Protein Preparation and Nano-Liquid Chromatography With Tandem Mass Spectroscopy (Micro-LC-MS/MS) Analysis

The workflow of protein preparation and proteomics analysis is shown in Figure 1 . The protein from tissue samples was extracted with lysis buffer (4% SDS and 100 mM HEPES, pH 7.6). The homogenate was sonicated for 10 min on ice. After centrifugation at 25,000 g for 30 min at 4°C, the supernatant was collected and stored at − 80°C. The total protein concentration was measured with a bicinchoninic acid (BCA) kit (23227, Pierce, Rockford, IL, USA).

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Figure 1

The workflow of proteomic strategy.

Equal protein samples from each of six fibrous meningiomas or four anaplastic meningioma tissues were combined into a single pool, as shown in Figure 1 . A mass of 200 mg of each pooled sample was reduced using reducing reagent at 60°C for 1h and alkylated with cysteine blocking reagent at room temperature for 10 min as described in the iTRAQ protocol (Applied Biosystems). Trypsin was added to the sample at a mass ratio of 1:50 (enzyme:protein) and incubated at 37°C overnight. The digested samples were labeled with 113 and 116 iTRAQ tags, respectively, as shown in Figure 1 , according to the manufacturer’s protocol (AB Sciex). The tagged peptides were dried via vacuum centrifugation and combined in one tube. The pooled sample was separated on apoly-LC SCX column (4.6 × 250 mm, 5 μm, 100 Å) using an Nexera XR instrument (LC-20ADXR, Shimadzu), and the labeled peptides were detected by ultraviolet radiation using SPD-20A (Shimadzu, Japan). In this study, a total of 55 fractions were collected, dried by speed vacuum centrifugation, and combined into 12 fractions according to the SCX chromatogram. Each fraction was injected onto a desalting column (10 × 0.3 mm, 5 μm-C18, 120 Å) and separated on an analytical column (0.3 × 150 mm, 3μmC18, 120 Å) using an Eksigent microLC instrument (Eksigent, Dublin, CA, USA). The samples separated via capillary high-performance liquid chromatography were subsequently analyzed using a Triple TOF 5600+ system (AbSciex, USA).

Protein identification and proteome annotation were performed using the ProteinPilot™ software package 5.0 (Applied Biosystems) and searched against the SwissProt database (March 2020). The following search parameters were utilized to analyze the MS/MS data: trypsin as the digestion enzyme with a maximum of two missed cleavages allowed; fixed modifications of carbamidomethyl (C) and iTRAQPlex (K and N-terminus); variable modifications of oxidation (M); peptide mass tolerance of ±20 ppm; fragment mass tolerance of ±0.1 Da; and peptide FDR ≤ 0.01.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository (28) with the dataset identifier PXD032342.

Bioinformatics Analysis

The differentially expressed proteins were analyzed via enrichment analyses using Gene Ontology (GO) (available at www.geneontology.org) for the identification of associated biological processes and molecular functions.

Cell Culture and Viability Measurement

The anaplastic meningioma cell line (CRL-3370) was obtained from the American Type Culture Collection. The cell culture medium consisted of DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). The cell line was maintained at 37°C in a humidified incubator with 5% CO₂.

Cell viability was determined using Cell Counting Kit-8 (CCK-8) (Biosharp, China, BS350B). Briefly, 5mg FK866 (Selleck, USA, S2799) was dissolved in 1.2771 ml dimethyl sulfoxide (DMSO) (Sigma, USA, D2650), then 10 mM FK866 was obtained. Then 10 mM FK866 was diluted with DMSO to obtain 10 μM FK866. After incubation with DMSO or different concentration of FK866 (25μM, 12.5μM, 2.5μM, 0.01μM, 0.005μM) for 24h, 48h or 72 h, 10 µl of CCK-8 reagent was added to each well, and the plates were further incubated in an incubator for 2h. Subsequently, the absorbance was measured at 450 nm by a microplate reader (Biotex, Synergy H1, USA). The cell viability (%) was calculated as follows: (average OD of treated groups at 24, 48 or 72 h/average OD of untreated groups at the same time point) x 100%.

Colony Formation Assay

The indicated cells were digested and resuspended and counted under a microscope. The cells were cultured in 6-cm plates at a density of 500 cells per well. The cells were cultured under normal culture conditions with DMSO or FK866 for 7days. The supernatant was removed, the cells were fixed with paraformaldehyde, and the cells were dyed with purple crystal. Then, the plates were washed with PBS twice. The whole field of view was photographed and counted. ImageJ (V1.48, NIH, Bethesda, MD, USA) was used for counting and measurement of colony mean size as described earlier (29). In brief, after converting the image to 8-bit format, the threshold was adjusted to exclude the dazzle. Then, colonies were counted and measured using the “Analyze Particles” function.

