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Method Article

Preparation Of Neovascular Tissues from Human Glioma Tissues for Quantitative Proteomics Analysis of Tumor Angiogenesis

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DOI:

10.3791/69547

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March 20th, 2026

 ,  ,  ,  ,  ,  ,  ,  , 
1Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University & Shandong Academy of Medical Science, 2Shandong Provincial Key Medical and Health Laboratory of Ovarian Cancer Multiomics, & Jinan Key Laboratory of Cancer Multiomics, Shandong First Medical University & Shandong Academy of Medical Science, 3School of Medicine, Wayne State University, 4Department of Pathology, The Second Xiangya Hospital, Central South University, 5Hunan Clinical Medical Research Center for Cancer Pathogenic Genes Testing and Diagnosis,, 6Department of The 1st General Surgery, The Third Xiangya Hospital, Central South University

In This Article

Summary

This protocol successfully prepared sufficient high-quality neovascular tissue from glioma samples using laser-capture microdissection for quantitative proteomics analysis of tumor angiogenesis, overcoming the limitations of studies that traditionally focus mainly on angiogenic factors and providing a large-scale proteomics profile of glioma angiogenesis.

Abstract

Glioma represents primary malignant tumors of the central nervous system with significant incidence and mortality. Tumor angiogenesis is the hallmark of glioma pathophysiology and is an important event in the tumor environment. The aberrant structure of tumor neovascularization serves dual roles: a nutrient supply source and a metastatic portal enabling tumor cell invasion into the bloodstream. Anti-angiogenesis therapy is a hot topic in the field of cancer. Most of the previous studies focus on the activating factors and inhibitory factors of tumor angiogenesis; however, this only clarifies tumor angiogenesis in a very limited range. Abnormal protein expression or post-translational modifications contribute to the abnormal morphology of blood vessels in glioma. This protocol used laser-capture microdissection (LCM) to isolate and purify an adequate amount of neovascular tissues from glioma tissues, which was used for isotope-labelled quantitative proteomics analysis to identify differentially expressed proteins in glioma neovascular tissues compared to controls. These data provide a crucial scientific basis for elucidating the pathological mechanisms of tumor angiogenesis, discovering anti-angiogenic therapeutic targets, and constructing tumor angiogenesis-based biomarkers.

Introduction

Gliomas are highly heterogeneous central nervous system(CNS) tumors originating from glial cells that provide structural and metabolic support to neurons1, and are histologically classified into subtypes: astrocytomas, oligodendrogliomas, and ependymomas2. According to the WHO grading system, glioma grades I and II are the low-grade gliomas (LGG), typically characterized by slow growth rates and potential long-term stability, often amenable to surgical resection based on anatomical location. In contrast, grades III and IV are the high-grade gliomas (HGG), predominantly undifferentiated and malignant with highly invasive behavior and poor prognosis3. Current treatments remain limited and largely ineffective; while maximal safe resection combined with postoperative radiotherapy/chemotherapy moderately improves prognosis, significant limitations and toxicities persist due to tumors' diffuse infiltrative growth and indistinct boundaries preventing radical resection4,5. Human gliomas, especially high grades of glioma, are highly vascular malignant tumors whose growth, proliferation, and invasion strongly depend on their abnormal neovascularization, which is a critical hallmark of glioma malignancy6,7. The targeting tumor angiogenesis study has significant scientific merits for therapeutic breakthroughs.

