微流體技術應用於個人化醫療-從腫瘤細胞的體外藥物測試到循環癌細胞的偵測與抓取

Microfluidic Toolbox for Personalized Medicine – from Ex-vivo Drug Testing of Solid Tumor to Detection and Retrieval of Circulating Tumor Cell

馬維遠(Wei-Yuan Ma)

指導教授 ： 胡文聰

國立臺灣大學/工學院/應用力學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.11287

摘要

循環腫瘤細胞於現階段被認為是一種具有檢測與診斷癌症潛力的指標。許多研究已驗證了循環腫瘤細胞的數量與患者的存活率有明顯的關係，但這種細胞本身極度稀少且異質性高，而現有大部份針對其純化之技術的靈敏度還需要被佐證的狀況下，這些循環腫瘤細胞計數的資訊目前於臨床上並不具有實質的效用。為了更加了解循環腫瘤細胞於癌症轉移的過程中所具有的臨床價值，對這些細胞進行定性分類、單細胞分析與分子分析的研究開始受到關注。本文展示一套搭配特殊設計平台的細胞回收與抓取系統，細胞株實驗結果證實，以密度將細胞分離的回收技術基礎之下，其細胞回收率不受細胞EpCAM表現量影響，平均回收率為89.7±4.7%，系統靈敏度以混入不同數量級細胞株(10-300顆)得到驗證。細胞抓取系統在適當的參數設定下其對於目標細胞的成功抓取率幾近100%且其抓取純率超越90%。
另一方面，為了因應對於稀少細胞如循環癌細胞或是檢體量受限情況之下的後續研究，本文設計並開發出利用微流體技術的孔盤，能將細胞在繁複的生物相關操作下有效保存高達99.2%，所需之檢體細胞量亦相較於傳統方式減少許多且無損測試的正確性。本文將其應用在癌細胞的藥物敏感度檢測上，經由細胞株(MCF7 and MDA-MB-231)以及實際臨床腫瘤檢體實驗後，驗證了此微流孔盤在細胞數量極少的情況下進行生物性測試實驗的可行性。藉由本研究所發展的全血中循環癌細胞回收系統以及微流孔盤，期望可以應用於癌症的診斷與治療，以提供臨床醫師在無論是病人的癌症管理方面或是藥物選用方面上有效的判斷依據。

關鍵字

微流體 ； 循環腫瘤細胞 ； 單細胞抓取 ； 化療敏感性檢測 ； 個人化癌症治療

並列摘要

Circulating tumor cell (CTC) now is considered as promising biomarker for cancer diagnosis and prognosis. Many studies had reported the overall survival of patients was related to the number of CTCs detected in whole blood. However, patient-derived CTC count may not enough for doctors to make an actionable decision since CTC possess high heterogeneity, yet the sensitivity of current technologies for CTC enrichment remains to be validated. To further investigate the role of CTC in metastasis process, there has been great interest in subtyping, single cell profiling and molecular analysis of CTC recently. This thesis presents a microfluidic-based, automatic, seamless system enabling single CTC isolation & retrieval from whole blood sample. Cell line results of isolation system showed the ability to recovery CTCs with 89.7 ± 4.7% efficiency from 2ml of human whole blood regardless of their EpCAM expression level. Sensitivity is achieved at low concentrations and recovery of cells through the range of 10 to 300 cells. Results of single cell retrieval system showed the successful retrieval rate reached nearly 100% of target cells and the average retrieval purity was over 90%.
On the other hand, for downstream cellular assay on rare cell or limited cell-supplied sample, this thesis presents a microfluidic-based, 96well-formatted plate with built-in micro gap to preserve these precious cells up to 99.2% during multiple assay/wash procedures. Comparing to traditional plate much less cell was needed when using this plate. Drug response profiling on limit cell-supplied tumor sample was performed as one application of this plate. Results on cell line (MCF7 and MDA-MB-231) and patient tumor sample showed the feasibility of cellular analysis in rare cell scenario using this plate.
In this thesis, an automatic CTC isolation & retrieval system and a micro-gap plate for cellular analysis of rare cell were presented. Solid evidence of feasibility, stability, and robustness of this whole system were also provided. Conceivably, this microfluidic-based system is applicable to personalized medicine, providing patient-derived information for doctors to tailor therapeutic interventions for patients.

並列關鍵字

Microfluidics ； Circulating tumor cells ； Single cell retrieving ； Chemosensitivity profiling ； Persoalized medicine

參考文獻

1. Chaffer, C. L. & Weinberg, R. A. A Perspective on Cancer Cell Metastasis. Science 331, 1559-1564 (2011).

3. Nagrath, S. et al. Isolation of rare circulating tumor cells in cancer patients by microchip technology. Nature 450, 1235-U1210 (2007).

4. Alix-Panabieres, C. & Pierga, J. Y. Circulating Tumor Cells : Liquid Biopsy. Bull. Cancer 101, 17-23 (2014).

5. Alix-Panabieres, C. and K. Pantel, Technologies for detection of circulating tumor cells: facts and vision. Lab Chip 14, 57-62 (2014).

6. Krebs, M.G., et al., Molecular analysis of circulating tumor cells-biology and biomarkers. Nature Reviews Clinical Oncology, 11, 129-144 (2014).