Cieslik Stefan A, Zafra Andrés G, Driemel Christiane, Sudarsanam Monica, Cieslik Jan-Philipp, Flügen Georg, Dizdar Levent, Krieg Andreas, Vaghiri Sascha, Ashmawy Hany, Fung Stephen, Wilms Miriam, Terstappen Leon W M M, Nanou Afroditi, Neubauer Hans, Rahbari Nuh N, Knoefel Wolfram T, Stoecklein Nikolas H, Neves Rui P L
Department of General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty of Heinrich-Heine University Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Germany.
Department of Obstetrics and Gynecology, University Hospital and Medical Faculty of the Heinrich-Heine University Düsseldorf, Moorenstr. 5, 40225, Düsseldorf, Germany.
J Exp Clin Cancer Res. 2025 Jan 8;44(1):9. doi: 10.1186/s13046-024-03259-6.
Circulating tumour cells (CTCs) and tumour-derived extracellular vesicles (tdEVs) have great potential for monitoring therapy response and early detection of tumour relapse, facilitating personalized adjuvant therapeutic strategies. However, their low abundance in peripheral blood limits their informative value. In this study, we explored the presence of CTCs and tdEVs collected intraoperatively from a tumour-draining vein (DV) and via a central venous catheter (CVC) prior to tumour resection.
CellSearch analyses of 395 blood samples from 306 patients with gastrointestinal tumours and 93 blood samples from healthy donors were used to establish and validate gates for the automated detection of CTCs and tdEVs with ACCEPT software and R scripts. The selected gate settings were applied to 227 samples of 142 patients with colorectal cancer (CRC) from two independent collectives. Phenotypic features were obtained via numeric analysis of their fluorescence signals (e.g. size, shape, and intensity) and were used for calculating diversity using Shannon index (SI) of clusters generated via the k-means algorithm after Uniform Manifold Approximation and Projection (UMAP) pre-processing, and standard deviation (SD).
CTCs and tdEVs were more abundant in the DV samples compared to CVC samples (p < 0.05). tdEVs were detected in higher numbers than CTCs in both compartments. Importantly, tdEVs in CVCs were associated with tumor spread, whereas CTCs in DVs were linked to tumor size. In both compartments, the prognostic value of tdEVs for overall survival (OS) surpassed that of CTCs, as demonstrated by univariate, multivariate, and Kaplan-Meier analyses. CTCs and tdEVs in DVs were phenotypically distinct, being larger, more eccentric, and displaying stronger cytokeratin intensities (p < 0.05) compared to those in CVC samples. Furthermore, increased diversity in CTC and tdEV phenotypes was significantly associated with shorter survival, validating the prognostic relevance of the SD-diversity metric.
Our study demonstrates that DV sampling significantly enhances the detection of prognostically relevant CTCs and tdEVs in CRC patients, underscoring the superior prognostic significance of tdEVs compared to CTCs. Importantly, the combined phenotypic diversity of both markers emerges as a more powerful biomarker than their enumeration alone. These findings suggest that comprehensive, automated analysis of CTCs and tdEVs in DVs may open new avenues for tailoring individualized therapies in CRC patients.
循环肿瘤细胞（CTCs）和肿瘤来源的细胞外囊泡（tdEVs）在监测治疗反应和早期检测肿瘤复发方面具有巨大潜力，有助于制定个性化辅助治疗策略。然而，它们在外周血中的低丰度限制了其信息价值。在本研究中，我们探讨了在肿瘤切除术前从肿瘤引流静脉（DV）和通过中心静脉导管（CVC）术中采集的CTCs和tdEVs的存在情况。
使用CellSearch分析来自306例胃肠道肿瘤患者的395份血样和来自健康供体的93份血样，通过ACCEPT软件和R脚本建立并验证用于自动检测CTCs和tdEVs的门控。将选定的门控设置应用于来自两个独立队列的142例结直肠癌（CRC）患者的227份样本。通过对其荧光信号进行数值分析（如大小、形状和强度）获得表型特征，并在均匀流形近似和投影（UMAP）预处理后，使用通过k均值算法生成的聚类的香农指数（SI）和标准差（SD）来计算多样性。
与CVC样本相比，DV样本中的CTCs和tdEVs更为丰富（p < 0.05）。在两个腔室中，检测到的tdEVs数量均高于CTCs。重要的是，CVC中的tdEVs与肿瘤扩散相关，而DV中的CTCs与肿瘤大小相关。在两个腔室中，单变量、多变量和Kaplan-Meier分析均表明，tdEVs对总生存期（OS）的预后价值超过CTCs。与CVC样本相比，DV中的CTCs和tdEVs在表型上有所不同，更大、更偏心，并且细胞角蛋白强度更强（p < 0.05）。此外，CTCs和tdEVs表型多样性的增加与较短的生存期显著相关，验证了SD多样性指标的预后相关性。
我们的研究表明，DV采样显著提高了CRC患者中具有预后相关性的CTCs和tdEVs的检测率，强调了tdEVs相对于CTCs的优越预后意义。重要的是，两种标志物的联合表型多样性作为一种生物标志物比单独计数更强大。这些发现表明，对DV中的CTCs和tdEVs进行全面、自动化分析可能为CRC患者量身定制个体化治疗开辟新途径。