Angiogenesis fundamentally involves endothelial cell proliferation, migration, and differentiation under specific stimuli, with research focusing on regulators: (i) inducers, including vascular endothelial growth factor (VEGF)8, vasorin (VASN)9, hypoxia-inducing factor 1 (HIF1)10, transforming growth factor β (TGF-β)11, endocan12, platelet-derived growth factor (PDGF)13, and epidermal growth factor (EGF)14; and (ii) inhibitors, including angiostatin15, endostatin16, platelet-activating factor receptor-1 (TSP-1)17, and tissue inhibitors of metalloproteinases (TIMPs)18. In phase I/II/III brain tumor clinical trials, most anti-angiogenic drugs target VEGF signaling due to its central role in angiogenesis, exemplified by bevacizumab, a monoclonal antibody that inhibits neovascularization through VEGF blockade19,20,21. However, recent phase II/III trials showed no significant overall survival benefit for advanced glioblastoma patients receiving bevacizumab plus lomustine versus lomustine alone19,20,21,22. Critically, anti-VEGF therapy improves vascular abnormalities but only suppresses angiogenesis without direct tumor cytotoxicity and may induce treatment resistance. Consequently, targeting a single or a few angiogenic regulators fails to achieve a radical cure; effective strategies require concurrent suppression of angiogenesis and blockade of tumor invasion and metastasis through vascular pathways. The diagnostic and prognostic value of proteins in gliomas is well-established, yet malignant glioma treatment has seen limited progress over the decades.

Proteomics can globally analyze all proteins in biological samples, including expression profiles, post-translational modifications (PTMs), and interactions, with mass spectrometry (MS)-based techniques, including top-down (TDP) and bottom-up (BUP) approaches. High-throughput MS enables the identification of tens of thousands of proteins per run, making it essential for studying tumor mechanisms and therapeutic target identification. Protein abundance and its PTMs can be identified in glioma tissues and liquid biopsies such as blood and its derivatives23,24,25, cerebrospinal fluid26, and urine27. For tumor vasculature, abnormal protein expressions and PTMs drive pathological vascular changes. The protein alterations in glioma neovascularization can be identified with proteomics, which drives the protocol development for reliable proteomic profiling of glioma neovascularization.

However, neovascular tissues are only one of the components in the entire glioma tissues. Isolation and purification of neovascular tissues from the entire glioma tissues is the key step for accurate quantitative proteomics analysis of glioma angiogenesis. Laser-capture microdissection (LCM)28,29,30,31 is an effective approach to isolate and purify neovascular tissues from entire glioma tissues. It is very hard to obtain a sufficient amount of neovascular tissue proteins for quantitative proteomics. In 2007, a research team tried to use LCM to separate neovascular tissues from grade IV glioma for proteomics analysis, which identified only four proteins with MS32. Subsequently, in 2012, they continuously used LCM to enrich neovascular tissues of grade IV gliomas for quantitative proteomics analysis, which only identified 29 proteins with MS33. Those two glioma angiogenesis proteomics studies show a very low proteomic throughput in glioma angiogenesis, which cannot represent the complex proteomic landscape of glioma angiogenesis.

This protocol developed a procedure that enables the acquisition of sufficient amounts of neovascular proteins for quantitative proteomics analysis of glioma angiogenesis, thereby constructing a large-scale proteomic landscape of tumor angiogenesis. This has significant scientific merit for the discovery of effective angiogenesis-based biomarkers and anti-angiogenesis therapeutic targets/drugs.

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Protocol

This protocol involving human tissue samples was approved by the Medical Ethics Review Committee of Shandong First Medical University (Approval no. R202211090133), and informed consent was obtained from patients and patient’s family. The glioma samples used in this study were all obtained from neurosurgical resected specimens of the neurosurgery department at Xiangya Hospital of Central South University, including 7 cases of astrocytoma grade III tissues. The normal control vessels (n = 3) were taken from microvessels within damaged brain tissue removed due to a cerebral trauma. All patients were first-time attendees without prior radiotherapy or chemotherapy before surgery, and they were all diagnosed by the pathology department of Xiangya Hospital, Central South University. Three normal control vascular tissues are obtained from neurosurgical resection of very tiny blood vessels from normal brain damaged by external force (without glioma) at Xiangya Hospital. The reagents and the equipment used are listed in the Table of Materials.

