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Updated: Oct 9, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Multi-Marker Immunofluorescent Staining and PD-L1 Detection on Circulating Tumour Cells from Ovarian Cancer Patients
Du-Bois Asante1,2, Michael Morici1,2, Ganendra R K A Mohan3
1School of Medical and Health Sciences, Edith Cowan University, Perth, WA 6027, Australia.
Abstract:
Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the Parsortix™ system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.007) and mesenchymal (p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1.
Insights
This study detected ovarian cancer circulating tumor cells (CTCs) using novel markers, including PD-L1. Researchers found CTCs in 63% of patients, with hybrid epithelial/mesenchymal phenotypes showing significant PD-L1 positivity.
Area of Science:
- Oncology
- Cell Biology
- Cancer Diagnostics
Background:
- Ovarian cancer (OC) circulating tumor cell (CTC) detection often misses mesenchymal cells.
- The presence of PD-L1 on OC CTCs remains largely uncharacterized.
Purpose of the Study:
- To develop and validate a method for detecting diverse ovarian cancer CTC phenotypes, including PD-L1 expression.
- To investigate the heterogeneity of CTCs in ovarian cancer patients.
Main Methods:
- Developed an antibody staining protocol targeting epithelial (CK, EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers.
- Utilized the Parsortix™ system for CTC enrichment from 16 OC patients.
- Characterized CTCs for PD-L1 expression and correlated it with cell phenotypes.
Main Results:
- CTCs were detected in 63% of ovarian cancer patients.
- Identified heterogeneous CTCs, including epithelial, mesenchymal, and hybrid phenotypes.
- Found PD-L1 expression on 39% of CTCs, significantly associated with hybrid phenotypes (p < 0.001).
Conclusions:
- The study successfully detected heterogeneous ovarian cancer CTCs, including those with mesenchymal and hybrid phenotypes.
- PD-L1 expression is present on a subset of OC CTCs, particularly those exhibiting epithelial-to-mesenchymal plasticity.
- Further research correlating CTC phenotypes with clinical outcomes is warranted to understand their role in OC progression and PD-L1 association.
