Name: Forfatter Spotlight: Lås op for mysterierne om orale potentielle maligniteter
Uploaded: 2023-08-11T15:00:00
Description: Watch this Scientific Journal Video about Unlocking the Mysteries of Oral Potential Malignancies at JoVE.com

Dette arbejde blev støttet af forskningsbevillinger fra National Nature Science Foundation of China (81671006, 81300894), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-038), National clinical key discipline construction project (PKUSSNKP-202102), Innovation Fund for Outstanding Doctoral Candidates of Peking University Health Science Center (BMU2022BSS001).

Denne undersøgelse blev godkendt af det institutionelle bedømmelsesudvalg for Peking University School and Hospital. Der blev indhentet informeret samtykke fra patienterne. De vævsprøver, der blev anvendt i denne undersøgelse, blev afidentificeret. Studieordningen er vist i figur 1.
1. Forberedelse af prøver
<ol><li>Skær formalinfikseret paraffinindlejret oralt submucøs fibrosevæv i kontinuerlige sektioner på 3 μm og 10 μm tykkelse p...

Optagelse af orale submukøse fibrose-vævsprøver til spatial-omics-analyse

<ol>
	<li>Cai, 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=Oral+submucous+fibrosis:+A+clinicopathological+study+of+674+cases+in+China.">Oral submucous fibrosis: A clinicopathological study of 674 cases in China.</a> Journal of Oral Pathology &amp; Medicine. 48 (4), 321-325 (2019).</li><li>Cai, X., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinicopathological+factors+associated+with+progression+of+oral+submucous+fibrosis:+A+population-based+retrospective+study.">Clinicopathological factors associated with progression of oral submucous fibrosis: A population-based retrospective study.</a> Oral Oncology. 130, 105949 (2022).</li><li>Ray, J. 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. H. <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+Review+on+etiopathogenesis,+diagnosis,+and+therapy.">Oral submucous fibrosis: A Review on etiopathogenesis, diagnosis, and therapy.</a> International Journal of Molecular Sciences. 20 (12), 2940 (2019).</li><li>Cai, 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=The+preliminary+exploration+of+immune+microenvironment+in+oral+leukoplakia+concomitant+with+oral+submucosal+fibrosis:+A+comparative+immunohistochemical+study.">The preliminary exploration of immune microenvironment in oral leukoplakia concomitant with oral submucosal fibrosis: A comparative immunohistochemical study.</a> Journal of Oral Pathology &amp; Medicine. , (2023).</li><li>Warnakulasuriya, 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=Oral+potentially+malignant+disorders:+A+consensus+report+from+an+international+seminar+on+nomenclature+and+classification,+convened+by+the+WHO+Collaborating+Centre+for+Oral+Cancer.">Oral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer.</a> Oral Diseases. 27 (8), 1862-1880 (2021).</li><li>Cai, 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=Development+and+validation+of+a+nomogram+prediction+model+for+malignant+transformation+of+oral+potentially+malignant+disorders.">Development and validation of a nomogram prediction model for malignant transformation of oral potentially malignant disorders.</a> Oral Oncology. 123, 105619 (2021).</li><li>Murthy, 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=Malignant+transformation+rate+of+oral+submucous+fibrosis:+A+systematic+review+and+meta-analysis.">Malignant transformation rate of oral submucous fibrosis: A systematic review and meta-analysis.</a> Journal of Clinical Medicine. 11 (7), 1793 (2022).</li><li>Kujan, O., Mello, F. 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. 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=Profiling+of+chromosomal+changes+in+potentially+malignant+and+malignant+oral+mucosal+lesions+from+South+and+Southeast+Asia+using+array-comparative+genomic+hybridization.">Profiling of chromosomal changes in potentially malignant and malignant oral mucosal lesions from South and Southeast Asia using array-comparative genomic hybridization.</a> Cancer Genomics &amp; Proteomics. 11 (3), 127-140 (2014).</li><li>Sun, 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=Spatially+resolved+metabolomics+to+discover+tumor-associated+metabolic+alterations.">Spatially resolved metabolomics to discover tumor-associated metabolic alterations.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (1), 52-57 (2019).</li><li>Ma, 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=Copy+number+alteration+profiling+facilitates+differential+diagnosis+between+ossifying+fibroma+and+fibrous+dysplasia+of+the+jaws.">Copy number alteration profiling facilitates differential diagnosis between ossifying fibroma and fibrous dysplasia of the jaws.</a> International Journal of Oral Science. 13 (1), 21 (2021).</li></ol>

Denne protokol rapporterede en pipeline til at fange OSF-vævsprøver med morfologisk og rumlig information til yderligere rumlige analyser gennem lasermikrodissektion. Fra de repræsentative resultater identificerede vi forskellige CNA-mønstre blandt forskellige morfologirelaterede prøver.
OSF, en type OPMD, er en almindelig precancerøs tilstand af oral pladecellekarcinom6. Genomisk ustabilitet er rapporteret at være forbundet med udvikling og ondartet transformati...

