Multi-Marker Immunofluorescent Staining and PD-L1 Detection on Circulating Tumour Cells from Ovarian Cancer Patients

SIMPLE SUMMARY: Circulating tumour cells (CTCs) have the potential to serve as a rich source of information for cancer diagnostic and therapeutic decisions. To fully exploit this minimally invasive diagnostic resource requires techniques that aid in enriching heterogenous populations of CTCs and markers to efficiently characterise these cells as tumour derived. In the present study we eva-luated the microfluidic enrichment of CTCs and a multi-marker staining methodology for the identification of heterogeneous CTCs in ovarian cancer (OC) patients and evaluation of PD-L1 expression. We showed, for the first time, the existence of hybrid CTCs with an epithelial/mesenchymal phenotype and their association with PD-L1 in OC. Incorporation of this method in future clinical trials can help predict immunotherapy responsiveness in OC patients. 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.