Name: Preparation Of Neovascular Tissues from Human Glioma Tissues for Quantitative Proteomics Analysis of Tumor Angiogenesis (Video) | JoVE Journal
Uploaded: 2026-03-20T14:20:00.000+00:00
Duration: 9 min 33 s
Description: Discover laser-capture microdissection and quantitative proteomics for profiling glioma neovascular tissues. This method identifies differentially expressed proteins to reveal tumor angiogenesis mechanisms and therapeutic targets.

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5 minutes

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).

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&#8217;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
<ol>
	<li>Prepare LCM slide: Manually coat polyethylene naphthalate (PEN) membrane on reusable metal slides, and store coated slides in -80 &#176;C. 
	NOTE: Pre-coated metal slides will be used to directly adhere the frozen tissue sections.</li>
	<li>Prepare toluidine blue staining solution (0.5%) : Dissolve 0.25 g toluidine blue in 40 mL ddH&#8322;O. Add one protease inhibitor tablet and mix until fully dissolved. Adjust volume to 50 mL with ddH&#8322;O, filter, and store on ice.</li>
	<li>Process tissue: Maintain cryostat at operating temperature (-25 &#176;C chamber/specimen head). Retrieve glioma tissue from -80 &#176;C storage, transfer on ice, remove blood with filter paper, and select appropriate tissue blocks for embedding.</li>
	<li>Embed tissue protocol : Pre-cool specimen disc. Apply the embedding medium and rapidly embed the tissue block. Freeze the entire block at -25 &#176;C for 10 min for hardening.</li>
	<li>Section tissue and screen vessels: Section tissues at 8 &#181;m 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&#8211;5 tissue sections and repeat the staining cycle until neovascular vessel regions appear.</li>
	<li>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 &#176;C.</li>
	<li>Fix LCM slide-mounted tissue sections in pre-chilled 70% ethanol (4 &#176;C, 1&#8211;2 min); drain and stain in 0.5% toluidine blue (4 &#176;C, 30 s); Drain stain, rinse twice in ddH&#8322;O (4 &#176;C; two separate wash baths), drain, incubate in protease inhibitor solution (30 s), and store at -80 &#176;C.</li>
	<li>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 &#181;L microcentrifuge tube collection device.</li>
	<li>Load the tissue sample-mounted LCM slices (stored at -80 &#176;C) on the prepared LCM instrument, and adjust the focus on the neovascular vessels in glioma tissues with the assistance of the compatible software.</li>
	<li>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.</li>
	<li>Invert and seal the collected neovascular vessel sample tube, and store it in a -80 &#176;C freezer.</li>
</ol>
2. Extraction of proteins from LCM-isolated glioma neovascular tissues and quantification of protein content 
<ol>
	<li>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.</li>
	<li>Resuspend LCM-collected glioma neovascular vessel tissues in extraction buffer. Sonicate on ice-water bath for 1 min for complete lysis.</li>
	<li>Centrifuge at 16,000 x g for 10 min, and transfer supernatant to new microcentrifuge tubes.</li>
	<li>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 &#176;C.</li>
	<li>Perform bicinchoninic acid (BCA) protein assay following the steps below:
	<ol>
		<li>Prepare working solution (Reagent A:B = 50:1).</li>
		<li>Prepare bovine serum albumin (BSA) standard (0.5 mg/mL).</li>
		<li>Add the corresponding solutions according to Table 1.</li>
		<li>Mix samples/standards with the working solution, and transfer to a 96-well plate.</li>
		<li>Incubate at 37 &#176;C for 30 min.</li>
		<li>Measure absorbance at 562 nm.</li>
		<li>Calculate concentrations against the standard curve.</li>
	</ol>
	</li>
</ol>
3. Using quantitative proteomics to analyze the prepared neovascular proteins
<ol>
	<li>Prepare tryptic peptide samples of the extracted neovascular proteins.
	<ol>
		<li>Add ice-cold acetone to each extracted protein sample (tumor; control) (sample: acetone = 1:5, v/v), mix thoroughly, and incubate at -20 &#176;C for 1 h. Centrifuge at 12,000 x g (4 &#176;C) for 15 min. Discard supernatant to remove protein extraction reagents (SDS, DTT, and Tris-HCL), and resuspend pellet in 20 &#181;L dissolution buffer with 1 &#181;L 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.</li>
