Tumor-derived neutrophil extracellular trap–associated DNA impairs treatment efficacy in breast cancer via CCDC25-dependent epithelial-mesenchymal transition
Heliang Li, Yetong Zhang, Jianghua Lin, Jiayi Zeng, Xinyan Liang, Linxi Xu, Jiang Li, Xiaoming Zhong, Xu Liu, Zhou Liu, Xinyu Yang, Yunyi Zhang, Shun Wang, Erwei Song, Man Nie, Linbin Yang
Heliang Li, Yetong Zhang, Jianghua Lin, Jiayi Zeng, Xinyan Liang, Linxi Xu, Jiang Li, Xiaoming Zhong, Xu Liu, Zhou Liu, Xinyu Yang, Yunyi Zhang, Shun Wang, Erwei Song, Man Nie, Linbin Yang
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Research Article Immunology Oncology

Tumor-derived neutrophil extracellular trap–associated DNA impairs treatment efficacy in breast cancer via CCDC25-dependent epithelial-mesenchymal transition

Abstract

Neutrophil extracellular traps (NETs) are associated with cancer progression; however, the functional role and clinical importance of NET-DNA in therapeutic resistance remain unclear. Here, we show that chemotherapy and radiotherapy provoke NET-DNA formation in primary tumor and metastatic organs in breast cancer patients and mouse models, and the level of NET-DNA correlates with treatment resistance. Mechanistically, the cathepsin C in tumor debris generated by anticancer therapy is phagocytosed by macrophages and drives CXCL1/2 and complement factor B production via activating the TLR4/NF-κB signaling pathway, subsequently promoting NETosis and impairing therapeutic efficacy. Importantly, we demonstrate that NET-DNA sensor CCDC25 is indispensable in NET-mediated treatment resistance by inducing cancer cell epithelial-mesenchymal transition via pyruvate kinase isoform M2–mediated STAT3 phosphorylation. Clinically, tumoral CCDC25 abundance is closely associated with poor prognosis in patients who underwent chemotherapy. Overall, our data reveal the mechanism of NET formation and elucidate the interaction of NET-CCDC25 in therapy resistance, highlighting CCDC25 as an appealing target for anticancer interventions.

Authors

Heliang Li, Yetong Zhang, Jianghua Lin, Jiayi Zeng, Xinyan Liang, Linxi Xu, Jiang Li, Xiaoming Zhong, Xu Liu, Zhou Liu, Xinyu Yang, Yunyi Zhang, Shun Wang, Erwei Song, Man Nie, Linbin Yang

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Figure 1

Chemotherapy and radiotherapy promote NET generation in breast cancer.

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Chemotherapy and radiotherapy promote NET generation in breast cancer. (...
(A) Workflow of scRNA-Seq of 4T1 tumors (n = 3/group). (B) Uniform manifold approximation and projection (UMAP) plot of cell subsets from vehicle (Veh) and Dox groups. The gray circle represents immune cells. (C) Stacked bar graph for percentage of immune cells (χ2 test). Bar colors represent corresponding subtypes described in B. (D) Expression levels of differentially expressed genes (DEGs) from GO and KEGG terms in neutrophils (adj. P < 0.05). Dot size and color indicate fraction of cells and average expression. (E) GSEA revealed enrichment of NET formation pathway in neutrophils from Dox group (permutation test). NOM, normalized. (F–I) Tumors, metastatic livers, lungs, and serum from 4T1-bearing mice were isolated and analyzed (n = 8/group). (F) Quantification of tumoral Ly6G+ cells (unpaired t test). (G) Representative H&E images and immunofluorescence images. Scale bars, 50 μm and 100 μm. * indicates areas magnified in the top-left insets. (H) Quantification of NET expression. Tumor, Mann-Whitney test. Liver and lung, unpaired t test. (I) Serum levels of MPO-DNA (Mann-Whitney test). (J and K) Quantification of NET expression in MCF-7 tumors (J) and MMTV-PyMT FVB/NJ mouse tumors (K) (n = 5/group, unpaired t test). (L) Schematic illustration. (M and N) Representative immunofluorescence images (M) and quantification (N) in paired pre- and post-NAC breast tumors (n = 81 pairs, Wilcoxon matched pairs signed-rank test). Scale bars, 50 μm and 1,000 μm. (O) Plasma levels of MPO-DNA in patients treated with or without chemotherapy (n = 40 and 41, respectively; Mann-Whitney test). (P) Quantification of NETs in untreated or radiotherapy-treated (radio) 4T1 tumors (n = 6/group, Mann-Whitney test). (Q) Quantification of neutrophil xCell score (GSE59733) from paired pre- and post-radio breast tumors (n = 9 pairs, paired t test) (22). (R) GSEA reveals enrichment of NET formation pathway in post-radio tumors (GSE59733) (22). Schematic illustration in A and L is created with BioRender.com. Data represent mean ± SD (F, H–K, and P) and mean ± SEM (O). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.