Name: Autor im Rampenlicht: Die Geheimnisse oraler potenzieller Malignome entschlüsseln
Uploaded: 2023-08-11T15:00:00
Description: Watch this Scientific Journal Video about Unlocking the Mysteries of Oral Potential Malignancies at JoVE.com

Diese Arbeit wurde durch Forschungsstipendien der National Nature Science Foundation of China (81671006, 81300894), des CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038), des National Clinical Key Discipline Construction Project (PKUSSNKP-202102) und des Innovation Fund for Outstanding Doctoral Candidates des Peking University Health Science Center (BMU2022BSS001) unterstützt.

Diese Studie wurde vom institutionellen Prüfungsausschuss der Peking University School and Hospital genehmigt. Von den Patienten wurde eine Einverständniserklärung eingeholt. Die in dieser Studie verwendeten Gewebeproben wurden deidentifiziert. Das Studienschema ist in Abbildung 1 dargestellt.
1. Vorbereitung der Probe
<ol><li>Schneiden Sie formalinfixiertes, paraffineingebettetes orales submuköses Fibrosegewebe in kontinuierliche Abschnitte...

Erfassung von Gewebeproben der oralen submukösen Fibrose für die Spatial-Omics-Analyse

<ol>
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G., Chatterjee, R., Chaudhuri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+submucous+fibrosis:+A+global+challenge.+Rising+incidence,+risk+factors,+management,+and+research+priorities.">Oral submucous fibrosis: A global challenge. Rising incidence, risk factors, management, and research priorities.</a> Periodontology 2000. 80 (1), 200-212 (2019).</li><li>Shih, Y. H., Wang, T. H., Shieh, T. M., Tseng, Y. 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W., Warnakulasuriya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+transformation+of+oral+submucous+fibrosis:+A+systematic+review+and+meta-analysis.">Malignant transformation of oral submucous fibrosis: A systematic review and meta-analysis.</a> Oral Diseases. 27 (8), 1936-1946 (2020).</li><li>Qin, X., Ning, Y., Zhou, L., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+submucous+fibrosis:+Etiological+mechanism,+malignant+transformation,+therapeutic+approaches+and+targets.">Oral submucous fibrosis: Etiological mechanism, malignant transformation, therapeutic approaches and targets.</a> International Journal of Molecular Sciences. 24 (5), 4992 (2023).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+and+spatial+dissection+of+precancerous+lesions+underlying+the+initiation+process+of+oral+squamous+cell+carcinoma.">Single-cell and spatial dissection of precancerous lesions underlying the initiation process of oral squamous cell carcinoma.</a> Cell Discovery. 9 (1), 28 (2023).</li><li>Zhu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delineating+the+dynamic+evolution+from+preneoplasia+to+invasive+lung+adenocarcinoma+by+integrating+single-cell+RNA+sequencing+and+spatial+transcriptomics.">Delineating the dynamic evolution from preneoplasia to invasive lung adenocarcinoma by integrating single-cell RNA sequencing and spatial transcriptomics.</a> Experimental &amp; Molecular Medicine. 54 (11), 2060-2076 (2022).</li><li>Ji, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+analysis+of+composition+and+spatial+architecture+in+human+squamous+cell+carcinoma.">Multimodal analysis of composition and spatial architecture in human squamous cell carcinoma.</a> Cell. 182 (2), 497-514 (2020).</li><li>Van den Bossche, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironment-driven+intratumoral+heterogeneity+in+head+and+neck+cancers:+clinical+challenges+and+opportunities+for+precision+medicine.">Microenvironment-driven intratumoral heterogeneity in head and neck cancers: clinical challenges and opportunities for precision medicine.