Abstract:

Objective To discuss the effect of exosome-induced macrophage polarization to M2 type on the biological behavior of human esophageal squamous cell carcinoma （ESCC） cells， and to clarify its molecular mechanism. Methods The data of esophageal cancer and normal esophageal tissue from The Cancer Genome Atlas （TCGA） database were analyzed； Gene Expression Profiling Interactive Analysis （GEPIA） was used to identify the enrichment of different subtypes of tumor-associated macrophages （TAMs） in esophageal cancer and normal tissues and the correlation between hypoxia-inducible factor 1α （HIF-1α） and related molecule expression in esophageal cancer； Tumor Immune Estimation Resource （TIMER） software was used to analyze the impact of TAMs on the survival indicators of esophageal cancer patients； single-sample gene set enrichment analysis （ssGSEA） was used to screen genes related to M2-type TAMs polarization. The Gene Expression Omnibus （GEO） database was searched with the keywords “ESCC” and “exosome” to obtain the GSE104926 dataset， which included plasma exosome gene expression profile data from ESCC patients and non-ESCC patients. GEO2R was used to analyze differential genes， exosome isolation kit was used to extract and purify exosomes produced by human ESCC cell line EC109. Transmission electron microscope （TEM） and nanoparticle tracking analysis （NTA） were used to detect the morphology and particle size of exosomes； Western blotting method was used to detect the expression of exosome-related proteins cluster of differentation（CD）9 and CD63 in the purified exosomes and the purified EC109 supernatant. Human peripheral blood mononuclear cell line THP-1 was induced into M0 macrophages using phorbol ester； immunofluorescence staining was used to observe the internalization process of exosomes by macrophages； flow cytometry was used to detect the expression of phenotypic markers CD80 and CD206 in macrophages after treated with EC109 exosomes and untreated M0 macrophages. Under hypoxic conditions， real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of HIF-1α mRNA and protein in macrophages before and after EC109 exosome treatment. The macrophages before and after exosome induction were co-cultured with EC109 cells； cell counting kit 8（CCK-8） method was used to detect the proliferation activity of the EC109 cells； flow cytometry was used to detect the apoptotic rate of the EC109 cells； Transwell chamber assay was used to detect the rate of invasion EC109 cells； scratch wound healing assay was used to detect the migration ability of the EC109 cells and the wound closure rate was calculated. Results The TCGA and GEO data analysis results showed that compared with normal esophageal tissue， the infiltration numbers of various subtypes of TAMs in esophageal cancer were significantly increased （P<0.05）； the infiltration number of M2-type TAMs was closely related to the disease-free interval（DFI） of esophageal cancer patients （P<0.05）. Biglycan （BGN） was a gene with increased expression in plasma exosomes of ESCC patients and significantly associated with M2-type TAMs polarization （P<0.05）. In esophageal cancer tissue， there were significant positive correlations between the expression levels of M2-type TAMs markers macrophage scavenger receptor 1 （MSR1）， biglycan（BGN） and HIF-1α （r>0， P<0.05）. The isolated EC109-derived exosomes exhibited a cup-shaped morphology with a size of （127.17±0.90） nm. Compared with extracted EC109 supernatant， the expression levels of CD9 and CD63 in the extracted EC109 exosomes were significantly increased （P<0.05）. The EC109 exosomes could be taken up and internalized by macrophages. Compared with untreated M₀ macrophages， the number of CD80^- and CD206⁺ cells in macrophages treated with EC109 exosomes was significantly increased （P<0.05）， and the expression levels of HIF-1α mRNA and protein in the cells were significantly increased （P<0.05）. Compared with M0 macrophage treatment group， after co-culture of exosome-induced macrophages with EC109 cells， the proliferation activity of the EC109 cells was increased （P<0.05）， the apoptotic rate was increased （P<0.05）， the rate of invasion cells was increased （P<0.05）， and the migration area was increased （P<0.05）. Conclusion The ESCC cell-derived exosomes may regulate macrophage polarization by activating HIF-1α through BGN， thereby promoting ESCC proliferation and invasion， and inhibiting apoptosis； its mechanism is related to the role of exosomes in signal transmission in the interaction between ESCC and TAMs.

Key words: Esophageal neoplasm, Exosome, Tumor-associated macrophage, Macrophage polarization, Hypoxia-inducible factor 1α, Biglycan