Circulating tumor cells (CTCs) hold immense promise for unraveling tumor heterogeneity and understanding treatment resistance. However, conventional methods, especially in cancers like non-small cell lung cancer (NSCLC), often yield low CTC numbers, hindering comprehensive analyses. This study addresses this limitation by employing diagnostic leukapheresis (DLA) to cancer patients, enabling the screening of larger blood volumes. To leverage DLA’s full potential, this study introduces a novel approach for CTC enrichment from DLAs. DLA was applied to six advanced stage NSCLC patients. For an unbiased CTC enrichment, a two-step approach based on negative depletion of hematopoietic cells was used. Single-cell (sc) whole-transcriptome sequencing was performed, and CTCs were identified based on gene signatures and inferred copy number variations. Remarkably, this innovative approach led to the identification of unprecedented 3,363 CTC transcriptomes. The extensive heterogeneity among CTCs was unveiled, highlighting distinct phenotypes related to the epithelial-mesenchymal transition (EMT) axis, stemness, immune responsiveness, and metabolism. Comparison with sc transcriptomes from primary NSCLC cells revealed that CTCs encapsulate the heterogeneity of their primary counterparts while maintaining unique CTC-specific phenotypes. In conclusion, this study pioneers a transformative method for enriching CTCs from DLA, resulting in a substantial increase in CTC numbers. This allowed the creation of the first-ever single-cell whole transcriptome in-depth characterization of the heterogeneity of over 3,300 NSCLC-CTCs. The findings not only confirm the diagnostic value of CTCs in monitoring tumor heterogeneity but also propose a CTC-specific signature that can be exploited for targeted CTC-directed therapies in the future. This comprehensive approach signifies a major leap forward, positioning CTCs as a key player in advancing our understanding of cancer dynamics and paving the way for tailored therapeutic interventions.

中文翻译：

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诊断性白细胞去除术揭示 NSCLC 循环肿瘤细胞的不同表型

循环肿瘤细胞 (CTC) 在揭示肿瘤异质性和了解治疗耐药性方面具有巨大前景。然而，传统方法，尤其是非小细胞肺癌 (NSCLC) 等癌症的方法，通常会产生较低的 CTC 数量，从而阻碍了综合分析。这项研究通过对癌症患者采用诊断性白细胞分离术 (DLA) 来解决这一局限性，从而能够筛查更大的血容量。为了充分发挥 DLA 的潜力，本研究引入了一种从 DLA 中富集 CTC 的新方法。 DLA 被应用于六名晚期 NSCLC 患者。为了实现无偏差的 CTC 富集，使用了基于造血细胞负耗竭的两步方法。进行了单细胞 (sc) 全转录组测序，并根据基因特征和推断的拷贝数变异来鉴定 CTC。值得注意的是，这种创新方法鉴定出了前所未有的 3,363 个 CTC 转录组。 CTC 之间广泛的异质性被揭示，突出了与上皮间质转化 (EMT) 轴、干性、免疫反应性和代谢相关的不同表型。与原代 NSCLC 细胞的 sc 转录组比较表明，CTC 封装了其原代对应物的异质性，同时保持了独特的 CTC 特异性表型。总之，这项研究开创了一种从 DLA 中富集 CTC 的变革性方法，从而导致 CTC 数量大幅增加。这使得能够创建第一个单细胞全转录组，深入表征 3,300 多个 NSCLC-CTC 的异质性。研究结果不仅证实了 CTC 在监测肿瘤异质性方面的诊断价值，还提出了 CTC 特异性特征，可用于未来针对 CTC 的靶向治疗。这种综合方法标志着一个重大飞跃，将 CTC 定位为增进我们对癌症动态的理解并为定制治疗干预措施铺平道路的关键参与者。