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

Summary

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.

Abstract

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.

Introduction

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 OSF^2,3. 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. ....

Protocol

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

1. Cut formalin-fixed paraffin-embedded oral submucous fibrosis tissues into continuous sections of 3 µm and 10 µm thickness on a microtome.
2. Unfold the sections in water and then fish out onto the slides. Fix the 3 µM and 10 µm sections on adhesion microscope slides (slide A) and PEN-membrane s....

Results

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

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesion microscope slides	CITOTEST	REF.188105
Div-haematoxylin	YiLi	20230326
Eosin solution	BASO	BA4098
Ethanol	PEKING REAGENT	No.32061
Harris hematoxylin dye solution	YiLi	20230326
Hot plate	LEICA	HI1220
Laser capture microdissection system	LEICA	LMD7	Machine
Laser microdissection microsystem	LEICA	8.2.3.7603	Software
Micromount mounting medium	LEICA	REF.3801731
Microscope cover glass	CITOTEST	REF.10212450C
Microtome	LEICA	RM2235
PCR tubes	AXYGEN	16421959
PEN-membrane slides	LEICA	No.11505158
Re-blue solution	YiLi	20230326
Ultrapure distilled water	Invitrogen	REF.10977-015
Xylene	PEKING REAGENT	No.33535