Bang-Christensen Sara R, Katerov Viatcheslav, Jørgensen Amalie M, Gustavsson Tobias, Choudhary Swati, Theander Thor G, Salanti Ali, Allawi Hatim T, Agerbæk Mette Ø
Centre for Medical Parasitology, Department for Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen and Department of Infectious Disease, Copenhagen University Hospital, 2200 Copenhagen, Denmark.
VarCT Diagnostics, Ole Maaløes Vej 3, 2200 Copenhagen, Denmark.
Cancers (Basel). 2021 Nov 23;13(23):5881. doi: 10.3390/cancers13235881.
Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT-qPCR using proprietary Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody-based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low-level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near-perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike-in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT-qPCR assay after rVAR2 capture ( < 0.01 for both primer targets, Mann-Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.
对血样中的循环肿瘤细胞（CTC）进行分析为癌症早期检测提供了一种非侵入性方法。然而，CTC的稀有性使得建立具有所需灵敏度和特异性的检测方法具有挑战性。我们将利用癌细胞结合重组疟疾VAR2CSA蛋白（rVAR2）的高灵敏度CTC捕获检测方法与结肠相关mRNA转录本（USH1C和CKMT1A）的检测相结合。癌细胞转录本通过使用专有的靶标富集长探针定量扩增信号（TELQAS）技术的RT-qPCR进行检测。我们使用掺入血液中的结直肠癌（CRC）细胞系对工作流程的每个步骤进行验证，并将其与基于抗体的细胞检测进行比较。USH1C和CKMT1A在健康结肠组织和CRC细胞系中表达，而在健康白细胞（WBC）中只能检测到低水平表达。qPCR反应对所有引物靶标的扩增效率接近完美，WBC cDNA的干扰最小。在rVAR2捕获后，使用RT-qPCR检测可以检测到3 mL血液中掺入的10个癌细胞，并与对照血液进行统计学区分（两个引物靶标的P值均<0.01，曼-惠特尼检验）。我们的结果为高灵敏度检测磁性富集的癌细胞提供了一个经过验证的工作流程。
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