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>
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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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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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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 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 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.

Author Spotlight: Unlocking the Mysteries of Oral Potential Malignancies 

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

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.

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...

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

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 ...

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

Research

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Medicine

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.

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.

972 Views.  Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices.  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.

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

Summary