Int J Stem Cells.  2022 Nov;15(4):415-421. 10.15283/ijsc22132.

Transcriptomic Comparison Analysis between Ameloblastoma and AM-1 Cell Line

• Li S
• Lee DJ
• Kim HY
• Harada H
• Jung YS
• Jung HS

• KMID: 2536211
• DOI: http://doi.org/10.15283/ijsc22132

Abstract

Cancer initiation and progression are profoundly along with the crosstalk between cancer cells and the surrounding stroma. Accumulating evidence has shown that the therapy targeting the extracellular matrix (ECM) would regress tumor growth and invasion in the most common carcinomas. However, it remains largely unexplored in several rare tumors like odontogenic tumors. Ameloblastoma (AM) is the representative odontogenic epithelial tumor in the jawbone, and it usually infiltrates into adjacent bone marrow and has unlimited growth capacity and a high potential for recurrence. This study aims to investigate the role of collagen-rich ECM during the invasion of AM. Transcriptomic analysis revealed that ECM- and epithelial-to-mesenchymal transition (EMT)-related genes were up-regulated in AM compared to ameloblastoma cell line, AM-1. Tumoroid forming analysis showed that Collagen-rich ECM is indispensable for AM progression, especially for aggressive growth patterns and collective invasion.

Keyword

AM-1; Ameloblastoma; Extracellular matrix; Collagen; Tumor invasiveness

Figure

• 

  Fig. 1 Transcriptomic comparison between ameloblastoma and AM-1 cell line. (a) Heatmap of whole transcriptome showing a significant difference in the transcriptome between three AM samples (AM#1, #2, #3) and AM-1 cell line. (b) Volcano plot of GO term of significantly down-regulated DEGs. The top five GO terms related to ECM (red), collagen (blue), and EMT (green) are indicated in different colors, and the top five GO IDs are selectively labeled. Cutoff values for DEG level in GO analysis were adjusted p-value <0.05 and |fold change| >2.0. (c∼e) Top five GO term lists and heatmaps of genes consisting of top three GO terms related to ECM, collagen, and EMT. In heatmaps, major genes of the tumor microenvironment are labelled.

• 

  Fig. 2 Type I collagen plays a crucial role in AM-1 tumoroid progression. (a) Brightfield image showing that round-shaped AM-1 tumoroids were observed in the Matrigel culture group. (b) Hematoxylin and eosin staining of a well-organized tumoroid structure, comprised of a heterogeneous cell population. (c) Col I was negatively expressed, and intense expression of Col IV was observed surrounding the edge of Matrigel-cultured tumoroids. (d) The terminal differentiation marker CK10 was strongly expressed in the tumoroid core, and p63 was intensely expressed in the basal cell layer. (e) MMP-9 showed a moderate expression in the basal cell layer. (f) E-cadherin was intensely expressed in the suprabasal layer and moderately expressed in the basal cell layer (indicated by a white dotted line). Vimentin was negatively expressed in the Matrigel culture group. (g) Net-like structures were observed in collagen gel-cultured (4 mg/ml) AM-1 tumoroids. (h) Hematoxylin and eosin staining of the invading strands from collagen gel-cultured tumoroids. (i) Immunohistochemistry staining of Col I and Col IV. Arrowheads indicate the rearranged fibrillar collagen fiber by adjacent AM-1 tumoroids, and Col IV was negatively expressed. (j) CK10 showed negative expression, and p63 showed a moderate expression. (k) Intense MMP-9 expression was observed in the majority of collagen-cultured AM-1 tumoroids. (l) E-cadherin showed a moderate expression, and vimentin was intensely expressed in the leading region of the invading tumor strand as indicated by an arrowhead. Nuclei were counterstained with TO-PRO-3 (TP-3). (m) Representative images of AM-1 cell cultured in Matrigel or varying concentrations of type I collagen gel (1, 2, and 4 mg/ml) obtained from 10 h of live-cell imaging. AM-1 cells were indicated with CMFDA dye (green). (n) Trajectory plots depicted the movement path of individual AM-1 cells (n=5). Through transformation, all trajectories started at the origin (x=0, y=0) in order to visualize the movements of all cells relative to each other. (o) Quantitative analysis of accumulation distance (μm) of Matrigel or collagen gel-cultured AM-1 cell. Scale bars: 100 μm. *p<0.05, ***p<0.001, and ****p<0.0001.

Reference

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