Abstract 764: Influence of platelets and neutrophils on circulating tumour cells
Authors: Ward Mark P. , Mohamed Bashir M. , Kane Laura , Bates Mark , Berghoff Janina , Spillane Cathy L. , Kelly Tanya , Kennedy John , Saadeh Feras A. , Hokamp Karsten , Gleeson Noreen , Sheils Orla , Martin Cara , Gallagher Michael , Hannify Sean , Dixon Eric P. , O'Toole Sharon A. , O'Leary John J. 
Published: September 2020

Abstract
Background: Circulating tumour cells (CTCs) are silent precursors of metastatic disease that utilise various mechanisms to survive in circulation and metastasise to distal sites. Classical CTC detection relies on EpCam affinity-based technologies; however, CTCs are highly heterogeneous and often undergo EMT. Recent research highlights the ability of platelets and neutrophils to ‘cloak' CTCs and crosstalk to aid in their proliferation and survival in circulation.
Aims: Assess the role of platelets and neutrophils in the characterisation of CTCs.
Methods: Cell lines: Breast cancer (MCF-7) and ovarian cancer (SKOV-3, OVCAR3, OAW42) cells were exposed to healthy donor isolated platelets and neutrophils for 24h. EMT and immune evasion gene assays, flow cytometry analysis and cell proliferation assays were performed following co-culture. Cell line-blood spike-in experiments were performed for gating strategy optimisation.Patient samples: Blood specimens were prospectively collected from breast and ovarian cancer patients (central venous and peripheral blood). CTCs and immune cells were then isolated using a ClearCell FX microfluidic device. FX isolated cells were immunophenotyped by either immunofluorescence (IF) or flow cytometry. Single cells were index sorted using BD FACS Melody for subsequent scRNAseq.
Results: Platelet cloaking altered EpCam, PLEK2, CCL2 and TWIST1 mRNA expression in MCF-7 cells. Platelet and neutrophil co-culture altered EpCam, PAI-1 and PD-L1 expression in ovarian cancer cell lines. Healthy neutrophils co-cultured with ovarian cancer cells increased cell viability, while induction of NETosis increased cancer cell proliferation. CTCs isolated from peripheral blood in breast and ovarian patients were EpCam/panCK+/CD45- by IF. Immunophenotyped CTCs were identified as EpCam+/E-Cadherin+/CD45-, N-Cadherin+/CD45- and as PD-L1+/EpCam+/E-Cadherin+/CD45- cells by flow cytometry. We also identified EpCam+/CD45+/CD66b+ cells in ovarian central blood. Isolated classical and non-classical CTCs were sorted based on their different immunophenotypes for downstream scRNAseq using BD Precise Whole Transcriptome Assays.
Conclusion: Platelets and neutrophils alter the expression of markers used in the identification of CTCs. Neutrophils were found to increase the proliferation of cancer cells as well as increase PD-L1 expression. We identified a heterogeneous population of CTCs across different patients and found that CTCs from central venous blood have increased numbers of non-classical CTCs (CD66b+/EpCam+ cells). Molecular scRNAseq signatures of CTC subsets form the basis for identifying the most clinically relevant CTCs in circulation. Our approach may improve diagnostic accuracy, allow for immunophenotyping of cloaked CTCs, and provide prognostic information.
Citation Format: Mark P. Ward, Bashir M. Mohamed, Laura Kane, Mark Bates, Janina Berghoff, Cathy L. Spillane, Tanya Kelly, John Kennedy, Feras A. Saadeh, Karsten Hokamp, Noreen Gleeson, Orla Sheils, Cara Martin, Michael Gallagher, Sean Hannify, Eric P. Dixon, Sharon A. O'Toole, John J. O'Leary. Influence of platelets and neutrophils on circulating tumour cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 764.