		<li>Add 2 &#181;L reducing reagent (DTT) to each sample, mix thoroughly, and incubate at 60 &#176;C for 1 h. Add 1 &#181;L cysteine-blocking reagent (iodoacetamide), then incubate at room temperature for 10 min.</li>
		<li>Add trypsin (protein: trypsin = 20:1, v/v) to each sample. Vortex-mix and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Reconstitute each iTRAQ reagent tube with 50 &#181;L isopropanol.</li>
		<li>Add tryptic peptide samples to the corresponding reagent tubes, incubate at room temperature for 1 h.</li>
		<li>Terminate reactions by adding 3&#215; volume ultrapure water, then incubate for 30 min at room temperature.</li>
		<li>Equally pool the labeled six samples, mix thoroughly, and lyophilize.</li>
	</ol>
	</li>
	<li>Use strong cation exchange (SCX) to fractionate the iTRAQ-labeled tryptic peptide sample.
	<ol>
		<li>Reconstitute lyophilized peptides in 2 mL Solution A (10 mM KH&#8322;PO&#8324;, 25% ACN, pH 2.7). Load onto a Sulfoethyl functionalized cation exchange column (4.6 &#215; 100 mm, 5 &#181;m, 200 &#197;).</li>
		<li>Elute with a constant flow rate of 1 mL/min using solution B (500 mM KCl, 10 mM KH&#8322;PO&#8324;, 25 % ACN, pH 2.7): 0 %&#8594;10 % (2 min) &#8594; 10 %&#8594;20 % (25 min) &#8594; 20 %&#8594;45 % (5 min) &#8594; 50 %&#8594;100 % (5 min). Monitor at 214 nm throughout the process, collect every minute per tube, for a total of 30 fractions.</li>
		<li>Concentrate each fraction by vacuum centrifugation, and then reconstitute with 40 &#181;L of 0.1% (v/v) trifluoroacetic acid (TFA). All 30 fractions are stored in a -80 &#176;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.</li>
	</ol>
	</li>
	<li>Use LC-MS/MS to analyze each fractionated peptide sample.
	<ol>
		<li>Use a C18 reversed-phase solid phase extraction cartridge (standard density, bed I.D. 7 mm, volume 3 mL) to desalt.</li>
		<li>Use a C18 reverse-phase liquid chromatography (LC) column (10 cm length, 75 &#181;m i.d., 3 &#181;m resin) to elute 10 &#181;L 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.</li>
		<li>Online input the LC-separated peptides into a high-resolution mass spectrometer for MS/MS analysis.</li>
		<li>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&#8217;s built-in module for database searching. 
		NOTE: The&#160;Uniprot human&#160;database (146,704 total sequences; downloaded July 20, 2025, from&#160;http://www.uniprot.org) is used with detailed search parameters (Table 3).</li>
		<li>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 &#60;0.01 to obtain high-confidence identifications.</li>
		<li>Identify the protein amino acid sequence with MS/MS data, quantify the protein abundance with iTRAQ reporter ion intensities.</li>
		<li>Calculate the experimental reproducibility to determine the cut-off value of differentially expressed proteins (DEPs) plus a significance p&#60;0,05. 
		NOTE: In this study, the cut-off value is determined as fold-change (FC) &#62; 1.4, which can effectively remove the systematic errors.</li>
	</ol>
	</li>
	<li>Analyze the functional characteristics of glioma neovascular DEPs.
	<ol>
		<li>Access the DAVID (Database for Annotation, Visualization and Integrated Discovery, version 6.7;&#160;https://david.ncifcrf.gov/) homepage and select Start Analysis in the menu bar to initiate analysis.</li>
		<li>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).</li>
		<li>For Step 2, select the identifier type matching the uploaded proteins; this study used&#160;UniProt Accession.</li>
		<li>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.</li>
		<li>Click on Submit List to submit the entries and initiate analysis. 
		NOTE: Analysis results are displayed on the right side of the &#8220;Annotation Summary Results&#8221; panel. Users can select outputs per analytical needs; this study specifically utilized the&#160;GOTERM_BP_FAT&#160;list to analyze biological processes involving DEPs.</li>
		<li>Obtain biological processes (BPs), cellular components (CCs), molecular functions (MFs), and KEGG pathways for glioma neovascular DEPs.</li>
	</ol>
	</li>
</ol>

