Ruan Haoyu, Zhou Yihang, Shen Jie, Zhai Yue, Xu Ying, Pi Linyu, Huang Ruofan, Chen Kun, Li Xiangyu, Ma Weizhe, Wu Zhiyuan, Deng Xuan, Wang Xu, Zhang Chao, Guan Ming
Department of Clinical Laboratory, Huashan Hospital, Fudan University, Shanghai, China.
Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Clin Transl Med. 2020 Dec;10(8):e246. doi: 10.1002/ctm2.246.
Brain metastases explain the majority of mortality associated with lung cancer, which is the leading cause of cancer death. Cytology analysis of the cerebrospinal fluid (CSF) remains the diagnostic gold standard, however, the circulating tumor cells (CTCs) in CSF (CSF-CTCs) are not well defined at the molecular and transcriptome levels.
We established an effective CSF-CTCs collection procedure and isolated individual CSF cells from five lung adenocarcinoma leptomeningeal metastases (LUAD-LM) patients and three controls. Three thousand seven hundred ninety-two single-cell transcriptomes were sequenced, and single-cell RNA sequencing (scRNA-seq) gene expression analysis was used to perform a comprehensive characterization of CSF cells.
Through clustering and expression analysis, we defined CSF-CTCs at the transcriptome level based on epithelial markers, proliferation markers, and genes with lung origin. The metastatic-CTC signature genes are enriched for metabolic pathway and cell adhesion molecule categories, which are crucial for the survival and metastases of tumor cells. We discovered substantial heterogeneity in patient CSF-CTCs. We quantified the degree of heterogeneity and found significantly greater among-patient heterogeneity compared to among-cell heterogeneity within a patient. This observation could be explained by spatial heterogeneity of metastatic sites, cell-cycle gene, and cancer-testis antigen (CTA) expression profiles as well as the proportion of CTCs displaying mesenchymal and cancer stem cell properties. In addition, our CSF-CTCs transcriptome profiling allowed us to determine the biomarkers during the progression of an LM patient with cancer of unknown primary site (CUP).
Our results will provide candidate genes for an RNA-based digital detection of CSF-CTCs from LUAD-LM and CUP-LM cases, and shed light on the therapy and mechanism of LUAD-LM.
脑转移是肺癌相关死亡的主要原因，而肺癌是癌症死亡的首要病因。脑脊液（CSF）细胞学分析仍是诊断的金标准，然而，CSF中的循环肿瘤细胞（CSF-CTCs）在分子和转录组水平上尚未得到充分定义。
我们建立了一种有效的CSF-CTCs收集程序，并从5例肺腺癌软脑膜转移（LUAD-LM）患者和3例对照中分离出单个CSF细胞。对3792个单细胞转录组进行了测序，并使用单细胞RNA测序（scRNA-seq）基因表达分析对CSF细胞进行了全面表征。
通过聚类和表达分析，我们基于上皮标志物、增殖标志物和具有肺源性的基因在转录组水平上定义了CSF-CTCs。转移性CTC特征基因在代谢途径和细胞粘附分子类别中富集，这对肿瘤细胞的存活和转移至关重要。我们发现患者CSF-CTCs存在显著的异质性。我们量化了异质性程度，发现患者间的异质性显著大于患者内细胞间的异质性。这一观察结果可以通过转移部位的空间异质性、细胞周期基因和癌胚抗原（CTA）表达谱以及显示间充质和癌症干细胞特性的CTCs比例来解释。此外，我们的CSF-CTCs转录组分析使我们能够确定一名原发部位不明癌症（CUP）的LM患者病程中的生物标志物。
我们的结果将为基于RNA的LUAD-LM和CUP-LM病例CSF-CTCs数字检测提供候选基因，并为LUAD-LM的治疗和机制提供线索。