</a> Drug Resistance Updates. 60, 100806 (2022).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+the+risk+assessment+of+oral+leukoplakia+through+morphology-related+copy+number+analysis.">Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis.</a> Science China. Life sciences. 64 (9), 1379-1391 (2021).</li><li>Watkins, T. B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+chromosomal+instability+and+karyotype+order+in+tumour+evolution.">Pervasive chromosomal instability and karyotype order in tumour evolution.</a> Nature. 587 (7832), 126-132 (2020).</li><li>Odell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+and+loss+of+heterozygosity+as+risk+markers+for+malignant+transformation+in+oral+mucosa.">Aneuploidy and loss of heterozygosity as risk markers for malignant transformation in oral mucosa.</a> Oral Diseases. 27 (8), 1993-2007 (2021).</li><li>Wood, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+road+to+invasion-examining+the+similarities+and+differences+in+the+genomes+of+associated+oral+pre-cancer+and+cancer+samples.">The genomic road to invasion-examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.</a> Genome Medicine. 9 (1), 53 (2017).</li><li>Venugopal, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+proteomics+based+on+2D+gel+electrophoresis+and+mass+spectrometry+with+validations:+Identification+of+a+biomarker+compendium+for+oral+submucous+fibrosis-An+indian+study.">Integrated proteomics based on 2D gel electrophoresis and mass spectrometry with validations: Identification of a biomarker compendium for oral submucous fibrosis-An indian study.</a> Journal of Personalized Medicine. 12 (2), 208 (2022).</li><li>Kundu, P., Pant, I., Jain, R., Rao, S. G., Kondaiah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+DNA+methylation+changes+in+oral+submucous+fibrosis.">Genome-wide DNA methylation changes in oral submucous fibrosis.</a> Oral Diseases. 28 (4), 1094-1103 (2022).</li><li>Cai, X., Zhang, H., Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-target+pharmacological+mechanisms+of+Salvia+miltiorrhiza+against+oral+submucous+fibrosis:+A+network+pharmacology+approach.">Multi-target pharmacological mechanisms of Salvia miltiorrhiza against oral submucous fibrosis: A network pharmacology approach.</a> Archives of Oral Biology. 126, 105131 (2021).</li><li>Xiao, X., Hu, Y., Li, C., Shi, L., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content+abnormality+in+oral+submucous+fibrosis+concomitant+leukoplakia:+A+preliminary+evaluation+of+the+diagnostic+and+clinical+implications.">DNA content abnormality in oral submucous fibrosis concomitant leukoplakia: A preliminary evaluation of the diagnostic and clinical implications.</a> Diagnostic Cytopathology. 48 (11), 1111-1114 (2020).</li><li>Zhou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+expression+profile+associated+with+malignant+progression+of+oral+submucous+fibrosis.">Long non-coding RNA expression profile associated with malignant progression of oral submucous fibrosis.</a> Journal of Oncology. 2019, 6835176 (2019).</li><li>Lunde, M. 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Dieses Protokoll berichtete über eine Pipeline zur Erfassung von OSF-Gewebeproben mit morphologischen und räumlichen Informationen für weitere räumlich-omische Analysen durch Lasermikrodissektion. Aus den repräsentativen Ergebnissen identifizierten wir unterschiedliche CNA-Muster in verschiedenen morphologiebezogenen Proben.
OSF, eine Form der OPMD, ist eine häufige Krebsvorstufe des oralen Plattenepithelkarzinoms6. Es wurde berichtet, dass genomische Instabilitä...

