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Background <p>Colorectal cancer (CRC) progression and metastasis are primarily driven by dynamic changes within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) exhibit significant heterogeneity within the TME and play a crucial role in regulating ferroptosis. Therefore, identifying novel ferroptosis-related biomarkers associated with CAFs could contribute to the development of personalized therapeutic strategies for cancer.</p> Methods <p>This study integrates single-cell sequencing data from 64 CRC samples and uses hierarchical clustering to identify cancer-associated fibroblast (CAF) subpopulations. We then combined spatial transcriptomics, hdWGCNA, CellChat, and Monocle analyses to characterize CAF subgroups. Single-cell and spatial transcriptomic data were integrated with Seurat to build a comprehensive spatial atlas. Multiplex immunohistochemical staining was used to analyze the spatial relationships of key genes with CAFs. Functional validation of these genes was performed with molecular docking, qPCR, Western blot, and both cellular and animal experiments.</p> Results <p>Single-cell data analysis identified five distinct CAF subpopulations, among which inflammatory cancer-associated fibroblasts (iCAFs) were significantly associated with the ferroptosis pathway and closely correlated with the proliferation and metastasis of malignant epithelial cells (<i>p</i> = 0.87). WGCNA revealed that the iCAFs-M10 module exhibited the strongest correlation with the expression of ferroptosis-related genes (cor = 0.6796). Further investigation identified ATG13 as a key hub gene, highly expressed in tumor tissues and significantly associated with poor patient prognosis. Spatial transcriptomics and multiplex immunohistochemistry (mIHC) results demonstrated that ATG13 was highly expressed in CAFs and co-localized with CAFs markers, such as α-SMA and Vimentin. In vitro and in vivo functional assays confirmed that ATG13 knockdown effectively inhibited the proliferation, migration, and invasion of colorectal cancer cells. Additionally, ferroptosis was induced upon ATG13 silencing, as evidenced by increased levels of GPX4, SLC7A11, ACSL4, MDA, Fe<sup>2</sup>⁺, GSH, and lipid peroxidation. In conclusion, this study reveals that ATG13, highly expressed in iCAFs, is associated with ferroptosis suppression in CRC, suggesting a potential CAF-tumor cell crosstalk axis that warrants further mechanistic investigation, thereby promoting the malignant progression of colorectal cancer. These findings provide potential targets for therapeutic strategies aimed at targeting the CAFs-ferroptosis axis.</p> Conclusion <p>This study identifies the iCAFs subpopulation as playing a key role in regulating ferroptosis in CRC, with ATG13 acting as a negative regulator of ferroptosis that promotes tumor progression. These findings provide new insights and potential targets for understanding the mechanisms of ferroptosis in CRC and for the development of targeted therapies.</p>

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Integrative single-cell and spatial transcriptomic analysis reveals that ATG13 enriched in cancer-associated fibroblasts is associated with the suppression of ferroptosis in colorectal cancer

  • 期刊论文

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  • 发表日期：

    2026年6月16日

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  • Tong Wang,
  • Jiaming Wu,
  • Peng Wang,
  • Jingjing Huang,
  • Lingfeng Guo,
  • Jialu Guan,
  • Shuo Ning,
  • Jingjing Wang,
  • Wenlu Liu,
  • Fengyu Tian,
  • Zhenghao Luan,
  • Zhongbing Wu,
  • Huayang Pan,
  • Desen Liang,
  • Dawei Wang

摘要

Background

Colorectal cancer (CRC) progression and metastasis are primarily driven by dynamic changes within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) exhibit significant heterogeneity within the TME and play a crucial role in regulating ferroptosis. Therefore, identifying novel ferroptosis-related biomarkers associated with CAFs could contribute to the development of personalized therapeutic strategies for cancer.

Methods

This study integrates single-cell sequencing data from 64 CRC samples and uses hierarchical clustering to identify cancer-associated fibroblast (CAF) subpopulations. We then combined spatial transcriptomics, hdWGCNA, CellChat, and Monocle analyses to characterize CAF subgroups. Single-cell and spatial transcriptomic data were integrated with Seurat to build a comprehensive spatial atlas. Multiplex immunohistochemical staining was used to analyze the spatial relationships of key genes with CAFs. Functional validation of these genes was performed with molecular docking, qPCR, Western blot, and both cellular and animal experiments.

Results

Single-cell data analysis identified five distinct CAF subpopulations, among which inflammatory cancer-associated fibroblasts (iCAFs) were significantly associated with the ferroptosis pathway and closely correlated with the proliferation and metastasis of malignant epithelial cells (p = 0.87). WGCNA revealed that the iCAFs-M10 module exhibited the strongest correlation with the expression of ferroptosis-related genes (cor = 0.6796). Further investigation identified ATG13 as a key hub gene, highly expressed in tumor tissues and significantly associated with poor patient prognosis. Spatial transcriptomics and multiplex immunohistochemistry (mIHC) results demonstrated that ATG13 was highly expressed in CAFs and co-localized with CAFs markers, such as α-SMA and Vimentin. In vitro and in vivo functional assays confirmed that ATG13 knockdown effectively inhibited the proliferation, migration, and invasion of colorectal cancer cells. Additionally, ferroptosis was induced upon ATG13 silencing, as evidenced by increased levels of GPX4, SLC7A11, ACSL4, MDA, Fe2⁺, GSH, and lipid peroxidation. In conclusion, this study reveals that ATG13, highly expressed in iCAFs, is associated with ferroptosis suppression in CRC, suggesting a potential CAF-tumor cell crosstalk axis that warrants further mechanistic investigation, thereby promoting the malignant progression of colorectal cancer. These findings provide potential targets for therapeutic strategies aimed at targeting the CAFs-ferroptosis axis.

Conclusion

This study identifies the iCAFs subpopulation as playing a key role in regulating ferroptosis in CRC, with ATG13 acting as a negative regulator of ferroptosis that promotes tumor progression. These findings provide new insights and potential targets for understanding the mechanisms of ferroptosis in CRC and for the development of targeted therapies.