The interplay between tumor derived FOXO1 and macrophages in the tumor microenvironment of esophageal squamous cell carcinoma

Abstract

Esophageal cancer is the sixth most common cancer worldwide with high mortality. Compared with esophageal adenocarcinoma, esophageal squamous cell carcinoma (ESCC) represents higher global incidences, especially in central and south Asian populations, which accounts for 80% of cases. Notwithstanding various therapies targeting ESCC, the five-year survival rate still remains around 9-27.1% and new therapeutic targets are waiting to be explored. As a rule, esophageal mucosa with chronic irritation and inflammation showed a propensity to develop into ESCC, indicating the crucial role of the microenvironment in tumor progression. In addition, infiltrating tumor-associated macrophages are seen as a poor prognostic factor in most cancers. This study therefore focused on the interaction between tumor cells and macrophages and sought to identify the key molecule involved in the process. In this study, the RNA sequencing was conducted in three paired ESCC tumor and non-tumor tissues and found that FOXO1 was upregulated in tumor tissues when compared with non-tumor tissues. Then more clinical samples were analyzed by immunohistochemistry (IHC) and qRT-PCR to confirm these results, and it was found that FOXO1 overexpression indicated a worse prognosis in patients with ESCC. FoxO1 is a member of the Forkhead Box O family (FOXO) of transcription factors, and research focusing on immunity revealed it could modulate downstream pro-inflammatory molecules. Through analyzing 22 kinds of immune cells infiltration of our RNA sequencing data and The Cancer Genome Atlas(TCGA) database, the results showed that increased FOXO1 expression positively correlated with the tumor infiltrating macrophages, especially M2-like macrophages. This result was further confirmed by IHC and qRT-PCR of clinical tissues and nude mice models. After successfully obtaining uncommitted macrophages (M0 macrophages) and M2 macrophages, the M2 migration assay was conducted and suggested that FOXO1 could promote M2 macrophages migration. For the results of qRT-PCR, western blot and ELISA suggested that CCL20 was upregulated by FOXO1. CCL20 recombinant protein and neutralizing antibody were used in M2 migration assay and confirmed that tumor derived FOXO1 could promote M2 macrophages migration by CCL20 secretion. Moreover, the co-culture experiment of tumor cells and M0 macrophages showed FOXO1 overexpressing cells could induce M0 differentiation towards M2 macrophages. RNA sequencing was performed to confirm the phenotypes of the induced M2-like macrophages. To explore what factors modulated by FOXO1 induce the M2 differentiation, qRT-PCR, ELISA and western blot were conducted and revealed that the augmented FOXO1 expression could promote the secretion of CSF-1. CSF-1 recombinant protein and neutralizing antibody confirmed that FOXO1 also could promote the production of CSF-1 in cancer cells to induce the M2 differentiation. The recruited M2 macrophages promoted tumor proliferation and migration via the FAK/PI3K/AKT pathway. Our study suggested FOXO1 might promote the establishment of a pro-tumorigenic microenvironment with the aid of increased M2 macrophages. Based on the above results, FOXO1 is a potential prognostic marker in patients with ESCC and might provide reference in treatment selection.