检测和分离临床局限性前列腺癌患者骨髓中的播散肿瘤细胞。
Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer.
机构信息
Division of Hematology and Oncology, University of Michigan School of Medicine, Ann Arbor, Michigan.
Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan.
出版信息
Prostate. 2019 Oct;79(14):1715-1727. doi: 10.1002/pros.23896. Epub 2019 Aug 26.
BACKGROUND
Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs.
METHODS
We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers.
RESULTS
At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%).
CONCLUSION
These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.
背景
已有报道称，局限性前列腺癌（PCa）患者的骨髓（BM）中存在播散性肿瘤细胞（DTCs）。然而，这些细胞的存在仍然存在争议，并且很少有可行的 DTC 分离方法。因此，我们试图开发新的方法来鉴定局限性 PCa 患者的 DTCs，如果检测到，则对其进行分析。
方法
我们使用荧光激活细胞分选（FACS）分离假定的 DTC 群体，该群体对 CD45、CD235a、碱性磷酸酶和 CD34 呈阴性，而对 EPCAM 呈强阳性。我们通过全基因组扩增后的批量细胞 RNA 测序（RNA-Seq）和全外显子组测序来检查肿瘤细胞含量。我们还用 α-EPCAM 免疫磁珠富集 BM DTCs，并进行用于 PCa 标志物的定量逆转录聚合酶链反应（qRT-PCR）。
结果
在每百万 BM 细胞中存在 4 个细胞的阈值下，10 例局限性 PCa 患者（17%）、8 例转移性 PCa 患者（疾病控制程度不同）和 1 例无已知癌症的患者存在假定的 DTC 群体，且与患者的血浆 PSA 值呈正相关。对上述阈值（3 例）和以下阈值（5 例）的样本中收集的假定 DTC 群体进行 RNA-Seq 分析显示，4 例局限性 PCa 患者中 4 例的 PCa 标志物基因表达增加，但 1 例正常供者中没有假定的 DTC 群体。全外显子组测序还显示，在 3 例局限性 PCa 患者中，2 例存在与 PCa 基因特征相关的单核苷酸多态性和结构变异。为了检查可能存在的污染细胞类型，我们使用髓样集落形成测定法、细胞涂片的差异计数以及使用 CIBERSORT 算法对 RNA-Seq 数据进行分析，结果最强烈地表明 B 细胞谱系是一种污染。最后，我们使用 EPCAM 富集和用于 PCa 标志物的 qRT-PCR 来估计 DTC 的流行率，在 44 个样本中的 21 个样本（47%）中发现了 DTC 的证据。
结论
这些数据支持局限性 PCa 患者的 BM 中存在 DTCs，并描述了一种新的 FACS 方法，用于分离和分析可行的 DTCs。
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