1. Using LCM to isolate neovascular tissues from human glioma tissues

  1. Prepare LCM slide: Manually coat polyethylene naphthalate (PEN) membrane on reusable metal slides, and store coated slides in -80 °C.
    NOTE: Pre-coated metal slides will be used to directly adhere the frozen tissue sections.
  2. Prepare toluidine blue staining solution (0.5%) : Dissolve 0.25 g toluidine blue in 40 mL ddH₂O. Add one protease inhibitor tablet and mix until fully dissolved. Adjust volume to 50 mL with ddH₂O, filter, and store on ice.
  3. Process tissue: Maintain cryostat at operating temperature (-25 °C chamber/specimen head). Retrieve glioma tissue from -80 °C storage, transfer on ice, remove blood with filter paper, and select appropriate tissue blocks for embedding.
  4. Embed tissue protocol : Pre-cool specimen disc. Apply the embedding medium and rapidly embed the tissue block. Freeze the entire block at -25 °C for 10 min for hardening.
  5. Section tissue and screen vessels: Section tissues at 8 µm thickness. Mount tissue sections on standard slides, stain tissue with toluidine blue staining solution, and screen neovascular vessels under optical microscopy.
    NOTE: If no vessels are detected, discard 3–5 tissue sections and repeat the staining cycle until neovascular vessel regions appear.
  6. Mount vessel-positive tissue section on the prepared LCM slide : Transfer vessel-positive sections onto PEN-coated LCM slides, seal, label, and store at -80 °C.
  7. Fix LCM slide-mounted tissue sections in pre-chilled 70% ethanol (4 °C, 1–2 min); drain and stain in 0.5% toluidine blue (4 °C, 30 s); Drain stain, rinse twice in ddH₂O (4 °C; two separate wash baths), drain, incubate in protease inhibitor solution (30 s), and store at -80 °C.
  8. Start the LCM instrument in advance, preheat for 30 min, use a solid-state infrared laser to preheat for another 30 min, and then install a 500 µL microcentrifuge tube collection device.
  9. Load the tissue sample-mounted LCM slices (stored at -80 °C) on the prepared LCM instrument, and adjust the focus on the neovascular vessels in glioma tissues with the assistance of the compatible software.
  10. Precisely outline the laser-marking line along the outer edge of the neovascular vessel (avoid the vascular wall), use the laser to burn and separate the neovascular vessels, and then collect them into the EP tube cap.
    NOTE: Use the second laser burn to ensure that the neovascular vessels have completely detached.
  11. Invert and seal the collected neovascular vessel sample tube, and store it in a -80 °C freezer.

2. Extraction of proteins from LCM-isolated glioma neovascular tissues and quantification of protein content

  1. Prepare protein extraction buffer containing 4% sodium dodecyl sulfate (SDS), 1 mM dithiothreitol (DTT), and 150 mM Tris-HCl (pH 8.0).
    NOTE: SDS is a hazardous dust that severely irritates eyes, skin, and the respiratory tract, which must be weighed in a fume hood; and DTT is an irritant, which must be weighed in a fume hood.
  2. Resuspend LCM-collected glioma neovascular vessel tissues in extraction buffer. Sonicate on ice-water bath for 1 min for complete lysis.
  3. Centrifuge at 16,000 x g for 10 min, and transfer supernatant to new microcentrifuge tubes.
  4. Aliquot minimal supernatant for protein quantification. Add DTT to the remaining supernatant (final concentration: 1 mM DTT), mix gently by pipetting to avoid bubbles. Aliquot and store at -80 °C.
  5. Perform bicinchoninic acid (BCA) protein assay following the steps below:
    1. Prepare working solution (Reagent A:B = 50:1).
    2. Prepare bovine serum albumin (BSA) standard (0.5 mg/mL).
    3. Add the corresponding solutions according to Table 1.
    4. Mix samples/standards with the working solution, and transfer to a 96-well plate.
    5. Incubate at 37 °C for 30 min.
    6. Measure absorbance at 562 nm.
    7. Calculate concentrations against the standard curve.