Die orale submuköse Fibrose (OSF) ist eine chronische, schleichende Erkrankung, die sich vor allem in der Wangenschleimhaut entwickelt und zu einer eingeschränkten Mundöffnung führt1. Während OSF eine multifaktorielle Erkrankung ist, ist das Kauen von Arekanüssen oder Betelnüssen die Hauptursache für OSF 2,3. Aufgrund dieser geographisch spezifischen Gewohnheit ist OSF überwiegend in Populationen in Südost- und Südasien konzentriert3. Zu den häufigsten histologischen Merkmalen der OSF gehören abnorme Kollagenablagerungen im Bindegewebe unter dem oralen Schleimhautepithel, Gefäßstenose und Okklusion1. OSF-Epithelgewebe kann mit Manifestationen von Atrophie oder Hyperplasie und sogar Dysplasie auftreten, wenn es mit oraler Leukoplakie einhergeht 4,5.
OSF wird von der Weltgesundheitsorganisation als eine häufige orale potenziell maligne Erkrankung (OPMD) definiert, die das Potenzial aufweist, zu einem oralen Plattenepithelkarzinom mit einer malignen Transformationsrate von 4%-6% fortzuschreiten6,7,8,9. Der Mechanismus, der der malignen Transformation von OSF zugrunde liegt, ist komplex10. Abnormales Wachstum des Epithels, einschließlich Dysplasie und Atrophie, erhöht das Potenzial für die Karzinogenese, und seneszente Fibroblasten im Stroma können an der malignen Progression von OSF beteiligt sein, indem sie den epithelial-mesenchymalen Übergang (EMT) durch reaktive Sauerstoffspezies (ROS) und andere Moleküle induzieren10.
Technologien für räumlich-omische Analysen generierten multi-omische Daten mit morphologischen und räumlichen Informationen, die Einblicke in Krebsmechanismen lieferten11,12,13. In dieser Arbeit stellen wir ein Protokoll vor, mit dem morphologiebezogene Zellpopulationen aus formalinfixiertem, paraffineingebettetem OSF-Gewebe mittels Lasermikrodissektion erfasst werden können. Multi-omic-Analysen dieser Proben können Herausforderungen in Bezug auf die Heterogenität innerhalb des Gewebes überwinden und das Verständnis der molekularen Pathologie und der Mechanismen der malignen Transformation bei OSF14 verbessern.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>CITOTEST</td>
			<td>REF.188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Div-haematoxylin</td>
			<td>YiLi</td>
			<td>20230326</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin solution</td>
			<td>BASO</td>
			<td>BA4098</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PEKING REAGENT</td>
			<td>No.32061</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin dye solution</td>
			<td>YiLi</td>
			<td>20230326</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>LEICA</td>
			<td>HI1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser capture microdissection system</td>
			<td>LEICA</td>
			<td>LMD7</td>
			<td>Machine</td>
		</tr>
		<tr>
			<td>Laser microdissection microsystem</td>
			<td>LEICA</td>
			<td>8.2.3.7603</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Micromount mounting medium</td>
			<td>LEICA</td>
			<td>REF.3801731</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>CITOTEST</td>
			<td>REF.10212450C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>LEICA</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>AXYGEN</td>
			<td>16421959</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN-membrane slides</td>
			<td>LEICA</td>
			<td>No.11505158</td>
			<td></td>
		</tr>
		<tr>
			<td>Re-blue solution</td>
			<td>YiLi</td>
			<td>20230326</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>REF.10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>PEKING REAGENT</td>
			<td>No.33535</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cells with morphological and spatial information from oral submucous fibrosis samples by laser capture microdissection

Die orale submuköse Fibrose (OSF) ist eine häufige Form einer potenziell bösartigen Erkrankung in der Mundhöhle. Die Atrophie des Epithels und die Fibrose der Lamina propria und der Submukosa sind häufig auf histopathologischen Objektträgern zu finden. Es wurde vorgeschlagen, dass epitheliale Dysplasie, epitheliale Atrophie und seneszente Fibroblasten mit der malignen Transformation von OSF assoziiert sind. Aufgrund der Heterogenität von potentiell malignen oralen Erkrankungen und oralen Plattenepithelkarzinomen ist es jedoch schwierig, die spezifischen molekularen Mechanismen der malignen Transformation bei OSF zu identifizieren. In dieser Arbeit stellen wir eine Methode vor, um eine kleine Anzahl von Epithel- oder Mesenchymzellen, die morphologische Daten und räumliche Informationen tragen, durch Laser-Capture-Mikrodissektion auf formalinfixierten, in Paraffin eingebetteten Gewebeobjektträgern zu erhalten. Mit einem Mikroskop können wir mikroskaliges (~500 Zellen) dysplastisches oder atrophisches Epithelgewebe und fibrotisches subepitheliales Gewebe präzise erfassen. Die extrahierten Zellen können durch Genom- oder Transkriptomsequenzierung ausgewertet werden, um genomische und transkriptomische Daten mit morphologischen und räumlichen Informationen zu erhalten. Dieser Ansatz beseitigt die Heterogenität der Massensequenzierung von OSF-Gewebe und die Interferenz, die durch Zellen in nicht läsionierten Bereichen verursacht wird, und ermöglicht eine präzise Spatial-Omics-Analyse von OSF-Gewebe.

