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Updated: Aug 26, 2026
Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence
Published on: August 14, 2019
Single-cell analysis and EpCAM-based microfluidic chip detection of PD-L1 on circulating tumor cells for prognostic
Lin Zhang1,2, Hongqing Lyu1,2, Xia Tong1,2
1Department of Gynecology, Jinyi Group Jinhua Maternal and Child Health Hospital, Jinhua, China.
Background:
The prognosis of epithelial ovarian cancer (EOC) after comprehensive treatment remains unsatisfactory. This study aimed to evaluate the feasibility of enriching and detecting circulating tumor cells (CTCs) in peripheral blood from EOC patients using EpCAM antibody-based microfluidic chips, investigate the prognostic value of CTC PD-L1 expression, and analyze the cellular heterogeneity of ovarian cancer through re-analysis of publicly available single-cell RNA sequencing data.
Methods:
Seventy-two EOC patients undergoing surgery at Jinhua Maternal and Child Health Hospital (2020.07-2023.12) were enrolled. CTCs were enriched and PD-L1 detected via CytoSorter with EpCAM-coated HB-Chip. Clinicopathological associations were tested by chi-square/Fisher's exact tests; paired CTC counts compared by Wilcoxon signed-rank test. Prognostic analyses used Kaplan-Meier curves (log-rank test) and Cox proportional hazards regression. Single-cell RNA-seq data (GEO: GSM8072347) were re-analyzed for tumor microenvironment PD-L1 expression patterns. qPCR and flow cytometry validated PD-L1 expression in three EOC cell lines.
Results:
Pre- and postoperative PD-L1+ CTC detection rates were 56.9% (41/72) and 45.8% (33/72), with significant postoperative decline (P < 0.0001). Pre-operative PD-L1+ CTC <2/4 mL predicted shorter PFS (median: 1.5 vs 3.3 months; P = 0.008), and remained independent in multivariate Cox regression (adjusted HR = 2.35, 95% CI: 1.39-3.96, P = 0.001), while FIGO stage III/IV was the strongest predictor (adjusted HR = 2.66, 95% CI: 1.55-4.57, P < 0.001). Total CTC count showed no PFS association (P = 0.858). scRNA-seq re-analysis identified distinct cell populations and CD274 expression patterns across the tumor microenvironment. IFN-γ-induced PD-L1 upregulation was confirmed by qPCR and surface protein validated by flow cytometry across EOC cell lines.
Conclusion:
The EpCAM antibody-based HB-Chip can achieve enrichment and detection of CTCs in peripheral blood from EOC patients. Pre-operative PD-L1+ CTC count is an independent prognostic factor for progression-free survival. CTC PD-L1 detection may assist in postoperative prognostic evaluation. Re-analysis of publicly available single-cell transcriptomic data further elucidated tumor microenvironment heterogeneity, and in vitro experiments provided supportive evidence for immune-related gene regulation in ovarian cancer cells.
