Yaojun Peng ¹ Lingxiong Wang ² Juan Yang ³ Qiyan Wu ² Xiaoxuan Sun ⁴ Jinying Zhang ⁵ Yanju Yu ⁶ Liping Zhang ¹ Jie Gao ⁷ Qing Zhou ⁸ Haiyan Zhu ¹ Fan Yin ^7*

• ¹ Department of Emergency, Chinese PLA General Hospital, Beijing, China
• ² Lab of the Oncology Department, The First Medical Center, Chinese PLA General Hospital, Beijing, China
• ³ Tianjin Fourth Central Hospital, Tianjin, Hebei, China
• ⁴ Department of Oncology Surgery, Tianjin Cancer Hospital Airport Free Trade Zone Hospital, Tianjin, China
• ⁵ Department of Basic Medicine, Medical School of Chinese PLA, Beijing, China
• ⁶ Institute of oncology, the Fifth Medical Centre, Chinese PLA General Hospital, Beijing, China
• ⁷ Department of Oncology, The Second Medical Center & National Clinical Research Center of Geriatric Disease, Chinese PLA General Hospital, Beijing, China
• ⁸ Department of Gastroenterology, The Second Medical Center & National Clinical Research Center of Geriatric Disease, Chinese PLA General Hospital, Beijing, China

Background Inhibition of indolamine-2,3-dioxygenase 1 (IDO1) has been proposed as a promising strategy for cancer immunotherapy; however, it has failed in clinical trials. Macrophages in the tumour microenvironment (TME) contribute to immune escape and serve as potential therapeutic targets. This study investigated the expression pattern of IDO1 in TME and its impact on prognosis and therapeutic response of patients with esophageal squamous cell carcinoma (ESCC). Methods RNA sequencing data from 95 patients with ESCC from The Cancer Genome Atlas (TCGA) database were used to explore the prognostic value of IDO1. Bioinformatics tools were used to estimate scores for stromal and immune cells in tumour tissues, abundance of eight immune cell types in TME, and sensitivity of chemotherapeutic drugs and immune checkpoint (IC) blockage. The results were validated using digitalized immunohistochemistry and multiplexed immunofluorescence in ESCC tissue samples obtained from our clinical center. Results TCGA and validation data suggested that high expression of IDO1 was associated with poor patient survival, and IDO1 was an independent prognostic factor. IDO1 expression positively correlated with macrophages in TME and PDCD1 within diverse IC genes. Single-cell RNA sequencing data analysis and multiplexed immunofluorescence verified the coexpression of IDO1 and PD-1 in tumor-associated macrophages (TAMs). Patients with high IDO1 expression showed increased sensitivity to various chemotherapeutic drugs, while were more likely to resist IC blockage. Conclusions This study identifies IDO1 as an independent prognostic indicator of OS in patients with ESCC, reveals a compelling connection of IDO1, PD-1, and TAMs, and explores the sensitivity of patients with high IDO1 expression to chemotherapeutic drugs and their resistance to IC blockade. These findings open new avenues for potential targets in ESCC immunotherapy.