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

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.