Western Blot Analysis

Tumor cells were lysed using RIPA buffer with a protease inhibitor cocktail (Millipore Sigma). Lysates were separated by SDS-PAGE (Bio-Rad) and transferred to PVDF membranes. The membranes were probed for NAMPT (Abcam, ab236874), STAT1 (CST, 14994), phosphorylated(p)-STAT1 (CST, 9167), PD-L1 (CST, 13684), B7-H3 (CST, 14058) and β-actin (Abcam, ab20272).

Immunohistochemical Staining

Immunohistochemical staining was performed on consecutive sections from xenograft tumors removed from nude mice treated with vehicle controls and FK866 (5 mg/kg by intraperitoneal (i.p.) injection once per day). The tumor samples were fixed in 10% buffered formalin and embedded in paraffin. The sections cut from paraffin embedded blocks were deparaffinized with xylene and ethanol, and then immersed for 40 min in 10 mM citric acid buffer (pH 6.0) or EDTA antigen retrieval solution (pH 9.0). The activity of endogenous peroxides was quenched for 20 min in 3% H₂O₂ in methanol. After rinsing in PBS, the sections were incubated at 4°C with anti-Ki-67 (Abcam, ab16667), anti-PD-L1 (CST, 13684) and anti-B7-H3 (CST, 14058) antibodies overnight. Then, a brown immunostain was developed using 3,3-diaminobenzidine tetrahydrochloride and hydrogen peroxidase, and counterstained with hematoxylin. The sections were then dehydrated, cleared with xylene and scanned with a digital slice scanning system (Leica, AT2, USA).

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was isolated from IOMM cell line using QIAzol Lysis Reagent (Qiagen Sciences, Maryland, USA). cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA). The cDNA was subsequently analyzed using QuantStudio 5 (Applied Biosystems, CA, USA). The amplification program was as follows: initial denaturation step at 95°C for 30 s, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. The expression of each specific gene was calculated relative to the expression of the internal reference gene GAPDH using the 2–ΔΔCt method. The primer sequences are shown in Supplementary Table 1 . All assays were performed in triplicate.

Staining With 5-ethynyl-2′-deoxyuridine (Edu)

An EdU (RiboBio, China, C10310-1) assay was used to determine the cell proliferation rate. Cells were seeded in 96-well plates (1×10³ cells/well) and cultured with DMEM (10% FBS) for 48h. Then, 100µl of EdU solution (50µM) was added to each well and incubated for 2h at 37°C. Then, the cells were fixed with 4% formaldehyde for 30min and permeabilized with 0.5% Triton X-100 for 10min at room temperature. Next the cells were washed with PBS for 5min and 1X ApolloR reaction cocktail was added to each well (100µl/well). Thirty minutes later, the cells were incubated with 100 µl Hoechst 33342 for 30min. Finally, the cells were observed with an inverted microscope (Zeiss, Axio Observer. ZI, USA).

Cell Cycle Assay

The Cell Cycle and Apoptosis Analysis Kit (Biosharp, China, BL114A) was used to detect cell cycle of IOMM cells treated with FK866 or DMSO. The cells were washed with cold PBS, fixed in 70% ethanol, and stored at 4°C for subsequent cell cycle analysis. The fixed cells were washed with PBS once and then re-suspended in 1 mL of PI staining reagent (50 mg/ml propidium iodide and 1 mg/ml RNAse in 1 ml of sodium citrate buffer, pH 7.4). The samples were incubated in the dark for 30 min before cell cycle analysis. The distribution of cells in the cell cycle was measured by an Amnis imaging flow cytometer (ImageStreamX MarkII), and quantitation of cell cycle distribution was performed using FlowJo 10.6.2 Software. The percentages of cells in the G1, S and G2 phases were calculated.

Xenograft Experiments

To construct the subcutaneous tumor xenograft mouse model, 2×10⁶ IOMM cells were suspended in 100 μL of solution (PBS) and injected subcutaneously into the right dorsal flank of 6–8-week-old female nude mice. Tumors were allowed to establish until the tumor size was measurable, and then the mice were randomly assigned to be treated for two weeks with vehicle controls or FK866 (5 mg/kg i.p. once per day). The dose of the compound was determined based on previous studies (30–32). FK866 was suspended in 45% propylene glycol + 5% Tween 80 + double distilled H₂O (ddH₂O). Tumor size was measured twice per week by two investigators as follows: tumor size (mm³) = [d² x D]/2, where d is the shortest diameter and D is the largest diameter. After two weeks of treatments, tumor samples were surgically removed and the tumor weights were measured as a surrogate for tumor burden.

