Fig. 1

PD-L1 CTC Assay Development (a) PD-L1-specific antibody and species-matched isotype control were tested in negative (Colo205) and high (H820) PD-L1-expressing cell lines. Individual cellular PD-L1 IF signal is quantified and plotted. No staining above background was seen with isotype control or in Colo205 stained with anti-PD-L1. b IFN-γ treatment increases PD-L1 expression in Colo205 and A549, while SU-DHL-1 is insensitive. c PD-L1 antibody was titrated in PD-L1 IF staining of high (H820), medium (H441), low (SU-DHL-1) and negative (Colo205, H23) PD-L1-expressing cell lines to determine assay sensitivity and dynamic range. At the optimal antibody concentration (1:2000 dilution), mean H820, H441 and SU-DHL-1 PD-L1 expression was determined to be 140-, 36- and 13-fold higher than mean background staining in negative controls. d Representative images of high, medium and negative PD-L1 expressing cell lines show membrane-localization of PD-L1 IF signal