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

SUBMITTER: Asante DB

PROVIDER: S-EPMC8699768 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPrevious studies have shown clinical relevance of programmed death-ligand 1 (PD-L1) and soluble PD-L1 (sPD-L1) in human cancers. However, still contradictory results exist. Our aim was evaluation of PD-L1-expressing monocytic myeloid-derived suppressor cells (M-MDSCs), monocytes/macrophages (MO/MA), tumour cells (TC) and immune/inflammatory cells (IC) as well as investigation of the sPD-L1 in ovarian cancer (OC) patients.MethodsThe group of 74 pretreatment women were enrollment to the study. The expression of PD-L1 on M-MDSCS and MO/MA was assessed by flow cytometry. The profile of sPD-L1 was examined with ELISA. The expression of PD-L1 in mononuclear cells (MCs) was analyzed using real time PCR. PD-L1 immunohistochemical analysis was prepared on TC and IC. An in silico validation of prognostic significance of PD-L1 mRNA expression was performed based microarray datasets.ResultsOC patients had significantly higher frequency of MO/MA versus M-MDSC in the blood, ascites and tumour (each p < 0.0001). In contrast, PD-L1 expression was higher on M-MDSCs versus MO/MA in the blood and ascites (each p < 0.0001), but not in the tumour (p > 0.05). Significantly higher accumulation of blood-circulating M-MDSC, MO/MA, PD-L1+M-MDSC, PD-L1+MO/MA and sPD-L1 was observed in patients versus control (p < 0.001, p < 0.05, p < 0.001, p < 0.001 and p < 0.0001, respectively). Accumulation of these factors was clinicopathologic-independent (p > 0.05). The expression of PD-L1 was significantly higher on IC versus TC (p < 0.0001) and was clinicopathologic-independent (p > 0.05) except higher level of PD-L1+TC in the endometrioid versus mucinous tumours. Interestingly, blood-circulating sPD-L1 positively correlated with PD-L1+M-MDSCs (p = 0.03) and PD-L1+MO/MA (p = 0.02) in the blood but not with these cells in the ascites and tumours nor with PD-L1+TC/IC (each p > 0.05). PD-L1 and sPD-L1 were not predictors of overall survival (OS; each p > 0.05). Further validation revealed no association between PD-L1 mRNA expression and OS in large independent OC patient cohort (n = 655, p > 0.05).ConclusionsAlthough PD-L1 may not be a prognostic factor for OC, our study demonstrated impaired immunity manifested by up-regulation of PD-L1/sPD-L1. Furthermore, there was a positive association between PD-L1+ myeloid cells and sPD-L1 in the blood, suggesting that sPD-L1 may be a noninvasive surrogate marker for PD-L1+myeloid cells immunomonitoring in OC. Overall, these data should be under consideration during future clinical studies/trials.

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

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