以癌症相关成纤维细胞 (CAF) 为主要细胞类型，脊索瘤组织中丰富的粘液样基质成分可能有助于其发生和进展。单细胞 RNA 测序 (scRNA-seq)、空间转录组学、批量 RNA-seq 和使用多重定量免疫荧光 (QIF) 来剖析脊索瘤中 CAF 的异质性、空间分布和临床意义。我们使用 scRNA 对来自三个原发性和三个复发性肿瘤样本以及三个髓核样本作为对照的 72097 个单细胞进行了测序。序列。我们在复发性肿瘤中发现了一个独特的 CAF 簇，它们高度表达缺氧基因，并且在内质网应激 (ERS) 中功能丰富。伪时间轨迹和细胞通讯分析表明，该ERS-CAF亚群起源于正常成纤维细胞，并与肿瘤和免疫细胞广泛相互作用。通过分析 126 名患者的大量 RNA-seq 数据，我们发现 ERS-CAF 特征评分与脊索瘤的侵袭和不良预后相关。通过将 scRNA-seq 的结果与空间转录组学相结合，我们证明了脊索瘤组织中 ERS-CAF 的存在，并揭示了这种 CAF 亚型与其周围的肿瘤细胞最接近。在随后涉及 105 名额外患者的 QIF 验证中，我们证实 ERS-CAF 在脊索瘤微环境中丰富且靠近肿瘤细胞。此外，ERS-CAF 密度及其与肿瘤细胞的距离均与肿瘤恶性表型和不良患者结局相关。这些发现描绘了脊索瘤的 CAF 景观，并可能为新型治疗方法的开发提供见解。© 作者2023 年。由牛津大学出版社代表神经肿瘤学会出版。版权所有。如需权限，请发送电子邮件至：journals.permissions@oup.com。

With cancer-associated fibroblasts (CAFs) as the main cell type, the rich myxoid stromal components in chordoma tissues may likely contribute to its development and progression.Single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, bulk RNA-seq and multiplexed quantitative immunofluorescence (QIF) were used to dissect the heterogeneity, spatial distribution and clinical implication of CAFs in chordoma.We here sequenced 72097 single cells from three primary and three recurrent tumor samples, as well as three nucleus pulposus samples as controls using scRNA-seq. We identified a unique cluster of CAF in recurrent tumors that highly expressed hypoxic genes and was functionally enriched in endoplasmic reticulum stress (ERS). Pseudotime trajectory and cell communication analyses showed that this ERS-CAF subpopulation was originated from normal fibroblasts and widely interacted with tumoral and immune cells. Analyzing the bulk RNA-seq data from 126 patients, we found that the ERS-CAF signature score was associated with the invasion and poor prognosis of chordoma. By integrating the results of scRNA-seq with spatial transcriptomics, we demonstrated the existence of ERS-CAF in chordoma tissues and revealed that this CAF subtype displayed the most proximity to its surrounding tumor cells. In subsequent QIF validation involving 105 additional patients, we confirmed that ERS-CAF was abundant in chordoma microenvironment and located close to tumor cells. Furthermore, both ERS-CAF density and its distance to tumor cells were correlated with tumor malignant phenotype and adverse patient outcomes.These findings depict the CAF landscape for chordoma and may provide insights into the development of novel treatment approaches.© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.