Oral submucous fibrosis (OSF) is a chronic, insidious disease that develops mainly in the buccal mucosa and results in restricted mouth opening1. While OSF is a multifactorial disease, areca nut or betel nut chewing is the main cause of OSF2,3. Because of this geographically specific habit, OSF is predominantly concentrated in populations in Southeast and South Asia3. The common histological features of OSF include abnormal collagen deposition in the connective tissue beneath the oral mucosal epithelium, vascular stenosis, and occlusion1. OSF epithelial tissue can present with manifestations of atrophy or hyperplasia and even dysplasia when concomitant with oral leukoplakia4,5.
OSF is defined by the World Health Organization as a common oral potentially malignant disorder (OPMD) that exhibits the potential to progress to oral squamous cell carcinoma with a malignant transformation rate of 4%-6%6,7,8,9. The mechanism underlying the malignant transformation of OSF is complex10. Abnormal growth of the epithelium, including both dysplasia and atrophy, increases the potential for carcinogenesis, and senescent fibroblasts in the stroma may be involved in the malignant progression of OSF by inducing epithelial-mesenchymal transition (EMT) through reactive oxygen species (ROS) and other molecules10.
Technologies for spatial-omic analyses generated multi-omic data with morphological and spatial information that have provided insights into cancer mechanisms11,12,13. Here, we present a protocol to capture morphology-related cell populations from formalin-fixed paraffin-embedded OSF tissue by laser microdissection. Multi-omic analyses of these samples can overcome challenges with intratissue heterogeneity and increase understanding of the molecular pathology and mechanisms of malignant transformation in OSF14.

Autor im Rampenlicht: Die Geheimnisse oraler potenzieller Malignome entschlüsseln 

Isolierung von Zellen mit morphologischen und räumlichen Informationen aus oralen submukösen Fibroseproben mittels Laser-Capture-Mikrodissektion

Our main research interests are the mechanisms of malignant transformation under the development of predictive models of oral, potentially malignant disorders such as oral leukoplakia and oral submucous fibrosis. Genomic transcriptomic, proteomic, and other multi-omic sequencing analysis under deep learning artificial intelligence models are used to study the progression from oral, potentially malignant disorders to oral cancer. Current sequencing and multi-omics analysis are mostly based on frozen samples, but oral, potentially malignant disorders are mostly formally fixed paraffin embedded tissue, often by biopsy, and they the need to develop experimental techniques based on paraffin samples.Paraffin tissue samples from oral, potentially malignant disorders, and the squamous cell carcinoma are available in large numbers and from a variety of sources, making them a very valuable resource for study. All protocol can help fully use these resources to better understand the mechanisms of disease development and malignant transformation.

Durch die Lasermikrodissektion von OSF-Geweben haben wir Proben von dysplastischem Epithel, Stroma unter dem dysplastischen Epithel, atrophischem Epithel und Stroma unter atrophischem Epithelgewebe entnommen (Abbildung 1). Durch die Extraktion von DNA und die Sequenzierung des gesamten Genoms mit geringer Tiefe waren wir in der Lage, morphologiebedingte Kopienzahlveränderungen (CNA) zu analysieren15. CNA ist eine häufige Form der genomischen Instabilität, die mit e...

Watch this Scientific Journal Video about Unlocking the Mysteries of Oral Potential Malignancies at JoVE.com

Die Laser-Capture-Mikrodissektion von oralem submukösem Fibrosegewebe ermöglicht die präzise Extraktion von Zellen aus histologischen Regionen von Interesse für die Analyse von Multi-Omics-Daten mit morphologischen und räumlichen Informationen.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...

