Integrative analyses of single-cell and bulk RNA sequencing reveal tumour microenvironment features associated with neoadjuvant immunochemotherapy response in oesophageal squamous cell carcinoma

Abstract

Background: Neoadjuvant chemotherapy combined with immunotherapy (NACI) has shown promise in oesophageal squamous cell carcinoma (ESCC). However, a significant proportion of patients exhibit resistance to NACI, and the underlying mechanisms remain unresolved.

Methods: We integrated single-cell RNA sequencing data, including seven patients with ESCC treated with NACI and 69 patients with ESCC treated with surgery alone. Bulk RNA sequencing data were obtained from a public database. Immunohistochemistry and multiplexed immunofluorescence staining were performed to verify the role of important immune cells and molecules in clinical treatment outcomes.

Results: Here, we profiled the transcriptomes of 512 736 cells from 76 patients with ESCC, revealing that the nonresponder baseline tumour microenvironment exhibited a relative absence of major histocompatibility complex II molecules expressed on CD20⁺B cells and a low expression of CXCL13 on CD4_Tfh and CD8_Tex cells. We also identified CD68⁺CD163⁺ macrophages that highly expressed the immunosuppressive LGALS9 gene and preferentially accumulated in the nonresponders after NACI treatment. In addition, nonresponders had a higher baseline fraction of POSTN⁺fibroblasts, which is associated with higher infiltration of CD68⁺CD163⁺ macrophages and lower infiltration of germinal centre B cells. Finally, we described the different characteristics of malignant epithelial cells from different pathological responses to tumours.

Conclusions: This study has unveiled a potential regulatory network among immune cells, stromal cells and malignant epithelial cells under different pathological response conditions and provides a valuable resource for discovering novel targeted therapies for ESCC.

Keywords

immunotherapy / neoadjuvant chemotherapy / oesophageal squamous cell carcinoma / single-cell RNA sequencing / tumour microenvironment

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2025 The Author(s). Clinical and Translational Discovery published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.