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<li>Mustafa, D. A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+glioma+neovascularization-related+proteins+by+using+MALDI-FTMS+and+nano-LC+fractionation+to+microdissected+tumor+vessels%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Identification of glioma neovascularization-related proteins by using MALDI-FTMS and nano-LC fractionation to microdissected tumor vessels.</a> Mol Cell Proteomics. 6, 1147-1157 (2007).</li>
<li>Mustafa, D. A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+proteome+comparison+between+physiological+angiogenesis+and+angiogenesis+in+glioblastoma%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">A proteome comparison between physiological angiogenesis and angiogenesis in glioblastoma.</a> Mol Cell Proteomics. 11 (6), M111.008466(2012).</li>
<li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accurate+qualitative+and+quantitative+proteomic+analysis+of+clinical+hepatocellular+carcinoma+using+laser+capture+microdissection+coupled+with+isotope-coded+affinity+tag+and+two-dimensional+liquid+chromatography+mass+spectrometry%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry.</a> Mol Cell Proteomics. 3 (4), 399-409 (2004).</li>
<li>Woo, J., Schoenfeld, M., Sun, X., Iraguha, T., Zhou, Z., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+paneth+cell-enriched+proteome+enabled+by+laser+capture+microdissection%5BTitle%5D)+AND+%22Proteome+Res%22%5BJournal%5D)">Mouse paneth cell-enriched proteome enabled by laser capture microdissection.</a> Proteome Res. 21 (10), 2435-2442 (2022).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+phosphoproteomics+reveals+molecular+pathway+network+alterations+in+human+early-stage+primary+hepatic+carcinomas%3A+potential+for+3P+medical+approach%5BTitle%5D)+AND+%22EPMA+J%22%5BJournal%5D)">Quantitative phosphoproteomics reveals molecular pathway network alterations in human early-stage primary hepatic carcinomas: potential for 3P medical approach.</a> EPMA J. 14 (3), 477-502 (2023).</li>
<li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitinomics+revealed+disease-+and+stage-specific+patterns+relevant+for+the+3PM+approach+in+human+sigmoid+colon+cancers%5BTitle%5D)+AND+%22EPMA+J%22%5BJournal%5D)">Ubiquitinomics revealed disease- and stage-specific patterns relevant for the 3PM approach in human sigmoid colon cancers.</a> EPMA J. 14 (3), 503-525 (2023).</li>
<li>Wen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+acetylomics+revealed+acetylation-mediated+molecular+pathway+network+changes+in+human+nonfunctional+pituitary+neuroendocrine+tumors%5BTitle%5D)+AND+%22Front+Endocrinol+%28Lausanne%29%22%5BJournal%5D)">Quantitative acetylomics revealed acetylation-mediated molecular pathway network changes in human nonfunctional pituitary neuroendocrine tumors.</a> Front Endocrinol (Lausanne). 12, 753606(2021).</li>
<li>Su, J., Yang, L., Sun, Z., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Personalized+drug+therapy%3A+Innovative+concept+guided+with+proteoformics%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Personalized drug therapy: Innovative concept guided with proteoformics.</a> Mol Cell Proteomics. 23 (3), 100737(2024).</li>
<li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+does+one-dimensional+gel+electrophoresis-based+western+blotting+data+really+mean+in+the+reality+of+proteoforms%5BTitle%5D)+AND+%22Talanta%22%5BJournal%5D)">What does one-dimensional gel electrophoresis-based western blotting data really mean in the reality of proteoforms.</a> Talanta. 295, 128266(2025).</li>
<li>Fang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteoformics%3A+Current+status+and+future+perspectives%5BTitle%5D)+AND+%22J+Proteomics%22%5BJournal%5D)">Proteoformics: Current status and future perspectives.</a> J Proteomics. 321, 105524(2025).</li>
<li>Lu, M., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+crucial+role+of+multiomic+approach+in+cancer+research+and+clinically+relevant+outcomes%5BTitle%5D)+AND+%22EPMA+J%22%5BJournal%5D)">The crucial role of multiomic approach in cancer research and clinically relevant outcomes.</a> EPMA J. 9 (1), 77-102 (2018).</li>
<li>Jia, W., Gong, X., Ye, Z., Li, N., Zhan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitroproteomics+is+instrumental+for+stratification+and+targeted+treatments+of+astrocytoma+patients%3A+Expert+recommendations+for+advanced+3PM+approach+with+improved+individual+outcomes%5BTitle%5D)+AND+%22EPMA+J%22%5BJournal%5D)">Nitroproteomics is instrumental for stratification and targeted treatments of astrocytoma patients: Expert recommendations for advanced 3PM approach with improved individual outcomes.</a> EPMA J. 14 (4), 673-696 (2023).</li>
<li>Gong, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glioma+angiogenesis+phosphoproteome+landscape+and+biomarker+sets+identified+with+phenome-centered+multiomics+toward+3P+medical+approaches%5BTitle%5D)+AND+%22EPMA+J%22%5BJournal%5D)">Glioma angiogenesis phosphoproteome landscape and biomarker sets identified with phenome-centered multiomics toward 3P medical approaches.</a> EPMA J. 16 (4), 831-856 (2025).</li>
</ol>

The authors declare no competing interests.

