Department of Surgical Oncology and General Surgery, The First Hospital of China Medical University, 155 N. Nanjing Street, Shenyang, Liaoning, 110001, PR China; Key Laboratory of Precision Diagnosis and Treatment of Gastrointestinal Tumors, Ministry of Education, China Medical University, No.77, Puhe Road, Shenyang, Liaoning, 110001, PR China; Institute of Health Sciences, China Medical University, No.77, Puhe Road, Shenyang, Liaoning, 110001, PR China.
College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan, 250014, PR China.
EBioMedicine. 2023 Apr;90:104522. doi: 10.1016/j.ebiom.2023.104522. Epub 2023 Mar 16.
Detecting free cancer cells from ascites and peritoneal lavages is crucial for diagnosing gastric cancer (GC). However, traditional methods are limited for early-stage diagnosis due to their low sensitivity.
A label-free, rapid, and high-throughput technique was developed for separating cancer cells from ascites and peritoneal lavages using an integrated microfluidic device, taking advantage of dean flow fractionation and deterministic lateral displacement. Afterward, separated cells were analyzed using a microfluidic single-cell trapping array chip (SCTA-chip). In situ immunofluorescence for EpCAM, YAP-1, HER-2, CD45 molecular expressions, and Wright-Giemsa staining were performed for cells in SCTA-chips. At last, YAP1 and HER-2 expression in tissues was analyzed by immunohistochemistry.
Through integrated microfluidic device, cancer cells were successfully separated from simulated peritoneal lavages containing 1/10,000 cancer cells with recovery rate of 84.8% and purity of 72.4%. Afterward, cancer cells were isolated from 12 patients' ascites samples. Cytological examinations showed cancer cells were efficiently enriched with background cells excluded. Afterwards, separated cells from ascites were analyzed by SCTA-chips, and recognized as cancer cells through EpCAM/CD45 expression and Wright-Giemsa staining. Interestingly, 8 out of 12 ascites samples showed HER-2 cancer cells. At last, the results through a serial expression analysis showed that YAP1 and HER-2 have discordant expression during metastasis.
Microfluidic Chips developed in our study could not only rapidly detect label-free free GC cells in ascites and peritoneal lavages with high-throughput, they could also analyze ascites cancer cells at the single-cell level, improving peritoneal metastasis diagnosis and investigation of therapeutic targets.
This research was supported by National Natural Science Foundation of China (22134004, U1908207, 91859111); Natural Science Foundation of Shandong Province of China (ZR2019JQ06); Taishan Scholars Program of Shandong Province tsqn (201909077); Local Science and Technology Development Fund Guided by the Central Government (YDZX20203700002568); Applied Basic Research Program of Liaoning Province (2022020284-JH2/1013).
从腹水和腹腔灌洗液中检测游离癌细胞对于诊断胃癌（GC）至关重要。然而，由于传统方法的灵敏度较低，对于早期诊断仍然具有一定局限性。
本研究开发了一种无标记、快速、高通量的技术，通过集成微流控装置，利用层流分离和定向横向位移从腹水和腹腔灌洗液中分离癌细胞。随后，使用微流控单细胞捕获阵列芯片（SCTA 芯片）对分离的细胞进行分析。对 SCTA 芯片中的细胞进行 EpCAM、YAP-1、HER-2、CD45 分子表达的免疫荧光原位染色和 Wright-Giemsa 染色。最后，通过免疫组织化学分析组织中 YAP1 和 HER-2 的表达。
通过集成微流控装置，成功地从含有 1/10000 个癌细胞的模拟腹腔灌洗液中分离出癌细胞，回收率为 84.8%，纯度为 72.4%。随后，从 12 名患者的腹水样本中分离出癌细胞。细胞学检查显示，背景细胞被有效富集，癌细胞被分离出来。之后，通过 SCTA 芯片对分离的腹水细胞进行分析，通过 EpCAM/CD45 表达和 Wright-Giemsa 染色将其识别为癌细胞。有趣的是，12 个腹水样本中有 8 个显示出 HER-2 阳性癌细胞。最后，通过一系列表达分析的结果表明，YAP1 和 HER-2 在转移过程中存在不一致的表达。
本研究开发的微流控芯片不仅可以快速高通量地检测腹水和腹腔灌洗液中无标记的游离 GC 细胞，还可以在单细胞水平上分析腹水癌细胞，提高腹膜转移诊断和治疗靶点研究的水平。
本研究得到了国家自然科学基金（22134004、U1908207、91859111）、山东省自然科学基金（ZR2019JQ06）、泰山学者工程专项（201909077）、中央引导地方科技发展专项资金（YDZX20203700002568）、辽宁省应用基础研究计划项目（2022020284-JH2/1013）的资助。
