Sánchez-Quesada Cristina, Toledo Estefanía, Jiménez-Moleón José Juan, Gaforio José Juan
Department of Health Sciences, Faculty of Health Sciences, University of Jaén, 23071 Jaén, Spain.
University Institute of Research on Olive and Olive Oils (INUO), University of Jaen, Campus las Lagunillas s/n, 23071 Jaén, Spain.
Biomolecules. 2025 May 15;15(5):723. doi: 10.3390/biom15050723.
Circulating tumor cells (CTCs) hold recognized prognostic value in various cancers, including breast cancer, where their presence correlates with survival outcomes. However, the typical 24 h window for blood processing and CTC isolation poses a logistical challenge, particularly for multicenter studies. This study aimed to evaluate cryopreservation at different stages of CTC isolation and immunocytological detection to extend the blood sample processing period. Using spiked peripheral blood samples with MDA-MB-231, SKBR3, and MCF7 breast cancer cell lines, four distinct cryopreservation points were assessed: following Ficoll gradient separation, immunomagnetic separation, cytocentrifugation, and cytokeratin labeling. Our findings demonstrated that cryopreservation of the mononuclear and granulocytic cell fraction after double-density Ficoll gradient separation was the only viable method for subsequent CTC detection. This approach allowed for consistent recovery of CK+ CTCs, with an average recovery rate of over 81% after one year of cryopreservation. In contrast, cryopreservation at later stages resulted in undetectable CTCs or only cellular debris. In conclusion, cryopreservation following density gradient centrifugation is a feasible strategy for delaying CTC isolation and immunocytological analysis in breast cancer research, facilitating its application in multicenter clinical trials.
循环肿瘤细胞（CTCs）在包括乳腺癌在内的多种癌症中具有公认的预后价值，其存在与生存结果相关。然而，血液处理和CTCs分离的典型24小时时间窗口带来了后勤方面的挑战，特别是对于多中心研究而言。本研究旨在评估CTCs分离和免疫细胞检测不同阶段的冷冻保存，以延长血样处理时间。使用添加了MDA-MB-231、SKBR3和MCF7乳腺癌细胞系的外周血样本，评估了四个不同的冷冻保存点：在Ficoll梯度分离、免疫磁珠分离、细胞离心涂片和细胞角蛋白标记之后。我们的研究结果表明，双密度Ficoll梯度分离后对单核细胞和粒细胞部分进行冷冻保存是后续CTCs检测的唯一可行方法。这种方法能够持续回收CK+ CTCs，冷冻保存一年后的平均回收率超过81%。相比之下，后期阶段的冷冻保存导致无法检测到CTCs或仅留下细胞碎片。总之，密度梯度离心后的冷冻保存是一种可行的策略，可用于在乳腺癌研究中延迟CTCs分离和免疫细胞分析，便于其在多中心临床试验中的应用。
