Name: 作者聚焦：揭开口腔潜在恶性肿瘤的神秘面纱
Uploaded: 2023-08-11T15:00:00
Description: Watch this Scientific Journal Video about Unlocking the Mysteries of Oral Potential Malignancies at JoVE.com

本研究得到了国家自然科学基金（81671006、81300894）、中国医学科学院医学创新基金（2019-I2M-5-038）、国家临床重点学科建设项目（PKUSSNKP-202102）、北京大学医学部优秀博士生创新基金（BMU2022BSS001）的资助。

本研究经北京大学附属学校机构评审委员会批准。已获得患者的知情同意。本研究中使用的组织样本被去标识化。研究方案如图 1所示。
1. 样品制备
<ol><li>在切片机上将福尔马林固定石蜡包埋的口腔粘膜下纤维化组织切成3μm和10μm厚度的连续切片。</li>
	<li>在水中展开各部分，然后捞到载玻片上。将3μM和10μm切片分别固定在粘附显微镜载玻...

捕获口腔粘膜下纤维化组织样本用于空间组学分析

<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. 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. 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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. 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该协议报告了一个管道，用于捕获具有形态和空间信息的OSF组织样本，以便通过激光显微切割进行进一步的空间组学分析。从代表性结果中，我们在各种形态相关样品中鉴定出不同的CNA模式。
OSF 是 OPMD 的一种，是口腔鳞状细胞癌6 的常见癌前病变。据报道，基因组不稳定性与 OPMD17,18 的发展和恶性转化有关。多项?...

口腔粘膜下纤维化 （OSF） 是一种慢性隐匿性疾病，主要发生在颊粘膜，导致张口受限1。虽然 OSF 是一种多因素疾病，但槟榔或咀嚼槟榔是 OSF的主要原因 2,3。由于这种地理上特定的习性，OSF主要集中在东南亚和南亚的人群中3。OSF 的常见组织学特征包括口腔黏膜上皮下方结缔组织中胶原蛋白异常沉积、血管狭窄和闭塞1。OSF上皮组织可表现为萎缩或增生，甚至在口腔白斑时出现异型增生4,5。
OSF 被世界卫生组织定义为一种常见的口腔潜在恶性疾病 （OPMD），具有进展为口腔鳞状细胞癌的潜力，恶性转化率为 4%-6%6,7,8,9。OSF恶性转化的机制是复杂的10。上皮细胞的异常生长，包括发育不良和萎缩，增加了致癌的可能性，基质中的衰老成纤维细胞可能通过活性氧 （ROS） 和其他分子诱导上皮-间充质转化 （EMT） 参与 OSF 的恶性进展10。
空间组学分析技术生成了具有形态学和空间信息的多组学数据，为癌症机制提供了见解11,12,13。在这里，我们提出了一种通过激光显微切割从福尔马林固定石蜡包埋的OSF组织中捕获形态相关细胞群的方案。对这些样本进行多组学分析可以克服组织内异质性的挑战，并增加对 OSF14 中分子病理学和恶性转化机制的理解。

<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

口腔粘膜下纤维化 （OSF） 是口腔中一种常见的潜在恶性疾病。上皮萎缩以及固有层和粘膜下层的纤维化经常在组织病理学载玻片上发现。上皮发育不良、上皮萎缩和衰老成纤维细胞被认为与 OSF 的恶性转化有关。然而，由于潜在恶性口腔疾病和口腔鳞状细胞癌的异质性，很难确定OSF中恶性转化的具体分子机制。在这里，我们提出了一种通过在福尔马林固定石蜡包埋的组织载玻片上进行激光捕获显微切割来获得少量携带形态学数据和空间信息的上皮或间充质细胞的方法。使用显微镜，我们可以精确捕获微尺度（~500 个细胞）发育不良或萎缩的上皮组织和纤维化上皮下组织。提取的细胞可以通过基因组或转录组测序进行评估，以获取具有形态学和空间学信息的基因组和转录组数据。这种方法消除了大量OSF组织测序的异质性和非病变区域细胞引起的干扰，从而可以对OSF组织进行精确的空间组学分析。

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.

作者聚焦：揭开口腔潜在恶性肿瘤的神秘面纱 

激光捕获显微切割从口腔粘膜下纤维化样本中分离具有形态学和空间信息的细胞

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.

通过对OSF组织进行激光显微切割，我们捕获了发育不良上皮、发育不良上皮下的基质、萎缩上皮和萎缩上皮组织下的基质样本（图1）。通过提取DNA和低深度全基因组测序，我们能够分析形态学相关的拷贝数改变（CNA）15。CNA 是一种常见的基因组不稳定形式，与 OPMD15,16 中恶性转化的风险增加有关。我们在4种样本?...

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

口腔粘膜下纤维化组织的激光捕获显微切割允许从感兴趣的组织学区域精确提取细胞，以分析具有形态学和空间信息的多组学数据。

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 ...

我们的主要研究兴趣是在口腔潜在恶性疾病（如口腔白斑和口腔粘膜下纤维化）的预测模型开发下的恶性转化机制。深度学习人工智能模型下的基因组转录组学、蛋白质组学和其他多组学测序分析用于研究从口腔、潜在恶性疾病到口腔癌的进展。目前的测序和多组学分析大多基于冷冻样本，但口腔、潜在恶性疾病大多是正式固定石蜡包埋的组织，通常通过活检，并且需要开发基于石蜡样本的实验技术。来自口腔、潜在恶性疾病和鳞状细胞癌的石蜡组织样本数量众多，来源广泛，使其成为非常有价值的研究资源。所有方案都有助于充分利用这些资源，以更好地了解疾病发展和恶性转化的机制。

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.

科研

医学

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.

用于苏木精-伊红染色的口腔粘膜下纤维化组织样品制备

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.