Unser Hauptforschungsinteresse gilt den Mechanismen der malignen Transformation unter der Entwicklung von Vorhersagemodellen für orale, potentiell maligne Erkrankungen wie orale Leukoplakie und orale submuköse Fibrose. Genomische Transkriptomik-, Proteom- und andere Multi-Omic-Sequenzierungsanalysen unter Deep-Learning-Modellen der künstlichen Intelligenz werden verwendet, um das Fortschreiten von oralen, potenziell bösartigen Erkrankungen zu Mundkrebs zu untersuchen. Derzeitige Sequenzierungen und Multi-Omics-Analysen basieren meist auf gefrorenen Proben, aber orale, potenziell maligne Erkrankungen sind meist formell fixiertes Paraffingewebe, oft durch Biopsie, und es besteht die Notwendigkeit, experimentelle Techniken auf der Grundlage von Paraffinproben zu entwickeln.Paraffingewebeproben von oralen, potenziell bösartigen Erkrankungen und dem Plattenepithelkarzinom sind in großer Zahl und aus einer Vielzahl von Quellen verfügbar, was sie zu einer sehr wertvollen Ressource für Studien macht. Alle Protokolle können dazu beitragen, diese Ressourcen voll auszuschöpfen, um die Mechanismen der Krankheitsentwicklung und der bösartigen Transformation besser zu verstehen.

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Medicine

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Forschung

Medizin

Oral Submucous Fibrosis Tissue Sample Preparation for Hematoxylin-Eosin Staining

To begin, using a microtome, cut the formalin fixed paraffin embedded oral submucous fibrosis tissues into continuous sections of three in 10 micrometers thickness, then unfold the sections in water. Place three micron sections on the adhesion microscope slide or slide A, and 10 microns on pen membrane slide or slide B.Place both slides on a hot plate at 60 degrees Celsius. After two hours, place slides A and B in xylene for 10 minutes, then hydrate the slides in graded ethanol concentrations for one minute in each concentration.Next, place the slides in ultrapure distilled water for 30 seconds. Soak slides in Harris hematoxylin dye solution for 90 seconds. Again wash the slides thrice in ultrapure distilled water for 30 seconds each.After that, submerge the slides in div-hematoxylin solution for two seconds. Return slides into ultrapure distilled water for 30 seconds, repeating three times. Next, immerse slides A and B in 1%re-blue solution for two seconds.After washing the slides with distilled water, place them in an eosin solution for two seconds. Again immerse slides in ultrapure distilled water for 30 seconds before dehydrating and increasing ethanol concentrations, then immerse slide A in xylene for three minutes. Finally, apply a drop of the mounting medium to slide A, and cover it with a glass cover.

Orale submuköse Fibrose Gewebeprobenvorbereitung für die Hämatoxylin-Eosin-Färbung

Laser Capture Microdissection of Oral Submucous Fibrosis Tissues for Morphology and Spatial Information Analysis

To begin, turn on the Laser Microdissection system and software. Place slide A containing stained three-micron sections of oral submucous fibrosis tissues on the slide stage. Observe the histological morphology to identify the area of interest for laser microdissection.Remove slide A and place slide B with the tissue side facing down on the slide stage. Next, click the Unload button to push out the collection device. Insert a PCR tube into the collection device and ensure the tube is fixed.Then click the Okay button. Select a cap by clicking the corresponding red circle, marking the collector device to tube caps, and wait until the selected circle turns green. Then click the Draw button and Draw plus Cut button and use the mouse to sketch the area of interest.Click on Start Cut to capture the area of interest, which will be collected by the cap. Then press the lower button to ensure the captured sample is collected. Click the specimen button to capture the next sample.Finally, click Unload to unload the PCR tube with the captured sample. In this study, using laser microdissection of oral submucous fibrosis, tissues of epithelium and stroma with different patterns are captured. Copy number alternations were present in epithelial samples but not in stroma samples.The copy number alternations pattern in the dysplastic epithelium was observed in chromosomes three and eight, while copy number alternations were detected at a lower frequency in chromosome eight in the atrophic epithelium.