Oral submucøs fibrose (OSF) er en kronisk, lumsk sygdom, der hovedsageligt udvikler sig i bukkalslimhinden og resulterer i begrænset mundåbning1. Mens OSF er en multifaktoriel sygdom, er arecanødder eller betelnødder den vigtigste årsag til OSF 2,3. På grund af denne geografisk specifikke vane er OSF overvejende koncentreret i befolkninger i Sydøst- og Sydasien3. De almindelige histologiske træk ved OSF inkluderer unormal kollagenaflejring i bindevævet under mundslimhindepitelet, vaskulær stenose og okklusion1. OSF-epitelvæv kan forekomme med manifestationer af atrofi eller hyperplasi og endda dysplasi, når det er samtidig med oral leukoplaki 4,5.
OSF er defineret af Verdenssundhedsorganisationen som en almindelig oral potentielt malign lidelse (OPMD), der udviser potentialet til at udvikle sig til oralt pladecellekarcinom med en ondartet transformationshastighed på 4% -6% 6,7,8,9. Mekanismen bag den ondartede transformation af OSF er kompleks10. Unormal vækst af epitelet, herunder både dysplasi og atrofi, øger potentialet for carcinogenese, og senescerende fibroblaster i stroma kan være involveret i den ondartede progression af OSF ved at inducere epitel-mesenkymal overgang (EMT) gennem reaktive iltarter (ROS) og andre molekyler10.
Teknologier til rumlige-omiske analyser genererede multi-omiske data med morfologisk og rumlig information, der har givet indsigt i kræftmekanismer11,12,13. Her præsenterer vi en protokol til at fange morfologirelaterede cellepopulationer fra formalinfikseret paraffinindlejret OSF-væv ved lasermikrodissektion. Multi-omiske analyser af disse prøver kan overvinde udfordringer med intratissue heterogenitet og øge forståelsen af molekylær patologi og mekanismer for ondartet transformation i OSF14.

<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

Oral submucøs fibrose (OSF) er en almindelig type potentielt ondartet lidelse i mundhulen. Atrofi af epitel og fibrose af lamina propria og submucosa findes ofte på histopatologiske dias. Epiteldysplasi, epitelatrofi og senescerende fibroblaster er blevet foreslået at være forbundet med den ondartede transformation af OSF. På grund af heterogeniteten af potentielt maligne orale lidelser og oralt pladecellekarcinom er det imidlertid vanskeligt at identificere de specifikke molekylære mekanismer for malign transformation i OSF. Her præsenterer vi en metode til at opnå et lille antal epitel- eller mesenkymale celler, der bærer morfologiske data og rumlig information ved laserfangstmikrodissektion på formalinfikserede paraffinindlejrede vævsdias. Ved hjælp af et mikroskop kan vi præcist fange mikroskala (~ 500 celler) dysplastisk eller atrofisk epitelvæv og fibrotisk subepitelvæv. De ekstraherede celler kan evalueres ved genom- eller transkriptomsekventering for at erhverve genomiske og transkriptomiske data med morfologisk og rumlig information. Denne tilgang fjerner heterogeniteten af bulk OSF-vævsekventering og interferensen forårsaget af celler i ikke-læsionerede områder, hvilket muliggør præcis rumlig omics-analyse af OSF-væv.

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.

Forfatter Spotlight: Lås op for mysterierne om orale potentielle maligniteter 

Isolering af celler med morfologisk og rumlig information fra orale submucøse fibroseprøver ved laserfangstmikrodissektion

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.

Ved at udføre lasermikrodissektion af OSF-væv fangede vi prøver af dysplastisk epitel, stroma under dysplastisk epitel, atrofisk epitel og stroma under atrofisk epitelvæv (figur 1). Ved at udvinde DNA og helgenomsekventering med lav dybde var vi i stand til at analysere morfologirelaterede kopinummerændringer (CNA)15. CNA er en almindelig form for genomisk ustabilitet forbundet med en øget risiko for ondartet transformation i OPMD15,16<su...

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

Laserfangstmikrodissektion af oral submucøs fibrosevæv muliggør præcis ekstraktion af celler fra histologiske områder af interesse for analyse af multi-omics-data med morfologisk og rumlig information.

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 ...

Vores primære forskningsinteresser er mekanismerne for ondartet transformation under udvikling af prædiktive modeller af orale, potentielt ondartede lidelser såsom oral leukoplaki og oral submucøs fibrose. Genomisk transkriptomisk, proteomisk og anden multi-omisk sekventeringsanalyse under dyb læring kunstig intelligens modeller bruges til at studere progressionen fra orale, potentielt ondartede lidelser til oral cancer. Nuværende sekventering og multi-omics analyse er for det meste baseret på frosne prøver, men orale, potentielt ondartede lidelser er for det meste formelt fikseret paraffinindlejret væv, ofte ved biopsi, og de behovet for at udvikle eksperimentelle teknikker baseret på paraffinprøver.Paraffinvævsprøver fra orale, potentielt ondartede lidelser og pladecellekarcinom er tilgængelige i stort antal og fra en række forskellige kilder, hvilket gør dem til en meget værdifuld ressource til undersøgelse. Alle protokoller kan hjælpe med fuldt ud at bruge disse ressourcer til bedre at forstå mekanismerne for sygdomsudvikling og ondartet transformation.

isolation-cells-with-morphological-spatial-information-from-oral

Research

JoVE Journal

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.

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.

Oral submucøs fibrose vævsprøve forberedelse til hæmatoxylin-eosin farvning

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.