Cancer-associated fibroblasts (CAFs) orchestrate a supportive niche that fuels cancer metastatic development in non-small cell lung cancer (NSCLC). Due to the heterogeneity and plasticity of CAFs, manipulating the activated phenotype of fibroblasts is a promising strategy for cancer therapy. However, the underlying mechanisms of fibroblast activation and phenotype switching that drive metastasis remain elusive. The clinical implications of fibroblast activation protein (FAP)-positive CAFs (FAP+CAFs) were evaluated based on tumor specimens from NSCLC patients and bioinformatic analysis of online databases. CAF-specific circular RNAs (circRNAs) were screened by circRNA microarrays of primary human CAFs and matched normal fibroblasts (NFs). Survival analyses were performed to assess the prognostic value of circNOX4 in NSCLC clinical samples. The biological effects of circNOX4 were investigated by gain- and loss-of-function experiments in vitro and in vivo. Fluorescence in situ hybridization, luciferase reporter assays, RNA immunoprecipitation, and miRNA rescue experiments were conducted to elucidate the underlying mechanisms of fibroblast activation. Cytokine antibody array, transwell coculture system, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the downstream effectors that promote cancer metastasis. FAP+CAFs were significantly enriched in metastatic cancer samples, and their higher abundance was correlated with the worse overall survival in NSCLC patients. A novel CAF-specific circRNA, circNOX4 (hsa_circ_0023988), evoked the phenotypic transition from NFs into CAFs and promoted the migration and invasion of NSCLC in vitro and in vivo. Clinically, circNOX4 correlated with the poor prognosis of advanced NSCLC patients. Mechanistically, circNOX4 upregulated FAP by sponging miR-329-5p, which led to fibroblast activation. Furthermore, the circNOX4/miR-329-5p/FAP axis activated an inflammatory fibroblast niche by preferentially inducing interleukin-6 (IL-6) and eventually promoting NSCLC progression. Disruption of the intercellular circNOX4/IL-6 axis significantly suppressed tumor growth and metastatic colonization in vivo. Our study reveals a role of the circRNA-induced fibroblast niche in tumor metastasis and highlights that targeting the circNOX4/FAP/IL-6 axis is a promising strategy for the intervention of NSCLC metastasis.

中文翻译：

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circNOX4通过FAP/IL-6轴激活炎症成纤维细胞微环境促进NSCLC肿瘤生长和转移

癌症相关成纤维细胞（CAF）精心策划了一个支持性生态位，促进非小细胞肺癌（NSCLC）的癌症转移发展。由于 CAF 的异质性和可塑性，操纵成纤维细胞的激活表型是一种有前途的癌症治疗策略。然而，成纤维细胞激活和表型转换驱动转移的潜在机制仍然难以捉摸。根据 NSCLC 患者的肿瘤标本和在线数据库的生物信息分析，评估了成纤维细胞激活蛋白（FAP）阳性 CAF（FAP+CAF）的临床意义。通过原代人 CAF 和匹配的正常成纤维细胞 (NF) 的 circRNA 微阵列筛选 CAF 特异性环状 RNA (circRNA)。进行生存分析以评估 circNOX4 在 NSCLC 临床样本中的预后价值。通过体外和体内功能获得和丧失实验研究了 circNOX4 的生物学效应。进行荧光原位杂交、荧光素酶报告基因测定、RNA 免疫沉淀和 miRNA 拯救实验来阐明成纤维细胞激活的潜在机制。采用细胞因子抗体阵列、Transwell共培养系统和酶联免疫吸附测定（ELISA）来研究促进癌症转移的下游效应器。FAP+CAF 在转移性癌症样本中显着富集，其较高的丰度与 NSCLC 患者较差的总生存率相关。一种新型的 CAF 特异性 circRNA circNOX4 (hsa_circ_0023988) 引发了从 NF 到 CAF 的表型转变，并促进 NSCLC 的体外和体内迁移和侵袭。临床上，circNOX4与晚期NSCLC患者的不良预后相关。从机制上讲，circNOX4 通过海绵 miR-329-5p 上调 FAP，从而导致成纤维细胞激活。此外，circNOX4/miR-329-5p/FAP 轴通过优先诱导白细胞介素 6 (IL-6) 激活炎症成纤维细胞生态位，并最终促进 NSCLC 进展。细胞间 circNOX4/IL-6 轴的破坏可显着抑制体内肿瘤生长和转移定植。我们的研究揭示了 circRNA 诱导的成纤维细胞微环境在肿瘤转移中的作用，并强调靶向 circNOX4/FAP/IL-6 轴是干预 NSCLC 转移的有前途的策略。