✍🏼 Mingxu Xie, Kai Yuan, Yongxin Zhang, Yating Zhang, Ruyi Zhang, Jiuhe Gao, Wenchao Wei, Lanping Jiang, Tianhui Li, Yanqiang Ding, Luyao Wang, Yufeng Lin, Chi Chun Wong, Jun Yu

🏠 Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK-Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China

📑 Cancer Cell (2025)

Abstract
Most colorectal cancer (CRC) patients do not respond to immune checkpoint blockade (ICB) therapy. Here, we identify Clostridium butyricum as a probiotic that boosts anti-PD-1 efficacy in CRC. In orthotopic allografts of microsatellite instability-high (MSI-H) and microsatellite stable (MSS) CRC, C. butyricum potentiates tumor suppressive effect of anti-PD-1, which is verified in AOM/DSS-induced CRC and germ-free mice. Single-cell RNA-seq reveals that C. butyricum activates cytotoxic CD8⁺ T lymphocytes (CTLs) and impairs tumor-associated macrophages (TAMs), especially in conjunction with anti-PD-1. Mechanistically, C. butyricum surface protein secD binds to CRC cell receptor glucose-regulated protein 78 (GRP78), which inactivates GRP78 and PI3K-AKT-NF-κB pathway, leading to reduced secretion of interleukin (IL)-6, an immunosuppressive cytokine that blunts CTLs and induces TAMs. Translational impact of C. butyricum in boosting anti-PD-1 efficacy is validated in huCD34⁺ humanized mice and autologous patient-derived CRC organoids-CTLs co-culture system. To summarize, C. butyricum is a promising adjuvant to augment ICB therapy.

How the WOLF was used in this study
The WOLF Cell Sorter (NanoCellect) was used as a critical enrichment step prior to single-cell RNA sequencing to ensure high-quality tumor-derived cell populations. Tumor tissues from CT26 orthotopic allografts were enzymatically digested into single-cell suspensions and stained with propidium iodide (PI) to exclude dead cells and FITC-conjugated anti-mouse CD45 to distinguish immune from non-immune cells. The WOLF Cell Sorter was then used to fluorescence-activate sort viable cells, removing dead cells and enabling clean separation of immune and non-immune compartments. This gentle, low-pressure sorting step ensured that only live, intact cells entered the 10× Genomics single-cell RNA-sequencing workflow, supporting accurate downstream classification of immune, epithelial, and tumor cell populations and robust differential gene expression analysis.