序列染色法應用於循環腫瘤細胞之分選

Abstract

根據 Globocan 2012 年統計,全球有將近 820 萬人死於癌症,而目 前認為癌症轉移與許多癌症患者的死亡有關。現今,從病人血液中取 出的循環腫瘤細胞已獲得許多關注,且被視為很有潛力能成為檢測癌 症的指標,如無惡化存活期(progression-free survival)和整體存活期(overall survival)皆有許多針對乳癌、前列腺癌與結腸直腸癌的臨床 試驗證實,因此研發了許多循環腫瘤細胞的純化與分離技術。然而, 由於循環腫瘤細胞之間的變異性極高,只有數量不足以在臨床應用上 提供足夠的資訊,因此針對循環腫瘤細胞做進一步的分析日趨重要。 本研究建構了一套整合現有細胞抓取系統的半自動化序列染色系 統,該系統重新設計整個染色過程,除了可獲得更正確的訊號,亦 可獲得循環腫瘤細胞更多的顯型,解決了多數實驗室的螢光顯微鏡 只有四個波段可觀察,且其中 PE 波段的訊號極有可能會有漏光現 象進而影響 FITC 的訊號之問題。此外,在辨別循環腫瘤細胞時需 要使用三個波段,導致只剩一個波段可用於進一步的分析。利用這 套系統,可以在四波段的螢光顯微鏡下獲得五個訊息,當中包含三 個辨別訊號以及兩個亞型訊號。實驗分析三種代表性細胞株 MCF-7、 AU565、MDA-MB-231 的特性後,相同總數而不同的混合比例去模擬 ER/HER2 亞型的情形。結果顯示該系統的回收率與染色效率與先前 的系統並沒有顯著差異,且 HER2 的表現相當穩定,然而 ER 會有 ±12.0% 的誤差。儘管如此,不同亞型的比例依然都可成功地預測與偵 測。藉由該多功能平台,我們提供了更多循環腫瘤細胞之亞型顯型訊 息,以利相關的後端研究。

According to Globocan, there were 8.2 million cancer deaths in 2012 worldwide, and metastasis was considered to be related to the death of many cancer patients. Nowadays circulating tumor cells (CTCs) obtained from blood samples has been considered as a promising indicator for prognosis such as progression-free survival (PFS) and overall survival (OS) of patients, and has been used and validated in many clinical trials in metastatic breast, prostate and colorectal cancers. Therefore, many technologies have been developed for CTCs enrichment and isolation. However, several studies show that CTC enumeration alone is not enough for clinical utility due to their high heterogeneity. Therefore, further analysis of enumerated CTCs has become increasingly important. This work presents a semi-automated sequential staining system combined with a single cell retrieval system as a competent multi-functional platform. It redesigned the staining procedure in order to obtain clearer signals and get more information about the phenotype of CTCs. Since in most cases, laboratories have only four-channel-fluorescence microscopes, where PE signal is likely to interfere with FITC signal due to its light leakage problem. Moreover, three channels may be used to identify CTCs, and only one channel remains for further information. By using this system, we may obtain five fluorescent signals in a four-channel-fluorescence microscope for both CTC detection and its subtype identification. Experiments characterized MCF-7, AU565, and MDA-MB-231 and used total about 50 cells to simulate ER/HER2 subtypes with different ratio of cell lines. Results show that recovery rate and staining efficiency in sequential staining system has no significant differences to the disk system. Also, it indicates that the performances were good for HER2 expression, while about SD=12.0% for ER expression. The overall proportion of mixed subtypes were successfully predicted and detected. With this multi-functional platform, we were able to provide more information about the subtype expression and may offer further analyses on CTCs.