Single-cell transcriptomics reveals hypoxia-driven iCAF_PLAU is associated with stemness and immunosuppression in anorectal malignant melanoma

Hao Zhang; Lin Gan; Xiaofei Duan; Baojing Tuo; Hao Zhang; Senbo Liu; Yugui Lian; Enjie Liu; Zhenqiang Sun

doi:10.1007/s00535-025-02273-5

Single-cell transcriptomics reveals hypoxia-driven iCAF_PLAU is associated with stemness and immunosuppression in anorectal malignant melanoma

J Gastroenterol. 2025 Jul 4. doi: 10.1007/s00535-025-02273-5. Online ahead of print.

Authors

Hao Zhang^{1

2}, Lin Gan^#^{1

3}, Xiaofei Duan¹, Baojing Tuo^{1

2}, Hao Zhang¹, Senbo Liu^{1

2}, Yugui Lian⁴, Enjie Liu⁵, Zhenqiang Sun^{6

7}

Affiliations

¹ Department of Colorectal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
² Henan Institute of Interconnected Intelligent Health Management, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
³ Department of Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
⁴ Department of Colorectal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China. lianyugui2010@163.com.
⁵ Department of Pathology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China. lianyugui2010@163.com.
⁶ Department of Colorectal Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China. fccsunzq@zzu.edu.cn.
⁷ Henan Institute of Interconnected Intelligent Health Management, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China. fccsunzq@zzu.edu.cn.

^# Contributed equally.

PMID: 40613916
DOI: 10.1007/s00535-025-02273-5

Abstract

Background: Anorectal malignant melanoma (ARMM) is a refractory malignancy that not only responds poorly to radiotherapy but also to immunotherapy. Tumor microenvironment (TME) components play an essential role in tumor progression and therapeutic response. However, TME characteristics of ARMM are not well understood.

Methods: We conducted a single-cell RNA sequencing on tumor and blood tissue from three ARMM patients, and combined with cutaneous melanoma datasets from the Gene Expression Omnibus database for a comprehensive joint analysis.

Results: Our findings revealed that cancer cells have four major patterns of chromosomal mutations. ARMM cancer cells exhibited marked intratumoral heterogeneity, and elevated angiogenesis, hypoxia, stemness, and epithelial-mesenchymal transition characteristics. We also identified the cancer stem cell subpopulation c5_Mel_CD55_VEPH1 in ARMM. Notably, we observed that CD8 + T cells in ARMM were poorly infiltrated and predominantly in a terminal exhausted state. Moreover, we identified a unique population of PLAU + fibroblast cells (iCAF_PLAU) in ARMM that likely have differentiated from myofibroblasts under hypoxic conditions. The iCAF_PLAU population enhances the stemness and aggressiveness of cancer cells through the ligand-receptor pairs WNT5A_FZD3_LRP6 and NRG1_ERBB3. In addition, iCAF_PLAU secretes CCL2, which binds to CCR1 on SPP1 + macrophages (TAM_SPP1) cells, leading to the activation of NFKBIA in TAM_SPP1 and subsequent upregulation of IL6, which may be linked to the exhaustion process of CD8 + T cells. Immunofluorescence staining confirmed the co-localization of iCAF_PLAU with TAM_SPP1 and TAM_SPP1 with CD8 + T cells.

Conclusion: Our data suggest a potential role of iCAF_PLAU in mediating cell-cell interactions within the TME of ARMM, highlighting potential therapeutic targets for this aggressive malignancy.

Keywords: Anorectal malignant melanoma; Hypoxia; Immunotherapy; ScRNA-seq; Tumor microenvironment.