Key Laboratory of Tumor Microenvironment and Immune Therapy of Zhejiang Province, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.
Cancer Center, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.
J Immunother Cancer. 2021 Oct;9(10). doi: 10.1136/jitc-2021-002875.
Neutrophils-linked premetastatic niche plays a key role in tumor metastasis, but not much is known about the heterogeneity and diverse role of neutrophils in niche formation. Our study focuses on the existence and biological function of a rarely delved subset of neutrophils, named as tumor-associated aged neutrophils (Naged, CXCR4CD62L), involved in premetastatic niche formation during breast cancer metastasis.
We explored the distributions of Naged in 206 patients and mice models (4T1 and MMTV-PyMT) by flow cytometry. The ability of Naged to form neutrophil extracellular traps (NETs) and promote tumor metastasis in patients and mice was determined by polychromatic immunohistochemistry, scanning electron microscopy and real-time video detection. Furthermore, the differences among tumor-associated Naged, Non-Naged and inflammation-associated aged neutrophils were compared by transcriptome, the biological characteristics of Naged were comprehensively analyzed from the perspectives of morphology, the metabolic capacity and mitochondrial function were investigated by Seahorse, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) and transmission electron microscopy (TEM). Finally, 120 patients' sample were applied to confirm the acceleration of Naged formation through secreted NAMPT, and the importance of blocking this pathway in mice was evaluated.
We find that Naged accumulate in the lung premetastatic niche at early stage of breast tumorigenesis in multiple mice models and also exist in peripheral blood and metastatic lung of patients with breast cancer. Naged exhibit distinct cell marker and morphological feature of oversegmented nuclei. Further transcriptome reveals that Naged are completely different from those of Non-Aged or inflammation-associated aged neutrophils and illustrates that the key transcription factor SIRT1 in Naged is the core to maintain their lifespan via mitophagy for their function. The responsible mechanism is that SIRT1 can induce the opening of mitochondrial permeability transition pore channels to release mitochondrial DNA and lead to the mitochondria-dependent vital NETs formation, rather than traditional Cit-Histone H3 dependent fatal-NETs. Further mechanically investigation found tumor derived NAMPT could induce Naged formation. Additionally, therapeutic interventions of Naged and its formation-linked pathways could effectively decrease breast cancer lung metastasis.
Naged exerts a vital role in breast cancer lung metastasis, and strategies targeting SIRT1-Naged-NETs axis show promise for translational application.
中性粒细胞相关的转移前龛在肿瘤转移中起着关键作用，但人们对龛内中性粒细胞的异质性和多样化作用知之甚少。我们的研究集中在一种很少被深入研究的中性粒细胞亚群的存在和生物学功能上，这种中性粒细胞被命名为肿瘤相关衰老中性粒细胞（Naged，CXCR4^CD62L），它参与乳腺癌转移过程中的转移前龛形成。
我们通过流式细胞术在 206 名患者和小鼠模型（4T1 和 MMTV-PyMT）中研究了 Naged 的分布情况。通过多色免疫组化、扫描电子显微镜和实时视频检测，确定了 Naged 形成中性粒细胞胞外陷阱（NETs）和促进患者和小鼠肿瘤转移的能力。此外，通过转录组比较了肿瘤相关的 Naged、非 Naged 和炎症相关衰老中性粒细胞之间的差异，从形态学、代谢能力和线粒体功能等方面全面分析了 Naged 的生物学特性，通过共免疫沉淀（Co-IP）、染色质免疫沉淀（ChIP）和透射电子显微镜（TEM）进行研究。最后，应用 120 名患者的样本证实了通过分泌的 NAMPT 加速 Naged 的形成，并在小鼠中评估了阻断该途径的重要性。
我们发现，Naged 在多种小鼠模型的乳腺癌早期肿瘤发生时积聚在肺部转移前龛中，也存在于乳腺癌患者的外周血和转移性肺中。Naged 表现出明显的细胞标记物和核分段的形态特征。进一步的转录组显示，Naged 与非衰老或炎症相关衰老中性粒细胞完全不同，并表明 Naged 中的关键转录因子 SIRT1 是通过线粒体自噬维持其寿命以发挥其功能的核心。负责的机制是，SIRT1 可以诱导线粒体通透性转换孔通道打开，释放线粒体 DNA，并导致依赖线粒体的重要 NETs 形成，而不是传统的 Cit-Histone H3 依赖性致命 NETs。进一步的机械研究发现，肿瘤衍生的 NAMPT 可以诱导 Naged 的形成。此外，针对 Naged 及其形成相关途径的治疗干预可以有效减少乳腺癌肺转移。
Naged 在乳腺癌肺转移中发挥着重要作用，靶向 SIRT1-Naged-NETs 轴的策略具有转化应用的前景。
