Dept. of Surgery, Washington University School of Medicine, St. Louis, MO, United States of America.
Dept. of Medicine, Washington University School of Medicine, St. Louis, MO, United States of America.
PLoS One. 2021 May 14;16(5):e0246139. doi: 10.1371/journal.pone.0246139. eCollection 2021.
Disseminated tumors cells (DTCs) present in the bone marrow (BM) are believed to be the progenitors of distant metastatic spread, a major cause of mortality in breast cancer patients. To better understand the behavior and therapeutic vulnerabilities of these rare cell populations, unbiased methods for selective cell enrichment are required. In this study, we have evaluated a microfluidic-based filtration system (ParsortixR, Angle PLC), previously demonstrated for use in circulating tumor cell (CTC) capture, to capture BM DTCs. Performance using BM samples was also compared directly to enrichment of CTCs in the peripheral blood (PB) from both metastatic and non-metastatic breast cancer patients. Although the non-specific capture of BM immune cells was significant, the device could routinely achieve significant cytoreduction of BM and PB WBCs and at least 1,000-fold enrichment of DTCs, based on labeled tumor cell spike-in experiments. Detection of previously characterized DTC-associated gene expression biomarkers was greatly enhanced by the enrichment method, as demonstrated by droplet digital PCR assay. Cells eluted from the device were viable and suitable for single cell RNA sequencing experiments. DTCs in enriched BM samples comprised up to 5% of the total cell population, allowing for effective single cell and population-based transcriptional profiling of these rare cells. Use of the Parsortix instrument will be an effective approach to enrich for rare BM DTCs in order to better understand their diverse molecular phenotypes and develop approaches to eradicate these cells to prevent distant disease development in breast cancer patients.
骨髓（BM）中存在的播散性肿瘤细胞（DTCs）被认为是远处转移扩散的起源，这是乳腺癌患者死亡的主要原因。为了更好地了解这些稀有细胞群体的行为和治疗弱点，需要使用无偏见的方法来选择性地富集细胞。在这项研究中，我们评估了一种基于微流控的过滤系统（ParsortixR，Angle PLC），该系统先前已被证明可用于循环肿瘤细胞（CTC）捕获，以捕获 BM DTCs。还直接比较了使用 BM 样本的性能与转移性和非转移性乳腺癌患者外周血（PB）中 CTC 的富集性能。尽管对 BM 免疫细胞的非特异性捕获很明显，但该设备通常可以实现 BM 和 PB WBC 的显著细胞减少，并且基于标记的肿瘤细胞掺入实验，至少可以富集 1000 倍的 DTC。通过液滴数字 PCR 分析，富集方法大大增强了先前表征的 DTC 相关基因表达生物标志物的检测。从设备洗脱的细胞具有活力，适合单细胞 RNA 测序实验。富集的 BM 样本中的 DTC 占总细胞群的 5%，允许对这些稀有细胞进行有效的单细胞和基于群体的转录谱分析。使用 Parsortix 仪器将是富集罕见 BM DTC 的有效方法，以更好地了解它们不同的分子表型，并开发消除这些细胞的方法，以防止乳腺癌患者发生远处疾病发展。
