Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Apr 15, 2024; 16(4): 1500-1513
Published online Apr 15, 2024. doi: 10.4251/wjgo.v16.i4.1500
Transcriptome sequencing reveals novel biomarkers and immune cell infiltration in esophageal tumorigenesis
Jian-Rong Sun, Dong-Mei Chen, Rong Huang, Rui-Tao Wang, Li-Qun Jia
Jian-Rong Sun, Rong Huang, Rui-Tao Wang, School of Clinical Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
Dong-Mei Chen, Li-Qun Jia, Integrated Chinese and Western Medicine Oncology, China-Japan Friendship Hospital, Beijing 100029, China
Author contributions: Sun JR and Jia LQ designed the study and modified the manuscript; Sun JR and Chen DM carried out the data extraction and statistical analysis; Sun JR, Chen DM, Huang R, and Wang RT drafted the manuscript; and all authors contributed to the article and approved the submitted version.
Supported by National Natural Foundation of China, No. 82174223; and 2019 Chinese and Western Medicine Clinical Collaborative Capacity Building Project for Major Difficult Diseases, No. 2019-ZX-005.
Institutional review board statement: The research involving human tissue samples was approved by the Institutional Ethics Committee of Cixian Cancer Hospital (2019-CXCH-19) and China-Japan Friendship Hospital (2019-190-K131).
Institutional animal care and use committee statement: All the animal procedures and experiments conducted in the current study were approved by the Animal Ethics Committee of the China-Japan Friendship Hospital (Permit No. ZRDWLL20230210).
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: The data used to support the findings of this study are available from the corresponding author upon request.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Li-Qun Jia, MD, Chief Doctor, Professor, Integrated Chinese and Western Medicine Oncology, China-Japan Friendship Hospital, No. 2 Yinghua East Street, Chaoyang District, Beijing 100029, China. liqun_jia@hotmail.com
Received: November 21, 2023
Peer-review started: November 21, 2023
First decision: December 27, 2023
Revised: January 7, 2024
Accepted: February 4, 2024
Article in press: February 4, 2024
Published online: April 15, 2024
Abstract
BACKGROUND

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, and its development comprises a multistep process from intraepithelial neoplasia (IN) to carcinoma (CA). However, the critical regulators and underlying molecular mechanisms remain largely unknown.

AIM

To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention.

METHODS

A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide (4NQO) to C57BL/6 mice. Moreover, we established a control group without 4NQO treatment of mice. Then, transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses, including low-grade IN (LGIN), high-grade IN (HGIN), and CA, and controlled normal tissue (NOR) samples. Differentially expressed genes (DEGs) were identified in the LGIN, HGIN, and CA groups, and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The CIBERSORT algorithm was used to detect the pattern of immune cell infiltration. Immunohistochemistry (IHC) was also conducted to validate our results. Finally, the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice.

RESULTS

Compared with those in the NOR group, a total of 681541, and 840 DEGs were obtained in the LGIN, HGIN, and CA groups, respectively. Using the intersection of the three sets of DEGs, we identified 86 genes as key genes involved in the development of ESCC. Enrichment analysis revealed that these genes were enriched mainly in the keratinization, epidermal cell differentiation, and interleukin (IL)-17 signaling pathways. CIBERSORT analysis revealed that, compared with those in the NOR group, M0 and M1 macrophages in the 4NQO group showed stronger infiltration, which was validated by IHC. Serum cytokine analysis revealed that, compared with those in the NOR group, IL-1β and IL-6 were upregulated, while IL-10 was downregulated in the LGIN, HGIN, and CA groups. Moreover, the expression of the representative key genes, such as S100a8 and Krt6b, was verified in external human samples, and the results of immunohistochemical staining were consistent with the findings in mice.

CONCLUSION

We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions. In addition, we found that macrophage infiltration and abnormal alterations in the levels of inflammation-associated cytokines, such as IL-1β, IL-6, and IL-10, in the peripheral blood may be closely associated with the development of ESCC.

Keywords: Esophageal squamous cell carcinoma, Intraepithelial neoplasia, Tumorigenesis, Transcriptome sequencing, Biomarkers, Immune cell infiltration, 4-nitroquinoline 1-oxid

Core Tip: The development of esophageal squamous cell carcinoma (ESCC) involves a stepwise progression from intraepithelial neoplasia to carcinoma. Examining the genes and immune cell infiltration in the microenvironment associated with the multi-stage progression of ESCC is essential for facilitating diagnosis and early intervention. A total of 86 crucial genes linked with ESCC development were discovered using transcriptome sequencing. These genes were enriched in pathways related to keratinization, epidermal cell differentiation, and interleukin (IL)-17 signaling. Additionally, the infiltration of macrophages and abnormal alterations of inflammation-associated cytokines (e.g., IL-1β, IL-6, and IL-10) in peripheral blood may be strongly linked with the development of ESCC.