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.&#8203;&#8203; Traditional anti-angiogenic therapies (such as VEGF inhibitors) show limited clinical efficacy due to target monotony and compen...

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 &#946; (TGF-&#946;)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.

<table><tbody><tr><td>96-Well Plate</td><td>Thermo Fisher Scientific (USA)</td><td>468667</td><td>Consumables</td></tr><tr><td>Acetone</td><td>NanJing Reagent (China)</td><td>C0720114023</td><td>Reagent</td></tr><tr><td>Acetonitrile</td><td>Merck (China)</td><td>CN34892</td><td>Reagent</td></tr><tr><td>BCA Protein Assay Kit</td><td>Abcam (UK)</td><td>ab102536</td><td>Reagent</td></tr><tr><td>Bovine serum albumin (BSA) standard</td><td>Solarbio (China)</td><td>PC0001-1</td><td>Reagent</td></tr><tr><td>centrifuge tube(50ML)</td><td>Kirgen (China)</td><td>KG2811</td><td>Consumables</td></tr><tr><td>Cryostat Microtome</td><td>Leica (Germany)</td><td>C1900</td><td>Equipment</td></tr><tr><td>Cysteine</td><td>Aladdin (China)</td><td>C108238</td><td>Reagent</td></tr><tr><td>DL-Dithiothreitol(DTT)</td><td>Amresco (USA)</td><td>D8220</td><td>Reagent</td></tr><tr><td>EASY-Spray C18 Reversed Phase HPLC Column</td><td>Thermo Scientific 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(China)</td><td>LCZYX-12</td><td>Consumables</td></tr><tr><td>Inverted Microscope&amp;nbsp;</td><td>Leica (Germany)</td><td>XDS-1</td><td>Equipment</td></tr><tr><td>Iodoacetamide</td><td>Solarbio (China)</td><td>I8010</td><td>Reagent</td></tr><tr><td>Isopropanol</td><td>Aladdin (China)</td><td>I112018</td><td>Reagent</td></tr><tr><td>iTRAQ reagents</td><td>Sigma-Aldrich (USA)</td><td>4381663</td><td>Reagent</td></tr><tr><td>KCl</td><td>Beyotime (China)</td><td>ST1596</td><td>Reagent</td></tr><tr><td>KH2PO4</td><td>ShangHai YuanYe (China)</td><td>S24278</td><td>Reagent</td></tr><tr><td>Laser-capture microdissection</td><td>Leica (Germany)</td><td>LMD 6000</td><td>Equipment</td></tr><tr><td>Laser-capture microdissection slide</td><td>Leica (Germany)</td><td>3800040</td><td>Consumables</td></tr><tr><td>Leica High-Profile Disposable Blades</td><td>Leica (Germany)</td><td>14035838926</td><td>Consumables</td></tr><tr><td>Liquid Nitrogen Container</td><td>KEYICRYO (China)</td><td>YDS-3</td><td>Equipment</td></tr><tr><td>Magnetic Hotplate Stirrer</td><td>ShanghaiSILE (China)</td><td>LAB-0002-0068-SHSL</td><td>Equipment</td></tr><tr><td>Microplate Reader</td><td>TECAN (Switzerland)</td><td>INFNITE F200</td><td>Equipment</td></tr><tr><td>PEN&amp;nbsp;Membrane</td><td>Leica (Germany)</td><td>11505189</td><td>Consumables</td></tr><tr><td>PH meter</td><td>Sartorius (USA)</td><td>PB-10</td><td>Equipment</td></tr><tr><td>Pipette</td><td>Eppendorf (Germany)</td><td>3123000268</td><td>Equipment</td></tr><tr><td>Pipette</td><td>Eppendorf (Germany)</td><td>3123000241</td><td>Equipment</td></tr><tr><td>Pipette</td><td>Eppendorf (Germany)</td><td>3123000225</td><td>Equipment</td></tr><tr><td>PolySULFOETHYL A</td><td>PolyLC Inc (USA)</td><td>104SE0303</td><td>Consumables</td></tr><tr><td>Protease inhibitor</td><td>Roche (Switzerland)</td><td>05892791001</td><td>Reagent</td></tr><tr><td>Refrigerated centrifuge</td><td>Beckman (USA)</td><td>AllegraTM X-22R</td><td>Equipment</td></tr><tr><td>Refrigerator</td><td>MEILING (China)</td><td>BCD-180KCT</td><td>Equipment</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Amresco (USA)</td><td>1027P033</td><td>Reagent</td></tr><tr><td>Staining Jar</td><td>Beyotime (China)</td><td>FG005</td><td>Consumables</td></tr><tr><td>Stainless Steel Scoop</td><td>Muzixing (China)</td><td>XYS-101</td><td>Consumables</td></tr><tr><td>Tips</td><td>Kirgen (China)</td><td>KG1313</td><td>Consumables</td></tr><tr><td>Toluidine blue staining solution</td><td>Sigma-Aldrich (USA)</td><td>198161-5G/198161-25G</td><td>Reagent</td></tr><tr><td>Trifluoroacetic acid</td><td>Aladdin (China)</td><td>T103293</td><td>Reagent</td></tr><tr><td>Tris-HCl</td><td>Amresco (USA)</td><td>T8230</td><td>Reagent</td></tr><tr><td>Trypsin-EDTA solution</td><td>Solarbio (China)</td><td>T1300</td><td>Reagent</td></tr><tr><td>Ultra-Low Temperature Freezer</td><td>Sanyo (Japan)</td><td>MDF-382E</td><td>Equipment</td></tr><tr><td>Ultra-pure Water Purifier</td><td>ELGA (UK)</td><td>PureLAB flex</td><td>Equipment</td></tr><tr><td>Ultrasonic Cell Disruptor</td><td>QSONICA (USA)</td><td>Q500</td><td>Equipment</td></tr><tr><td>Vacuum freeze dryer</td><td>Thermo (USA)</td><td>SNL 315SV</td><td>Equipment</td></tr><tr><td>Vortex mixer</td><td>Crystal Technology &amp;amp; Industries (USA)</td><td>HYQ-3110</td><td>Equipment</td></tr><tr><td>Water bath incubator</td><td>Shanghai Yiheng Technology Instrument (China)</td><td>HWS-26</td><td>Equipment</td></tr><tr><td>Weighing Paper</td><td>Cole-parmer (USA)</td><td>133802</td><td>Consumables</td></tr></tbody></table>

