We integrated single-cell RNA sequencing data from seven datasets (192 ESCC patients, >440k cells) and spatial transcriptomics to delineate the tumor microenvironment (TME) landscape of esophageal squamous cell carcinoma (ESCC) during progression and immunotherapy response. B-lineage cells decreased with tumor progression but accumulated in immunotherapy-resistant cases. Resistant tumors harbored a malignant subpopulation with elevated cholesterol biosynthesis. Spatial and communication analyses revealed that these cholesterol-biosynthetic tumor cells interacted with germinal center B cells in tertiary lymphoid structures. Mechanistically, tumor-derived MIF disrupted B cell immunity by competing with CXCL12–CXCR4 signaling through MIF–CXCR4 binding.

MIF-expressing tumor cells mediate immunotherapeutic resistance in esophageal squamous cell carcinoma

Jing Song¹, Xiaomei Song², Yue Xie¹, Hong Guo² , Yupeng Cun¹ 

1. Pediatric Research Institute, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Cognitive Development and Learning and Memory Disorders, National Clinical Research Center for Child Health and Disorders, Children’s Hospital of Chongqing Medical University, No. 136 Zhongshan 2nd Road, Yuzhong District, 400014, Chongqing, China.
2. Department of Gastroenterology, Chongqing General Hospital, Chongqing University, 118 Xingguang Avenue, Yubei District, 401147, Chongqing, China.

Background: Despite the use of immunotherapy in esophageal squamous cell carcinoma (ESCC), treatment failure occurs occasionally in patients, yet the underlying mechanisms remain poorly understood.

Methods: We conducted large-scale single-cell RNA sequencing (scRNA-seq) data analysis, which integrated seven independent datasets from 192 ESCC patients to yield over 440,000 high-quality single cells, to systematically characterize the tumor microenvironment (TME) landscape during ESCC progression and immunotherapy response. Additionally, we performed high-resolution spatial transcriptomics (stRNA-seq) using the 10x Visium HD platform on paired pre- and post-treatment tissues from two patients (one immunotherapy responder and one non-responder), which enhanced the findings from the scRNA-seq data and mapped therapy-induced TME at the spatial level. Multiplex immunohistochemistry was employed based on seven patients to confirm distinct patterns of intercellular crosstalk underlying differential therapeutic outcomes.

Results: In scRNA-seq data, we found that B lineage cells were reduced during ESCC progression but were enriched in immunotherapy-resistant patients. Further analysis of malignant ESCC cells suggested that immunotherapy resistance might be associated with a subpopulation of tumor cells exhibiting aberrantly elevated cholesterol biosynthesis. Cell communication analysis of scRNA-seq and stRNA-seq data collectively revealed that immunotherapy resistance was linked to cellular crosstalk between cholesterol-biosynthetic tumor cells and germinal center (GC) B cells within tertiary lymphoid structures. Notably, single-cell, spatial data, and multiplex immunohistochemistry demonstrated that cholesterol biosynthesis-associated ESCC cells express elevated levels of MIF. This disrupts GC reactions by competing with the CXCL12-CXCR4 signaling axis via MIF-CXCR4 interactions, thereby impairing B cell-mediated immunity.

Conclusions: MIF⁺ tumor cells in GCs may be a biomarker for predicting immunotherapy resistance in ESCC.

Keywords: esophageal squamous cell carcinoma, immunotherapy, B cell, cholesterol biosynthesis, ScRNA-seq, spatial RNA sequencing