Workflow for evaluation of PD-L1 expression on patient CTC and matched tumor biopsy. (A) CTC Workflow. ① Blood is collected from cancer patients and processed through Vortex technology to enrich for CTCs. ② Blood is diluted 10X with PBS and ③ injected through the microfluidic device with syringe pumps. ④ Purified cells are collected into a 96 well-plate, where they are ⑤ stained with immunofluorescence markers and imaged. ⑥ Fluorescence intensity can be analyzed and PD-L1 gene expression quantified. (B) Tumor biopsy workflow: In parallel of the CTC workflow, lung biopsies were analyzed for PD-L1 expression. While biopsy provides information on the intra-tumor heterogeneity, only the CTCs present in a blood draw can cover both intra and inter-tumor heterogeneity.