preparation of neovascular tissues from human glioma tissues for quantitative proteomics analysis of tumor angiogenesis

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

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.

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...

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.

Glioma represents primary malignant tumors of the central nervous system with significant incidence and mortality. Tumor angiogenesis is the hallmark of ...

We perform proteomic profiling of glioma vasculature across tumor grades to identify biomarkers and neuropathic targets, reviving mechanisms driving angiogenesis and tumor progression. Existing methods face protein degradation in complete lysates, scarce grade one samples, and nonvascular contamination drawing LCM. This protocol optimizes handling and improves vascular protein profiling accuracy.To begin, obtain polyethylene naphtholate membrane and reusable slides. Manually coat the membrane onto the reusable slide and store the coated slide at minus 80 degrees Celsius. Maintain the cryostat at minus 25 degrees Celsius for both the chamber and specimen head.Retrieve glioma tissue from minus 80 degrees Celsius storage and transfer it on ice. Remove residual blood with filter paper. Apply embedding medium to a pre-cool disc and rapidly embed the tissue block.Freeze the entire block at minus 25 degrees Celsius for 10 minutes to harden. After sectioning the tissue, mount the tissue sections onto a standard slide. Stain the sections with the 0.5%toluidine blue solution and screen neovascular vessels under an optical microscope.Then transfer the vessel positive tissue section onto the polyethylene naphtholate coated laser, capture micro dissection slide. Seal the slide, label it, and store at minus 80 degrees Celsius. Fix the slide mounted tissue, stain it, and cryopreserve the processed sample at minus 80 degrees Celsius.Start the laser capture microdissection instrument in advance and preheat for 30 minutes. Use the solid state infrared laser to preheat for another 30 minutes, and install a 500 microliter, micro-centrifuged tube collection device. Next, load the tissue sample mounted laser capture, micro dissection slide onto the prepared instrument, and adjust the focus on neovascular vessels in glioma tissues, using the compatible software.Outline the laser marking line precisely along the outer edge of the neovascular vessel, while avoiding the vascular wall. Use the laser to burn and separate the neovascular vessel and collect the isolated vessel into the micro-centrifuged tube cap. Invert and seal the collected neovascular vessel sample tube and store the tube in a minus 80 degrees Celsius freezer.Resuspend the collected tissue in the protein extraction buffer. Sonicate the suspension in an ice water bath for one minute to achieve complete lysis. Centrifuge the lysate at 16, 000 G for 10 minutes, and transfer the supernatant to a new micro-centrifuged tube.Aliquot a minimal volume of the supernatant for protein quantification, using the by synkinetic acid assay. Add dithioerythritol to the remaining supernatant to a final concentration of one millimolar, and mixed gently by pipetting to avoid bubble formation. After aliquoting the treated supernatant, store at minus 80 degrees Celsius.Add ice cold acetone to each extracted protein sample at a sample to acetone ratio of one to five volume per volume. After mixing, incubate the sample at minus 20 degrees Celsius for one hour, and then centrifuge at 12, 000 G at four degrees Celsius for 15 minutes. Discard the supernatant to remove sodium dodecyl sulfate, dithioerythritol, and tris-hydrochloride.Re-suspend the pellet in 20 microliters of dissolution buffer containing one microliter of 1%sodium dodecyl sulfate. Next, add two microliters of dithioerythritol reducing reagent to the sample and mix