mRNA expression of ST3GAL1 and ST3GAL3 was observed in the malignant meningioma cell lines—HKBMM and IOMM-Lee (Fig. 7B).

Altered fucosyltransferase expression in meningiomas

Lewis antigens are terminal fucosylated carbohydrate motifs decorating cell surface N- and O-linked glycoproteins by capping with fucosyltransferases (FUTs). Additionally, overexpression of different FUTs during malignant cell transformation are associated with the acquisition of an increased proliferative capacity and a pro-survival phenotype (Lv et al., 2023). To address the complex structures of the O-linked glycosylation in benign and malignant meningiomas, the expressions of fucosyltransferases (FUTs) were determined. Fucosylation of the mucin-type O-linked glycosylation may generate the complex structure, i.e., Sialyl-Lewis X (sLex) and Sialyl-Lewis A (sLea) in meningiomas. Thus, the expression of nine fucosyltransferases (FUTs) were investigated (viz., FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, and FUT9). In the GEO database, there was no respective significantly different expression of FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, or FUT9 in grade I vs grade II & III meningiomas (Fig. 8A). However, the gene expression experiments demonstrated that FUT1 and FUT8 were highly expressed in the malignant meningioma—HKBMM—vs the primary benign meningiomas—SUT-MG12 and SUT-MG14 (Fig. 8B). Due to the high expression of FUT1 and FUT8 in HKBMM cell lines, we removed FUT1 and FUT8 results from the analysis. We found that the respective expression of FUT2, FUT3, FUT6, and FUT7 was also increased in the malignant meningiomas—HKBMM and IOMM-Lee (Fig. 8C).