Name: Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection (Video) | JoVE Journal
Uploaded: 2023-08-11T15:00:00.000+00:00
Duration: 5 min 42 s
Description: Learn laser capture microdissection for isolating cells from oral submucous fibrosis samples with preserved morphological and spatial information. This technique enables multi-omics analysis of paraffin-embedded tissues.

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5 minutes

This work was supported by research grants from the 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).

This study was approved by the institutional review board of Peking University School and Hospital. Informed consent was obtained from the patients. The tissue samples used in this study were deidentified. The study scheme is shown in Figure 1.
1. Sample preparation
<ol>
	<li>Cut formalin-fixed paraffin-embedded oral submucous fibrosis tissues into continuous sections of 3 &#181;m and 10 &#181;m thickness on a microtome.</li>
	<li>Unfold the sections in water and then fish out onto the slides. Fix the 3 &#181;M and 10 &#181;m sections on adhesion microscope slides (slide A) and PEN-membrane slides (slide B), respectively.</li>
	<li>Place the slides on a hot plate at 60 &#176;C for 2 h.</li>
</ol>
2. Hematoxylin-eosin staining
<ol>
	<li>Place slides A and B in xylene (CAUTION) for 10 min and repeat this step thrice.</li>
	<li>Hydrate slides A and B in graded ethanol solutions in the order of 100%, 100%, 90%, 80%, and 70% for 1 min in each concentration.</li>
	<li>Place slides A and B in ultrapure distilled water for 30 s</li>
	<li>Place slides A and B in Harris hematoxylin dye solution for 90 s.</li>
	<li>Place slides A and B in ultrapure distilled water for 30 s and repeat this step thrice.</li>
	<li>Place slides A and B in div-hematoxylin solution (CAUTION) for 2 s.</li>
	<li>Place slides A and B in ultrapure distilled water for 30 s and repeat this step thrice.</li>
	<li>Place slides in A and B in 1% re-blue solution for 2 s.</li>
	<li>Place slides A and B in ultrapure distilled water for 30 s and repeat this step thrice.</li>
	<li>Place slides in eosin solution for 2 s.</li>
	<li>Place slides A and B in ultrapure distilled water for 30 s and repeat this step thrice.</li>
	<li>Dehydrate the slides in graded ethanol solutions in the order of 70%, 80%, 90%, 100%, and 100% for 2 s in each concentration.</li>
	<li>Place slide A in xylene for 3 min and repeat this step twice.</li>
	<li>Add a drop of mounting medium (CAUTION) to slide A and cover it with a cover glass.</li>
</ol>
3. Observation of histological morphology and laser capture microdissection
<ol>
	<li>Initiate the laser microdissection system and software (Figure 2).</li>
	<li>Place slide A on the slide stage and observe the histological morphology to identify the area of interest for laser microdissection.</li>
	<li>Remove slide A and place slide B inversely on the slide stage.</li>
	<li>Click the Unload button (Figure 2) to push out the collection device. Insert a polymerase chain reaction (PCR) tube into the collection device and ensure that the tube is held fixed. Click the OK button (Figure 3).</li>
	<li>Select a cap by clicking the corresponding red circle marking the Collector Device: Tube Caps. The selected circle will turn green.</li>
	<li>Click the Draw button and Draw + Cut button and use the mouse to sketch the area of interest.</li>
	<li>Click the Start Cut button to capture the area of interest; the captured sample will be collected by the cap (Figure 4).</li>
	<li>Click the Lower button to ensure that the captured sample is collected (Figure 5).</li>
	<li>Click the Specimen button to capture the next sample.</li>
	<li>Click the Unload button to unload the PCR tube with the captured sample.</li>
</ol>

