Cancer-associated fibroblasts enhance colorectal cancer lymphatic metastasis via CLEC11A/LGR5-mediated WNT pathway activation
Chuhan Zhang, Teng Pan, Yuyuan Zhang, Yushuai Wu, Anning Zuo, Shutong Liu, Yuhao Ba, Benyu Liu, Shuaixi Yang, Yukang Chen, Hui Xu, Peng Luo, Quan Cheng, Siyuan Weng, Long Liu, Xing Zhou, Jingyuan Ning, Xinwei Han, Jinhai Deng, Zaoqu Liu
Chuhan Zhang, Teng Pan, Yuyuan Zhang, Yushuai Wu, Anning Zuo, Shutong Liu, Yuhao Ba, Benyu Liu, Shuaixi Yang, Yukang Chen, Hui Xu, Peng Luo, Quan Cheng, Siyuan Weng, Long Liu, Xing Zhou, Jingyuan Ning, Xinwei Han, Jinhai Deng, Zaoqu Liu
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Research Article Gastroenterology Oncology

Cancer-associated fibroblasts enhance colorectal cancer lymphatic metastasis via CLEC11A/LGR5-mediated WNT pathway activation

Abstract

Hypoxia in the tumor microenvironment promotes lymphatic metastasis, yet the role of cancer-associated fibroblasts (CAFs) in this process remains insufficiently elucidated in colorectal cancer (CRC). In this study, we developed a large language model–based cellular hypoxia–predicting classifier to identify hypoxic CAFs (HCAFs) at single-cell resolution. Our findings revealed that HCAFs enhance CRC lymphatic metastasis by secreting CLEC11A, a protein that binds to the LGR5 receptor on tumor cells, subsequently activating the WNT/β-catenin signaling pathway. This promotes epithelial-mesenchymal transition and lymphangiogenesis, facilitating the spread of tumor cells via the lymphatic system. Furthermore, we demonstrate that the hypoxia-induced transcription factor HIF1A regulates the conversion of normoxic CAFs to HCAFs, driving CLEC11A expression and promoting metastasis. In vivo and vitro experiments confirmed the pro-metastatic role of CLEC11A in CRC, with its inhibition reducing lymphatic metastasis. This effect was markedly reversed by targeting the LGR5 receptor on tumor cells or inhibiting the WNT/β-catenin pathway, further elucidating the underlying mechanisms of CLEC11A-driven metastasis. These findings underscore the potential of targeting the CLEC11A-LGR5 axis to prevent lymphatic dissemination in CRC. Our study highlights the role of HCAFs in CRC progression and reveals mechanisms of lymphatic metastasis for intervention.

Authors

Chuhan Zhang, Teng Pan, Yuyuan Zhang, Yushuai Wu, Anning Zuo, Shutong Liu, Yuhao Ba, Benyu Liu, Shuaixi Yang, Yukang Chen, Hui Xu, Peng Luo, Quan Cheng, Siyuan Weng, Long Liu, Xing Zhou, Jingyuan Ning, Xinwei Han, Jinhai Deng, Zaoqu Liu

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Figure 4

Hypoxia-activated HIF1A in CAFs transcriptionally enhanced the expression of CLEC11A.

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Hypoxia-activated HIF1A in CAFs transcriptionally enhanced the expressio...
(A–C) VECTOR (A) and Monocle (B and C) analyses of the transition from NCAFs to HCAFs. UMAP, uniform manifold approximation and projection. (D) Changes in gene expression (left) and pathway activity correlation (right) along Monocle pseudotime. (E) Gene Ontology Biological Process enrichment analysis of pseudotime-associated genes. (F) Gene set enrichment analysis revealed the association of pseudotime-associated genes related to hypoxia and EMT pathways. NES, normalized enrichment score. (G) GeneSwitches analysis identifying key transcription factors involved in the transition from NCAFs to HCAFs. TFs, transcription factors. (H) Significant (P value < 0.05) differences in transcription factor activity between NCAFs and HCAFs. (I) HIF1A regulon activity, expression, and pseudotime correlation. (J and K) Box plot showing differences in HIF1A regulatory specificity (J) and expression (K) between NCAFs and HCAFs. (L) The downstream target gene network of HIF1A. (M) Expression dynamics of HIF1A and CLEC11A along Monocle pseudotime. (N and O) Correlation of HIF1A and CLEC11A in single-cell (N) and bulk transcriptomic datasets (O). (P) ChIP-qPCR analysis showing significant enrichment of HIF1A at the promoter region of CLEC11A (n = 4 per group). (Q) Luciferase assay showing that HIF1A enhances WT over mutant CLEC11A promoter activity (n = 4 per group). (R) WB analysis of CLEC11A and HIF1A protein levels in CAFs. (S) RT-qPCR analysis of CLEC11A mRNA levels in CAFs (n = 4 per group). All data are presented as means ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, by Spearman’s rank correlation test (D, I, and O), Pearson’s correlation test (N), hypergeometric test with Benjamini-Hochberg correction (E), permutation test with Benjamini-Hochberg correction (F), empirical Bayes moderated t test with Benjamini-Hochberg correction (H), Mann-Whitney U test (J, K, and P), and 1-way ANOVA with Tukey’s post test (Q and S).