Abstract
Circulating tumor cells (CTCs) are important biomarkers for the diagnosis, prognosis, and treatment of cancer. However, because of their extreme rarity, a more precise technique for isolating CTCs is required to gain deeper insight into the characteristics of cancer. This study compares the performance of a lateral magnetophoretic microseparator (“CTC-μChip”), as a representative microfluidic device, and AdnaTest ProstateCancer (Qiagen), as a commercially available specialized method, for isolating CTCs from the blood of patients with prostate cancer. The enumeration and genetic analysis results of CTCs isolated via the two methods were compared under identical conditions. In the CTC enumeration experiment, the number of CTCs isolated by the CTC-μChip averaged 17.67 CTCs/mL, compared to 1.56 CTCs/mL by the AdnaTest. The number of contaminating white blood cells (WBCs) and the CTC purity with the CTC-μChip averaged 772.22 WBCs/mL and 3.91%, respectively, whereas those with the AdnaTest averaged 67.34 WBCs/mL and 1.98%, respectively. Through genetic analysis, using a cancer-specific gene panel (AR (androgen receptor), AR-V7 (A\\androgen receptor variant-7), PSMA (prostate specific membrane antigen), KRT19 (cytokeratin-19), CD45 (PTPRC, Protein tyrosine phosphatase, receptor type, C)) with reverse transcription droplet digital PCR, three genes (AR, AR-V7, and PSMA) were more highly expressed in cells isolated by the CTC-μChip, while KRT19 and CD45 were similarly detected using both methods. Consequently, this study showed that the CTC-μChip can be used to isolate CTCs more reliably than AdnaTest ProstateCancer, as a specialized method for gene analysis of prostate CTCs, as well as more sensitively obtain cancer-associated gene expressions.
Highlights
Circulating tumor cells (CTCs) are an emerging biomarker in cancer biology and clinical research as they can reveal the landscape of cancer genes [1,2,3]
Along with CTC enumeration, the CTCs and white blood cells (WBCs) isolated by the two methods were lysed to analyze the gene expression levels of the selected five genes that reflect reactivity to androgen hormones (AR and androgen receptor splice variant 7 (AR-V7)), prostate cancer progression (PSMA), epithelial phenotype (KRT19), and leukocyte-specific marker (CD45)
CTCs and WBCs isolated by the CTC-μChip were clearly dyed and classified with anti-pan-cytokeratin Alexa 488 and anti-CD45 Alexa 647 antibodies, respectively, whereas cells isolated by the AdnaTest were surrounded by 4.8 μm diameter magnetic beads, making them difficult to distinguish (Figure 3)
Summary
Circulating tumor cells (CTCs) are an emerging biomarker in cancer biology and clinical research as they can reveal the landscape of cancer genes [1,2,3]. CTCs are acquired through blood-based liquid biopsy, enabling real-time monitoring of cancer owing to minimal invasiveness [4]. Macroscale techniques, such as magnetic-activated cell sorting (MACS) [5], CellSearch [6], CellCollector [7], ISET [8], and AdnaTest [9], are conventionally used to isolate CTCs owing to their simplicity, availability, and well-established procedures [10,11]. This study compares the performance of a lateral magnetophoretic microseparator (“CTCμChip”) [32,33,34], as a representative microfluidic device, and the AdnaTest ProstateCancer, as a commercially available specialized method, for isolating CTCs from the blood of patients with prostate cancer. Cancer-associated genes in the CTCs isolated via the two methods were measured using reverse transcription droplet digital PCR (RT-ddPCR), thereby directly comparing their accuracy for CTC-based genetic analysis
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