Oyama Rintaro, Mori Masataka, Matsumiya Hiroki, Nemoto Yukiko, Nishizawa Natsumasa, Honda Yohei, Kanayama Masatoshi, Taira Akihiro, Kuwata Taiji, Takenaka Masaru, Kuroda Koji, Yoneda Kazue, Ohnaga Takashi, Endo Motoyoshi, Tanaka Fumihiro
School of Medicine, Second Department of Surgery, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-Ku Kitakyushu-Shi, Fukuoka, 807-8555, Japan.
School of Medicine, Department of Molecular Biology, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-Ku Kitakyushu-Shi, Fukuoka, 807-8555, Japan.
Sci Rep. 2025 Aug 11;15(1):29306. doi: 10.1038/s41598-025-14826-y.
Detecting rare circulating tumor cells (CTCs), malignant cells of primary site origin, in the bloodstream is difficult. We previously constructed a polymeric microfluidic device with high capture efficiency for lung cancer cell lines using antibodies against the epithelial cell adhesion molecule (EpCAM). In this study, we investigated a method for extracting DNA from single cells captured in a microfluidic device and performed targeted sequencing using the Cancer Hotspot Panel v2. Additionally, we employed a fixation method, which enabled more efficient sequencing of the EpCAM-chip-captured cells than existing fixation methods. We used blood samples obtained from three patients with lung cancer to assess the clinical applicability of our method. Directly sequenced samples revealed better coverage uniformity than samples subjected to whole-genome sequencing. Direct sequencing of cells fixed with preserver fluid and 100% ethanol was performed accurately with high coverage uniformity. Our method demonstrated a sensitivity of 99.4%, specificity of 99.5%, and area under the curve of 1 (allele frequency cut-off, 18%). Our approach suggests that single cells captured in a microfluidic device are sufficient for genetic mutation analysis.
在血液中检测罕见的循环肿瘤细胞（CTC），即源自原发部位的恶性细胞，并非易事。我们之前构建了一种聚合物微流控装置，该装置利用抗上皮细胞粘附分子（EpCAM）抗体对肺癌细胞系具有高捕获效率。在本研究中，我们研究了一种从微流控装置中捕获的单细胞提取DNA的方法，并使用癌症热点Panel v2进行靶向测序。此外，我们采用了一种固定方法，与现有固定方法相比，该方法能使EpCAM芯片捕获的细胞进行更高效的测序。我们使用从三名肺癌患者获得的血样来评估我们方法的临床适用性。直接测序的样本比进行全基因组测序的样本显示出更好的覆盖均匀性。用保存液和100%乙醇固定的细胞进行直接测序，准确性高且覆盖均匀性好。我们的方法显示出99.4%的灵敏度、99.5%的特异性和1的曲线下面积（等位基因频率截止值为18%）。我们的方法表明，微流控装置中捕获的单细胞足以进行基因突变分析。