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

<ol><li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+submucous+fibrosis%3A+A+clinicopathological+study+of+674+cases+in+China%5BTitle%5D)+AND+%22Journal+of+Oral+Pathology+%26+Medicine%22%5BJournal%5D)">Oral submucous fibrosis: A clinicopathological study of 674 cases in China.</a> Journal of Oral Pathology & Medicine. 48 (4), 321-325 (2019).</li>
<li>Cai, X., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinicopathological+factors+associated+with+progression+of+oral+submucous+fibrosis%3A+A+population-based+retrospective+study%5BTitle%5D)+AND+%22Oral+Oncology%22%5BJournal%5D)">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/pubmed?term=((Oral+submucous+fibrosis%3A+A+global+challenge.+Rising+incidence%2C+risk+factors%2C+management%2C+and+research+priorities%5BTitle%5D)+AND+%22Periodontology+2000%22%5BJournal%5D)">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/pubmed?term=((Oral+submucous+fibrosis%3A+A+Review+on+etiopathogenesis%2C+diagnosis%2C+and+therapy%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">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/pubmed?term=((The+preliminary+exploration+of+immune+microenvironment+in+oral+leukoplakia+concomitant+with+oral+submucosal+fibrosis%3A+A+comparative+immunohistochemical+study%5BTitle%5D)+AND+%22Journal+of+Oral+Pathology+%26+Medicine%22%5BJournal%5D)">The preliminary exploration of immune microenvironment in oral leukoplakia concomitant with oral submucosal fibrosis: A comparative immunohistochemical study.</a> Journal of Oral Pathology & Medicine. , (2023).</li>
<li>Warnakulasuriya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+potentially+malignant+disorders%3A+A+consensus+report+from+an+international+seminar+on+nomenclature+and+classification%2C+convened+by+the+WHO+Collaborating+Centre+for+Oral+Cancer%5BTitle%5D)+AND+%22Oral+Diseases%22%5BJournal%5D)">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/pubmed?term=((Development+and+validation+of+a+nomogram+prediction+model+for+malignant+transformation+of+oral+potentially+malignant+disorders%5BTitle%5D)+AND+%22Oral+Oncology%22%5BJournal%5D)">Development and validation of a nomogram prediction model for malignant transformation of oral potentially malignant disorders.</a> Oral Oncology. 123, 105619(2021).</li>
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<li>Kujan, O., Mello, F. W., Warnakulasuriya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Malignant+transformation+of+oral+submucous+fibrosis%3A+A+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Oral+Diseases%22%5BJournal%5D)">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/pubmed?term=((Oral+submucous+fibrosis%3A+Etiological+mechanism%2C+malignant+transformation%2C+therapeutic+approaches+and+targets%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">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/pubmed?term=((Single-cell+and+spatial+dissection+of+precancerous+lesions+underlying+the+initiation+process+of+oral+squamous+cell+carcinoma%5BTitle%5D)+AND+%22Cell+Discovery%22%5BJournal%5D)">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/pubmed?term=((Delineating+the+dynamic+evolution+from+preneoplasia+to+invasive+lung+adenocarcinoma+by+integrating+single-cell+RNA+sequencing+and+spatial+transcriptomics%5BTitle%5D)+AND+%22Experimental+%26+Molecular+Medicine%22%5BJournal%5D)">Delineating the dynamic evolution from preneoplasia to invasive lung adenocarcinoma by integrating single-cell RNA sequencing and spatial transcriptomics.</a> Experimental & Molecular Medicine. 54 (11), 2060-2076 (2022).</li>
<li>Ji, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodal+analysis+of+composition+and+spatial+architecture+in+human+squamous+cell+carcinoma%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Microenvironment-driven+intratumoral+heterogeneity+in+head+and+neck+cancers%3A+clinical+challenges+and+opportunities+for+precision+medicine%5BTitle%5D)+AND+%22Drug+Resistance+Updates%22%5BJournal%5D)">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/pubmed?term=((Improvement+in+the+risk+assessment+of+oral+leukoplakia+through+morphology-related+copy+number+analysis%5BTitle%5D)+AND+%22Science+China.+Life+sciences%22%5BJournal%5D)">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/pubmed?term=((Pervasive+chromosomal+instability+and+karyotype+order+in+tumour+evolution%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Aneuploidy+and+loss+of+heterozygosity+as+risk+markers+for+malignant+transformation+in+oral+mucosa%5BTitle%5D)+AND+%22Oral+Diseases%22%5BJournal%5D)">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/pubmed?term=((The+genomic+road+to+invasion-examining+the+similarities+and+differences+in+the+genomes+of+associated+oral+pre-cancer+and+cancer+samples%5BTitle%5D)+AND+%22Genome+Medicine%22%5BJournal%5D)">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/pubmed?term=((Integrated+proteomics+based+on+2D+gel+electrophoresis+and+mass+spectrometry+with+validations%3A+Identification+of+a+biomarker+compendium+for+oral+submucous+fibrosis-An+indian+study%5BTitle%5D)+AND+%22Journal+of+Personalized+Medicine%22%5BJournal%5D)">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/pubmed?term=((Genome-wide+DNA+methylation+changes+in+oral+submucous+fibrosis%5BTitle%5D)+AND+%22Oral+Diseases%22%5BJournal%5D)">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/pubmed?term=((Multi-target+pharmacological+mechanisms+of+Salvia+miltiorrhiza+against+oral+submucous+fibrosis%3A+A+network+pharmacology+approach%5BTitle%5D)+AND+%22Archives+of+Oral+Biology%22%5BJournal%5D)">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/pubmed?term=((DNA+content+abnormality+in+oral+submucous+fibrosis+concomitant+leukoplakia%3A+A+preliminary+evaluation+of+the+diagnostic+and+clinical+implications%5BTitle%5D)+AND+%22Diagnostic+Cytopathology%22%5BJournal%5D)">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/pubmed?term=((Long+non-coding+RNA+expression+profile+associated+with+malignant+progression+of+oral+submucous+fibrosis%5BTitle%5D)+AND+%22Journal+of+Oncology%22%5BJournal%5D)">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/pubmed?term=((Profiling+of+chromosomal+changes+in+potentially+malignant+and+malignant+oral+mucosal+lesions+from+South+and+Southeast+Asia+using+array-comparative+genomic+hybridization%5BTitle%5D)+AND+%22Cancer+Genomics+%26+Proteomics%22%5BJournal%5D)">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 & Proteomics. 11 (3), 127-140 (2014).</li>
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</ol>

The authors declare no conflict of interest.

This protocol reported a pipeline to capture OSF tissue samples with morphological and spatial information for further spatial-omic analyses through laser microdissection. From the representative results, we identified different CNA patterns among various morphology-related samples.
OSF, a type of OPMD, is a common precancerous condition of oral squamous cell carcinoma6. Genomic instability has been reported to be associated with the development and malignant transforma...

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.

<table><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

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.

By performing laser microdissection of OSF tissues, we captured samples of dysplastic epithelium, stroma beneath the dysplastic epithelium, atrophic epithelium, and stroma beneath atrophic epithelial tissue (Figure 1).Through extracting DNA and low-depth whole genome sequencing, we were able to analyze morphology-related copy number alterations (CNA)15. CNA is a common form of genomic instability associated with an increased risk of malignant transformation in OPMD<su...

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

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Learn laser capture microdissection for isolating cells from oral submucous fibrosis samples with preserved morphological and spatial information. This technique enables multi-omics analysis of paraffin-embedded tissues.

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection (Video) | JoVE Journal

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

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