使用光声流细胞测定卵巢癌检测
Summary
提出了利用定制的光声流系统和靶向叶酸封铜硫化纳米粒子检测循环卵巢肿瘤细胞的协议。
Abstract
许多研究表明，循环肿瘤细胞（CTCs）的枚举可能显示作为卵巢癌的预后工具的希望。目前检测CTC的策略包括流式细胞测量、微流体装置和实时聚合酶链反应（RT-PCR）。尽管最近取得了一些进展，早期卵巢癌转移的检测方法仍然缺乏临床翻译所需的敏感性和特异性。本文提出了一种利用定制三维（3D）打印系统（包括流动室和注射器泵）的光声流细胞测定卵巢循环肿瘤细胞的新方法。该方法利用叶酸封铜硫化纳米颗粒（FA-CuS NPs）通过PAFC靶向SKOV-3卵巢癌细胞。这项工作证明了这些造影剂对卵巢癌细胞的亲和力。结果表明，通过荧光显微镜进行NP表征、PAFC检测和NP吸量，从而证明这种新型系统在生理相关浓度下检测卵巢CC的潜力。
Introduction
卵巢癌是最致命的妇科恶性肿瘤之一，2018年全球估计有184，800人死亡。多项研究表明卵巢癌进展（即转移）与CTCs2、3、4之间存在的相关性。CTC检测和分离的最常见方法是利用细胞搜索系统，该系统以EpCam受体5为目标。EpCam表达，然而，在上皮到间质过渡，这已经牵连在癌症转移6。尽管取得了进步，但目前的临床技术仍然受到低准确性、高成本和复杂性的影响。由于这些缺点，发现和枚举卵巢CTC的新技术已成为研究的一个重要领域。
最近，PAFC成为非侵入性检测癌细胞、分析纳米材料以及鉴定细菌7、8、9的有效方法。PAFC 不同于传统的荧光流细胞测量，它利用光声检测流中的分析物。当激光被引起热弹性膨胀的材料吸收时，会产生光声效应，产生声波，超声波可以由超声波传感器10、11探测到。PAFC 与传统流式细胞测定方法相比具有的优势包括简单、易于翻译到临床设置，以及检测在患者样本12、13中前所未有的深度的 CTC。最近的研究已经利用PAFC系统检测细胞使用内源和外源造影14，15。近红外（NIR）光吸收造影剂，如丁氰化绿色染料，和金属NPs（如黄金和CuS）已用于细胞和组织的选择标记，结合光声成像16，17，18。由于生物组织中近红外光的穿透深度提高，吸收剂的光声检测可以在更深的临床应用中进行。由于其在诊所使用的巨大潜力，靶向NIR造影剂与PAFC的结合引起了对检测高氯联产委的极大兴趣。
PAFC 与靶向造影剂相结合，为高通量分析患者样本提供了改进的方法，提高了 PC 的准确性和有针对性的检测。CTC的主要检测策略之一是特定靶向感兴趣的细胞上的膜蛋白。卵巢CTC的一个显著特征是位于其外膜19上的叶酸受体的过度表达。叶酸受体靶向是鉴定血液中卵巢CTC的理想策略，因为内源性细胞具有较高的叶酸受体表达，一般是发光的，对血液接触有限。硫化铜（CuSNPs）最近被公认为能够靶向癌细胞21上表达的叶酸受体。这些NP造影剂结合其生物相容性、易于合成和吸收在近红外的深层，为利用PAFC检测卵巢CTC的理想靶向策略。
这项工作描述了FA-CuS NPs的制备及其用于检测光声流系统中的卵巢癌细胞。CuS NPs 用叶酸进行修饰，以专门针对卵巢 CTC，并在使用 1，053 nm 激光刺激时发出光声信号。结果表明，在PAFC系统中用这些光声造影剂孵育的卵巢癌细胞被成功检测。这些结果表明，卵巢癌细胞的检测浓度下降到1细胞/μL，荧光显微镜证实SKOV-3卵巢癌细胞22成功吸出这些颗粒。这项工作详细介绍了FA-CuS NPs合成、荧光显微镜样品制备、光声流系统构建以及卵巢癌细胞的光声检测。该方法表明，利用FA-CuS NPs成功识别了流动中的卵巢CTC。今后的工作将侧重于该技术在卵巢癌转移的早期发现方面的临床应用。
Protocol
1. 纳米粒子合成和功能化 注：FA-CuS NPs的合成是使用一个从先前发布的协议21改编的一罐合成方法实现的。注意：所有合成应发生在通风的化学烟气罩中。 在合成之前，通过 0.2 μm 无菌过滤器过滤大约 300 mL 的去离子 （DI） 水。 用洗涤剂溶液清洁 250 mL 玻璃圆底瓶，然后用 DI 水冲洗。将 0.0134 g 的 CuCl2添加到 100 mL 的 DI 水中，以创建…
Representative Results
图1A显示了合成纳米粒子的典型TEM图像。典型纳米粒子的平均尺寸约为 8.6 nm ± 2.5 nm。纳米粒子测量在ImageJ中进行。阈值和分水岭函数用于分离粒子进行测量。每个粒子的水平和垂直直径是垂直测量并进一步平均。对于 DLS，图 1B显示了具有代表性的测量值。这些颗粒的平均流体动力学直径为73.6纳米。硫…
Discussion
该协议是利用PAFC和目标CuS造影剂检测卵巢CTC的一种简单方法。已经探索出许多检测卵巢CTC的方法，包括微流体装置、RT-PCR和荧光流细胞测定仪23、24、25。这些范围的复杂性、成本和准确性，限制了它们在临床环境中的有效性。PAFC 引入了与这些传统检测 CTC 方法相比的若干优点，包括检测患者样本中的 CTC 的能力，以及将其易?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢玛德琳·豪厄尔在合成方面给予的帮助，马修·切斯特帮助设计流系统，伊森·马沙尔帮助SolidWorks。
Materials
| 0.025% Trypsin With EDTA | Corning | 25-053-Cl | |
| 0.2 µm 1000 mL Vacuum Filtration Unit | VWR | 10040-440 | For filtering larger volumes of DI water. |
| 0.2 µm sterile syringe filter | VWR | 28145-477 | |
| 3D Printed Tank | Custom-made | ||
| Acquisition Card | National Instruments | PXIe-5170R | 250 MS/s, 8-Channel, 14-bit |
| Alconox | Sigma-Aldrich | 242985-1.8KG | Detergent used for cleaning glassware. |
| Amicon Ultra-15 Centrifugal Filters | Millipore | UFC903024 | |