3. Using quantitative proteomics to analyze the prepared neovascular proteins

  1. Prepare tryptic peptide samples of the extracted neovascular proteins.
    1. Add ice-cold acetone to each extracted protein sample (tumor; control) (sample: acetone = 1:5, v/v), mix thoroughly, and incubate at -20 °C for 1 h. Centrifuge at 12,000 x g (4 °C) for 15 min. Discard supernatant to remove protein extraction reagents (SDS, DTT, and Tris-HCL), and resuspend pellet in 20 µL dissolution buffer with 1 µL 1% SDS.
      NOTE: Use acetone with adequate ventilation while wearing safety goggles, keep away from ignition sources, and dispose of waste in a dedicated, spark-proof container.
    2. Add 2 µL reducing reagent (DTT) to each sample, mix thoroughly, and incubate at 60 °C for 1 h. Add 1 µL cysteine-blocking reagent (iodoacetamide), then incubate at room temperature for 10 min.
    3. Add trypsin (protein: trypsin = 20:1, v/v) to each sample. Vortex-mix and incubate at 37 °C overnight.
  2. Use Isobaric targs for relative and absolute quantification (iTRAQ) reagents to label the prepared tryptic peptides.
    NOTE: In this protocol, six-plex iTRAQ reagents (plex tags 113, 114, 115, 116, 117, and 118) is used to label tryptic peptide samples (tumor; control), each type of sample is labeled three times (Table 2).
    1. Reconstitute each iTRAQ reagent tube with 50 µL isopropanol.
    2. Add tryptic peptide samples to the corresponding reagent tubes, incubate at room temperature for 1 h.
    3. Terminate reactions by adding 3× volume ultrapure water, then incubate for 30 min at room temperature.
    4. Equally pool the labeled six samples, mix thoroughly, and lyophilize.
  3. Use strong cation exchange (SCX) to fractionate the iTRAQ-labeled tryptic peptide sample.
    1. Reconstitute lyophilized peptides in 2 mL Solution A (10 mM KH₂PO₄, 25% ACN, pH 2.7). Load onto a Sulfoethyl functionalized cation exchange column (4.6 × 100 mm, 5 µm, 200 Å).
    2. Elute with a constant flow rate of 1 mL/min using solution B (500 mM KCl, 10 mM KH₂PO₄, 25 % ACN, pH 2.7): 0 %→10 % (2 min) → 10 %→20 % (25 min) → 20 %→45 % (5 min) → 50 %→100 % (5 min). Monitor at 214 nm throughout the process, collect every minute per tube, for a total of 30 fractions.
    3. Concentrate each fraction by vacuum centrifugation, and then reconstitute with 40 µL of 0.1% (v/v) trifluoroacetic acid (TFA). All 30 fractions are stored in a -80 °C freezer.
      NOTE: TFA is highly corrosive and toxic, causes severe skin and eye damage, and irritates the respiratory system; it must be handled in a fume hood with appropriate PPE that includes acid-resistant gloves, goggles, and a lab coat, and waste must be collected as acidic waste.
  4. Use LC-MS/MS to analyze each fractionated peptide sample.
    1. Use a C18 reversed-phase solid phase extraction cartridge (standard density, bed I.D. 7 mm, volume 3 mL) to desalt.
    2. Use a C18 reverse-phase liquid chromatography (LC) column (10 cm length, 75 µm i.d., 3 µm resin) to elute 10 µL of each fraction with solution C (0.1 % Formic acid) and a gradient of solution D (80 % acetonitrile and 0.1 % Formic acid) at a rate of 250 nL/min for 140 min.
    3. Online input the LC-separated peptides into a high-resolution mass spectrometer for MS/MS analysis.
    4. Convert raw spectrum files (.raw) generated by Q Exactive to .mgf format with Proteome Discoverer 1.4, and then submit these .mgf files to the MASCOT 2.2 server the software’s built-in module for database searching.
      NOTE: The Uniprot human database (146,704 total sequences; downloaded July 20, 2025, from http://www.uniprot.org) is used with detailed search parameters (Table 3).
    5. Retrieve the resulting search files (.dat) from the MASCOT server back into Proteome Discoverer 1.4. Data for filtration at a false discovery rate (FDR) threshold of <0.01 to obtain high-confidence identifications.
    6. Identify the protein amino acid sequence with MS/MS data, quantify the protein abundance with iTRAQ reporter ion intensities.
    7. Calculate the experimental reproducibility to determine the cut-off value of differentially expressed proteins (DEPs) plus a significance p<0,05.
      NOTE: In this study, the cut-off value is determined as fold-change (FC) > 1.4, which can effectively remove the systematic errors.
  5. Analyze the functional characteristics of glioma neovascular DEPs.
    1. Access the DAVID (Database for Annotation, Visualization and Integrated Discovery, version 6.7; https://david.ncifcrf.gov/) homepage and select Start Analysis in the menu bar to initiate analysis.
    2. Select the left panel serves as the upload interface; enter protein or gene IDs into the Step 1 input box as prompted (utilizing copy-paste functionality).
    3. For Step 2, select the identifier type matching the uploaded proteins; this study used UniProt Accession.
    4. For Step 3, select gene list or background settings: Gene enrichment analysis requires choosing a background gene set. The system defaults to the entire genome of the species, or users may upload a custom protein/gene list as the background.
    5. Click on Submit List to submit the entries and initiate analysis.
      NOTE: Analysis results are displayed on the right side of the “Annotation Summary Results” panel. Users can select outputs per analytical needs; this study specifically utilized the GOTERM_BP_FAT list to analyze biological processes involving DEPs.
    6. Obtain biological processes (BPs), cellular components (CCs), molecular functions (MFs), and KEGG pathways for glioma neovascular DEPs.