thoroughly. Incubate at 60 degrees Celsius for one hour, then add one microliter of iodoacetamide cysteine blocking reagent and incubate at room temperature for 10 minutes.Add trypsin to each sample at a protein to trypsin ratio of 20 to one volume per volume. Vortex mix the sample and incubate it at 37 degrees Celsius overnight. Reconstitute each iTRAQ sample with 50 microliters of isopropanol.Add the corresponding triptych peptide sample to the reagent tube, and incubate at room temperature for one hour. After incubation, add three times the reaction volume of ultrapure water to terminate the reaction, and incubate at room temperature for 30 minutes. Equally pool six labeled samples, mix thoroughly, and lyophilize.Reconstitute the lyophilized peptides in two milliliters of solution A, containing 10 millimolar, potassium dihydrogen phosphate, and 25%acetonitrile at pH 2.7. After running the sample through a sulfoethyl functionalized cation exchange column, collect fractions using suitable buffer gradients into separate tubes. Finally, concentrate each fraction by vacuum centrifugation.Reconstitute each fraction with 40 microliters of 0.1%volume per volume trichloroacetic acid, and store all fractions at minus 80 degrees Celsius. Perform LCMSMS to analyze each fractionated peptide sample. The mass deviation of identified peptides followed a normal distribution, centered around zero.The peptide length distribution showed that the vast majority of peptides had lengths of eight to 20 amino acid residues. The distribution of relative mass of all identified proteins shows that almost 100%of the protein molecular weights are distributed between zero to 200 kilodaltons. And most of the protein isoelectric points are distributed between four to 10.Quantitative analysis identified 371 differentially expressed proteins in glioma neovascular tissues compared to controls, including 170 upregulated proteins and 201 downregulated proteins. Gene ontology analysis identified 76 statistically significant cellular components, 75 statistically significant biological processes, and 46 statistically significant molecular functions among the 371 differentially expressed proteins. Kyoto Encyclopedia of genes and genomes pathway analysis identified 23 statistically significant signaling pathway changes among the 371 differentially expressed proteins.Protein protein interaction network analysis identified 25 hub differentially expressed proteins, using four algorithms. This protocol enables, analyze our differential protein expression in glioma vasculature, identifying grade specific angiogenesis related proteins and quantifying protein omic genes, using air tract technology. An important challenge is to locate tumor neovascularization under an optical microscope and collect the dissected vessels accurately.Our future studies will analyze grade specific angiogenesis protein omics, PTM omics, pathway harps, and multi omics integrated biomarkers and drug targets.

preparation-neovascular-tissues-from-human-glioma-tissues-for

Research

JoVE Journal

Cancer Research

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

We present a 3D glioblastoma migration model using patient derived magnetic gliomaspheres generated from newly diagnosed and recurrent tumors. This platform allows visualization of patient specific migration dynamics and assessment of therapeutic efficacy. The assay can be multiplexed post migration to evaluate protein localization and expression patterns.

Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions

Discover laser-capture microdissection and quantitative proteomics for profiling glioma neovascular tissues. This method identifies differentially expressed proteins to reveal tumor angiogenesis mechanisms and therapeutic targets.

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

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