循环肿瘤细胞（CTC）是潜在的癌症生物标志物，但目前的单细胞分辨率CTC分析方法有限。在这里，我们描述了HNSCC中CTCs的高维单细胞质谱蛋白质分析。通过使用41种抗体进行了髓质微流探针（Parsortix）富集的14例未接受治疗的HNSCC患者的CTC质谱分析。除了多种信号通路激活状态的多个磷酸化蛋白质的翻译后修饰状况，还对免疫细胞系谱、上皮间质转化（EMT）、干性、增殖和免疫检查点的表达进行了评估。比较了与患者相匹配的肿瘤基因表达和CTC的EMT谱。使用标准的CTC RNA测序作为基准比较了质谱数据。13/14例患者的CTC计数为2-24个CTCs/ml血液。无监督的聚类将CTCs分为上皮细胞、早期EMT和晚期EMT组，这些组在信号通路激活状态上有所不同。患者特异性的CTC聚类模式分为免疫检查点低和高组。患者的肿瘤和CTC的EMT谱有所不同。质谱蛋白质分析优于批量RNA测序，能够检测CTCs并表征细胞表型。我们证明质谱蛋白质分析可以实现对CTCs进行高维蛋白质表征，鉴定常见CTC亚群，有潜力用于新型生物标志物的开发和免疫检查点抑制剂治疗分层。© 2023. 作者（们）。

Circulating tumour cells (CTCs) are a potential cancer biomarker, but current methods of CTC analysis at single-cell resolution are limited. Here, we describe high-dimensional single-cell mass cytometry proteomic analysis of CTCs in HNSCC.Parsortix microfluidic-enriched CTCs from 14 treatment-naïve HNSCC patients were analysed by mass cytometry analysis using 41 antibodies. Immune cell lineage, epithelial-mesenchymal transition (EMT), stemness, proliferation and immune checkpoint expression was assessed alongside phosphorylation status of multiple signalling proteins. Patient-matched tumour gene expression and CTC EMT profiles were compared. Standard bulk CTC RNAseq was performed as a baseline comparator to assess mass cytometry data.CTCs were detected in 13/14 patients with CTC counts of 2-24 CTCs/ml blood. Unsupervised clustering separated CTCs into epithelial, early EMT and advanced EMT groups that differed in signalling pathway activation state. Patient-specific CTC cluster patterns separated into immune checkpoint low and high groups. Patient tumour and CTC EMT profiles differed. Mass cytometry outperformed bulk RNAseq to detect CTCs and characterise cell phenotype.We demonstrate mass cytometry allows high-plex proteomic characterisation of CTCs at single-cell resolution and identify common CTC sub-groups with potential for novel biomarker development and immune checkpoint inhibitor treatment stratification.© 2023. The Author(s).