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Results

A sufficient amount of LCM-isolated protein samples was obtained for quantitative proteomics

To obtain reliable proteomic profiles of differentially expressed proteins in glioma neovascular tissues, this study selected the most prevalent glioma subtype, astrocytoma Grade III, and isolated neovascular tissues using LCM due to the availability of enriched neovascular vessels. Moreover, quantitative proteomics requires ≥400 µg protein per sample. This study used LCM technology to...

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Discussion

LCM combined with isotope-labelling quantitative proteomics identified the first large-scale proteomic profile of glioma angiogenesis

Tumor angiogenesis is a critical pathological hallmark of gliomas, closely associated with their high invasiveness and therapeutic resistance6,7.​​ Traditional anti-angiogenic therapies (such as VEGF inhibitors) show limited clinical efficacy due to target monotony and compen...

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Disclosures

The authors declare no competing interests.

Acknowledgements

This work was supported by the China National Nature Scientific Funds (81272798 to X.Z., and 82203592 to N.L.), the Shandong Provincial Natural Science Foundation (ZR2022QH112 to N.L.), Shandong Provincial Taishan Scholar Engineering Project Special Funds (NO.tstp20221143 to X.Z.), the China National High-end Foreign Expert Talent Program (H20240743; Foreign Expert: X.Z.), and Shandong First Medical University Scientific Research Cultivation Project in Emerging Strategic Fields (202403).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
96-Well PlateThermo Fisher Scientific (USA)468667Consumables
AcetoneNanJing Reagent (China)C0720114023Reagent
AcetonitrileMerck (China)CN34892Reagent
BCA Protein Assay KitAbcam (UK)ab102536Reagent
Bovine serum albumin (BSA) standardSolarbio (China)PC0001-1Reagent
centrifuge tube(50ML)Kirgen (China)KG2811Consumables
Cryostat MicrotomeLeica (Germany)C1900Equipment
CysteineAladdin (China)C108238Reagent
DL-Dithiothreitol(DTT)Amresco (USA)D8220Reagent
EASY-Spray C18 Reversed Phase HPLC ColumnThermo Scientific (USA)ES800AConsumables
Electronic BalanceSartorius (USA)BSA224SEquipment
Embedding mediumLeica (Germany)39475237Consumables
Empore SPE Cartridges C18Sigma-Aldrich (USA)66872-UConsumables
Eppendorf TubeKirgen (China)KG2211Consumables
Filter PaperCytiva (USA)Grade42Consumables
Formic acidAladdin (China)F112032Reagent
