Plasma was isolated from samples by centrifugation for 20 minutes at 300 ×g before CTC enrichment with anti-EpCAM coated magnetic beads (Appendix 1 ). The CTC capture process was carried out using anti-EpCAM beads in a modified protocol developed in our laboratory (31 (link)). Captured cells were immunostained with antibody cocktail containing three mixed pan-cytokeratin antibodies to ensure broad cytokeratin coverage, CD45, CD16, and CD66b antibodies to exclude hematopoietic cells and anti-PD-L1 antibody (28.8) to detect PD-L1 expression as detailed in Appendix 1 .
In parallel, another blood sample was processed using the Parsortix system at 99-mbar through a 6.5-µm cassette (Figure 1 ). Enriched cells were harvested according to the manufacturer’s instructions and fixed for 10 minutes at room temperature with 4% paraformaldehyde (PFA). A total of 8–9 mL of blood was processed through each method. To increase the numbers of markers to be interrogated, such as EpCAM expression separate from cytokeratins, vimentin, and PD-L1 expression (29 (link)), we adapted the quenching and re-staining protocol described by Adams et al. (32 (link)). This protocol utilises borohydride to quench fluorescent signals after an initial round of immunostaining followed by a second round of staining for additional markers, allowing for multi-phenotype analysis of CTCs. The PD-L1 detection, quenching, and restaining methods were standardised using MCF7, MCF7 induced with IFN-γ, MDA-MB-231 cell lines spiked into white blood cells (WBCs) from healthy control donors as detailed in Appendix 1 .
In parallel, another blood sample was processed using the Parsortix system at 99-mbar through a 6.5-µm cassette (
