Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, United States.
Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC 27599, United States; Joint Department of Biomedical Engineering, North Carolina State University, Raleigh, NC 27695, United States.
Biosens Bioelectron. 2014 Apr 15;54:476-83. doi: 10.1016/j.bios.2013.11.019. Epub 2013 Nov 18.
Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.
循环肿瘤细胞 (CTCs) 是癌症进展和转移潜力的重要生物标志物。外周血中 CTCs 的稀有性促使人们开发出高特异性分离这些肿瘤细胞的技术；然而，在对 CTCs 进行计数后，用于分离靶 CTCs 的技术有限。本文描述了一种使用称为微托盘的可释放微结构阵列从全血中捕获和分离活肿瘤细胞的策略。通过用纤连蛋白、Matrigel 或抗上皮细胞黏附分子 (EpCAM) 对微托盘表面进行功能化，实现了对有核细胞或表达 EpCAM 的细胞的特异性捕获。聚丙烯酸的表面接枝，随后通过蛋白 A/G 的共价结合，可在微托盘表面实现 EpCAM 抗体的有效捕获。使用抗 EpCAM 涂层的微托盘阵列时，MCF-7 细胞（人乳腺癌腺癌）的保留效率达到 90±8%。为了证明在血液存在的情况下肿瘤细胞在微托盘阵列上的保留效率，将 MCF-7 细胞混入全血中，然后加入涂有纤连蛋白、Matrigel 或抗 EpCAM 的小阵列 (71 mm(2))。这些方法可从≤10 µL 的全血中以高于 85%的效率捕获 MCF-7 细胞。此外，将 MCF-7 细胞与 1 mL 血液混合并加载到大阵列 (7171 mm(2)) 上时，也可以以高效率 (>97%)捕获，通过激光方法从阵列上分离出来，并证明在从阵列上移除后可获得高的集落形成率 (>85%)。该技术的临床应用通过从植入原发性人胰腺肿瘤的小鼠血液中捕获、分离和成功培养 CTC 得到了证明。从血液中直接捕获和分离活肿瘤细胞，然后进行分析或培养，将成为癌症细胞特征分析的有力工具。