| Amicon Ultra-4 Centrifugal Filters | Millipore | UFC803024 | |
| Bright-Line Hematocytometer | Hausser Scientific | 1492 | |
| Copper(II) Chloride | ACROS ORGANICS | 206532500 | |
| Coupling Objective | Thorlabs | LMH-10x-532 | To couple pulsed light to optical fiber. |
| Coupling Stage | Newport | F-91-C1-T | Stage for coupling pulsed light to objective. Holds FP-1A and LMH-10x-532 |
| CPX Series Digital Ultrasonic Cleaning Bath | Fisherbrand | Model CPX3800 | |
| Data Acquisition software | National Instruments | NI LabVIEW 2017 (32-bit) | LabVIEW used to synchronize laser pulses with data acquisition. |
| Data Processing Software | Mathworks | Matlab R2016a | Reconstructions and graphs produced using Matlab software. |
| FBS | Sigma-Aldrich | F2442-500ML | |
| Fiber Chuck | Newport | FPH-DJ | Used to hold the bare fiber. |
| Fiber Coupler | Newport | FP-1A | 3-Axis stage for positioning fiber chuck and optical fiber at the focus of the objective. |
| Folic Acid | Sigma-Aldrich | F7876-10G | |
| Formvar Coated TEM Grids | Electron Microscopy Sciences | FCF300-CU-SB | |
| Masterflex Tubing | Cole Parmer | EW-96420-14 | |
| McCoy's 5A Medium | ATCC | 30-2007 | |
| Norm-Ject 10 mL Syringes | HENKE SASS WOLF | 4100-X00V0 | |
| Optical Fiber | Thorlabs | FG550LEC | Used to expose sample to pulsed light. |
| PBS | Alfa Aesar | J62036 | |
| Penicillin Streptomycin | GIBCO | 15140-122 | |
| Pulsed Laser | RPMC Lasers Inc | Quantus-Q1D-1053 | Pulsed laser source with specifications 1053 nm, 8 ns pulse, 10 Hz maximum. |
| Pulser/Receiver | Olympus | 5077PR | Receives, filters, and amplifies photoacoustic signals. Operated with 59 dB Gain. |
| Quartz Capillary Tube | Sutter Instrument | QF150-75-10 | |
| RPMI Midum 1640 (1X) Folic Acid Free | Gibco | 27016-021 | |
| Silicone | Momentive Performance Materials, Inc. | GE284 | |
| SKOV-3 Cells | ATCC | HTB-77 | |
| Sodium Bicarbonate | Sigma-Aldrich | S5761 | |
| Sodium Carbonate | Sigma-Aldrich | S7795-500G | |
| Sodium Hydroxide Beads | BDH | BDH9292-500G | |
| Sodium Sulfide Nonahydrate | Sigma-Aldrich | 431648-50G | |
| Syringe Pumps | New Era Pump Systems Inc | DUAL-1000 | |
| Texas Red-X-Succinimydl ester | Invitrogen | 1949071 | |
| Transducer | Olynmpus | V214-BB-RM | Ultrasound detector with central frequency of 50 MHz and -6 dB fractional bandwidth of 82%. |
| Trypan Blue Solution .4% | Amresco | K940-100ML | |
| Tween 20 | Sigma-Aldrich | P7949-100ML | |
| Ultrasound Gel | Parker Laboratories Inc. | Aquasonic 100 | Ultrasound gel for transducer coupling |
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Cite This Article
Lusk, J. F., Miranda, C., Smith, B. S. Ovarian Cancer Detection Using Photoacoustic Flow Cytometry. J. Vis. Exp. (155), e60279, doi:10.3791/60279 (2020).