The Inhibition of NAMPT by FK866 Suppressed the Proliferation of IOMM Cells In Vitro

In this study, the inhibitory effect of FK866 on the viability of IOMM cells was investigated at different concentrations (0-25 μM) ( Figure 4A ). The viability of the IOMM cells was detected at different times (24, 48 and 72 h). A dose-dependent inhibition of cell growth was observed with increasing FK866 concentration at 24, 48 and 72 h. We also thought that a high concentration of FK866 induces toxicity in normal cells. We therefore chose the dose of 2.5 μM in subsequent in vitro studies to examine the effect of FK866 on IOMM cells. A significant difference was observed between the DMSO group and the FK866-treated groups at 2.5 μM after 48 h of incubation (p=0.0022) ( Figure 4B ). The effect of FK866 on IOMM proliferation was also detected by colony formation and EdU assays. The colony formation assays revealed that FK866 inhibited colony formation of IOMM cells ( Figure 4C ). Colony numbers were significantly different between the FK866‐treated group and DMSO-treated group (p=0.0032) ( Figure 4D ). The results of the EdU assay are shown in Figures 4E, F . The number of cells in the proliferative phase was significantly between the FK866‐treated group and the DMSO-treated group (p=0.0025). There were fewer cells in the proliferative phase in the FK866-treated group than in the DMSO-treated group. In summary, FK866 inhibited the proliferation of IOMM cells.

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Figure 4

(A, B) Inhibitory effects of FK866 on IOMM cells as demonstrated using the CCK-8 assay. The cells were treated with various concentrations of FK866 (0–25 µM) for 24, 48 and 72 h. (C, D) The proliferation of IOMM cells treated with FK866 or DMSO was detected by colony formation assay. (E, F) Percentage of EdU positive cells among IOMM cells. **p < 0.01, ****p < 0.0001.

The Inhibition of NAMPT Was Effective Against Cell Cycle Progression

To understand how FK866 influenced the proliferation of the IOMM cell line, flow cytometry was used to analyze the effect of FK866 on the cell cycle. The percentage of cells in the G2/M phase was significantly different between the FK866-treated group and the DMSO-treated group (p= 0.0025) ( Figures 5A, B ). The proportion of cells in the G2/M phase among FK866-treated IOMM cells had increased. In addition, a significant difference in the number of cells in the S phase was also observed between the FK866-treated group and the DMSO-treated group (p=0.0041) ( Figures 5A, B ). FK866 reduced the number of cells in the S phase.

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Figure 5

(A, B) Distribution of the cell cycle in IOMM cells treated with FK866 or DMSO. (C, D) Changes in NAMPT (NAMPT/GAPDH) and STAT1 (STAT1/GAPDH) mRNA relative expression in IOMM cells treated with FK866 or DMSO. (E, F) Changes in STAT1 (STAT1/β-actin) and p-STAT1 (p-STAT1/β-actin) protein relative expression in IOMM cells treated with FK866 or DMSO. (G, H) Changes in PD-L1 (PD-L1/β-actin) and B7-H3 (B7-H3/β-actin) protein relative expression in IOMM cells treated with FK866 or DMSO. *p < 0.05, **p < 0.01, ***p < 0.001.

The NAMPT Inhibitor-FK866-Suppressed Immune Checkpoint Expression and Proliferation of IOMM Cells by Downregulating the Expression of STAT1

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was preformed to detect the mRNA relative expression of NAMPT and STAT1. The results showed that FK866 can elevate the mRNA expression of NAMPT ( Figure 5C ) and reduce the mRNA expression of STAT1 ( Figure 5D ). And the mRNA relative expression of NAMPT and STAT1 showed significant differences between the DMSO group and the FK866 group (p=0.0003 and p=0.0022). Western blotting was preformed to detect the expression of STAT1 and p-STAT1. A significant difference was observed between the DMSO group and the FK866 group (p=0.0019 and p=0.0353) ( Figure 5F ). The results showed that FK866 reduced the expression of STAT1 and p-STAT1 ( Figure 5E ). Intriguingly, STAT1 can promote the expression of the immune checkpoint (PD-L1) (27, 34–36), which can lead to immune escape (26). Therefore, we performed Western blotting to detect the expression of immune checkpoint proteins. The expression of PD-L1 and B7-H3, indeed, showed significant differences between the DMSO group and the FK866 group (p=0.0053 and p=0.0250) ( Figure 5H ). FK866 may decrease the expression of PD-L1 and B7-H3 by downregulating STAT1 and p-STAT1 ( Figure 5G ).