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

doi:10.4251/wjgo.v16.i4.1500

Transcriptome sequencing reveals novel biomarkers and immune cell infiltration in esophageal tumorigenesis

World J Gastrointest Oncol. 2024 Apr 15;16(4):1500-1513. doi: 10.4251/wjgo.v16.i4.1500.

Authors

Jian-Rong Sun¹, Dong-Mei Chen², Rong Huang¹, Rui-Tao Wang¹, Li-Qun Jia³

Affiliations

¹ School of Clinical Medicine, Beijing University of Chinese Medicine, Beijing 100029, China.
² Integrated Chinese and Western Medicine Oncology, China-Japan Friendship Hospital, Beijing 100029, China.
³ Integrated Chinese and Western Medicine Oncology, China-Japan Friendship Hospital, Beijing 100029, China. liqun_jia@hotmail.com.

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