Williams Amber L, Fitzgerald Jessica E, Ivich Fernando, Sontag Eduardo D, Niedre Mark
Department of Bioengineering, Northeastern University, Boston, MA, United States.
Department of Electrical and Computer Engineering, Northeastern University, Boston, MA, United States.
Front Oncol. 2020 Nov 6;10:601085. doi: 10.3389/fonc.2020.601085. eCollection 2020.
Circulating tumor cells (CTCs) are widely studied using liquid biopsy methods that analyze fractionally-small peripheral blood (PB) samples. However, little is known about natural fluctuations in CTC numbers that may occur over short timescales , and how these may affect detection and enumeration of rare CTCs from small blood samples.
We recently developed an optical instrument called "diffuse flow cytometry" (DiFC) that uniquely allows continuous, non-invasive counting of rare, green fluorescent protein expressing CTCs in large blood vessels in mice. Here, we used DiFC to study short-term changes in CTC numbers in multiple myeloma and Lewis lung carcinoma xenograft models. We analyzed CTC detections in over 100 h of DiFC data, and considered intervals corresponding to approximately 1%, 5%, 10%, and 20% of the PB volume. In addition, we analyzed changes in CTC numbers over 24 h (diurnal) periods.
For rare CTCs (fewer than 1 CTC per ml of blood), the use of short DiFC intervals (corresponding to small PB samples) frequently resulted in no detections. For more abundant CTCs, CTC numbers frequently varied by an order of magnitude or more over the time-scales considered. This variance in CTC detections far exceeded that expected by Poisson statistics or by instrument variability. Rather, the data were consistent with significant changes in mean numbers of CTCs on the timescales of minutes and hours.
The observed temporal changes can be explained by known properties of CTCs, namely, the continuous shedding of CTCs from tumors and the short half-life of CTCs in blood. It follows that the number of cells in a blood sample are strongly impacted by the timing of the draw. The issue is likely to be compounded for multicellular CTC clusters or specific CTC subtypes, which are even more rare than single CTCs. However, we show that enumeration can in principle be improved by averaging multiple samples, analysis of larger volumes, or development of methods for enumeration of CTCs directly .
循环肿瘤细胞（CTC）通过分析微量外周血（PB）样本的液体活检方法得到广泛研究。然而，对于短时间内CTC数量的自然波动情况以及这些波动如何影响从小血样中检测和计数罕见CTC，人们了解甚少。
我们最近开发了一种名为“漫射流式细胞术”（DiFC）的光学仪器，它能够独特地对小鼠大血管中表达绿色荧光蛋白的罕见CTC进行连续、非侵入性计数。在此，我们使用DiFC研究多发性骨髓瘤和Lewis肺癌异种移植模型中CTC数量的短期变化。我们分析了超过100小时的DiFC数据中的CTC检测情况，并考虑了对应于PB体积约1%、5%、10%和20%的时间段。此外，我们分析了24小时（昼夜）周期内CTC数量的变化。
对于罕见的CTC（每毫升血液中少于1个CTC），使用短的DiFC时间段（对应小PB样本）常常导致无法检测到。对于数量较多的CTC，在考虑的时间尺度上，CTC数量常常变化一个数量级或更多。CTC检测中的这种变化远远超过泊松统计或仪器变异性所预期的。相反，数据与几分钟和几小时时间尺度上CTC平均数量的显著变化一致。
观察到的时间变化可以用CTC的已知特性来解释，即肿瘤中CTC的持续脱落以及CTC在血液中的短半衰期。由此可知，血样中的细胞数量受到采血时间的强烈影响。对于多细胞CTC簇或特定CTC亚型，这个问题可能会更加复杂，因为它们比单个CTC更为罕见。然而，我们表明，原则上可以通过对多个样本进行平均、分析更大体积的样本或开发直接计数CTC的方法来改进计数。