Glass BeakerSHUBO (China)1015479Consumables
Glass Graduated CylinderSHUBO (China)1601Consumables
‌High-Resolution Liquid Chromatography-Mass SpectrometryThermo Scientific (USA)Q-ExactiveEquipment
Insulated ContainerFhefon (China)LCZYX-12Consumables
Inverted Microscope Leica (Germany)XDS-1Equipment
IodoacetamideSolarbio (China)I8010Reagent
IsopropanolAladdin (China)I112018Reagent
iTRAQ reagentsSigma-Aldrich (USA)4381663Reagent
KClBeyotime (China)ST1596Reagent
KH2PO4ShangHai YuanYe (China)S24278Reagent
Laser-capture microdissectionLeica (Germany)LMD 6000Equipment
Laser-capture microdissection slideLeica (Germany)3800040Consumables
Leica High-Profile Disposable BladesLeica (Germany)14035838926Consumables
Liquid Nitrogen ContainerKEYICRYO (China)YDS-3Equipment
Magnetic Hotplate StirrerShanghaiSILE (China)LAB-0002-0068-SHSLEquipment
Microplate ReaderTECAN (Switzerland)INFNITE F200Equipment
PEN MembraneLeica (Germany)11505189Consumables
PH meterSartorius (USA)PB-10Equipment
PipetteEppendorf (Germany)3123000268Equipment
PipetteEppendorf (Germany)3123000241Equipment
PipetteEppendorf (Germany)3123000225Equipment
PolySULFOETHYL APolyLC Inc (USA)104SE0303Consumables
Protease inhibitorRoche (Switzerland)05892791001Reagent
Refrigerated centrifugeBeckman (USA)AllegraTM X-22REquipment
RefrigeratorMEILING (China)BCD-180KCTEquipment
Sodium dodecyl sulfate (SDS)Amresco (USA)1027P033Reagent
Staining JarBeyotime (China)FG005Consumables
Stainless Steel ScoopMuzixing (China)XYS-101Consumables
TipsKirgen (China)KG1313Consumables
Toluidine blue staining solutionSigma-Aldrich (USA)198161-5G/198161-25GReagent
Trifluoroacetic acidAladdin (China)T103293Reagent
Tris-HClAmresco (USA)T8230Reagent
Trypsin-EDTA solutionSolarbio (China)T1300Reagent
Ultra-Low Temperature FreezerSanyo (Japan)MDF-382EEquipment
Ultra-pure Water PurifierELGA (UK)PureLAB flexEquipment
Ultrasonic Cell DisruptorQSONICA (USA)Q500Equipment
Vacuum freeze dryerThermo (USA)SNL 315SVEquipment
Vortex mixerCrystal Technology & Industries (USA)HYQ-3110Equipment
Water bath incubatorShanghai Yiheng Technology Instrument (China)HWS-26Equipment
Weighing PaperCole-parmer (USA)133802Consumables

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Glioma AngiogenesisLaser Capture MicrodissectionTumor VasculatureDifferential Protein ExpressionLCMSMS AnalysisProtein Interaction NetworkTumor BiomarkersAnti-Angiogenesis Therapy
Preparation Of Neovascular Tissues from Human Glioma Tissues for Quantitative Proteomics Analysis of Tumor Angiogenesis
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