Published online Apr 15, 2024. doi: 10.4251/wjgo.v16.i4.1500
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
Processing time: 141 Days and 12.4 Hours
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.
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.
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.
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.
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.
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.