High-content immunofluorescence assay detecting PD-L1 expression changes in head and neck cancer patient-derived cultures

Clinical uses of monoclonal antibodies against immune checkpoint molecules, such as the Program Death-Ligand 1 (PD-L1) or Program Death Protein-1 (PD-1), has transformed cancer therapy across pan-cancers. In addition to antibody therapies, there is a growing interest in identifying small molecules that could modulate PD-L1 levels in cancer cells. Yet, most current PD-L1 assays are not robust enough to be developed for drug screening purposes. Here, we report the development of a sensitive PD-L1 immunofluorescence assay that can capture PD-L1 expression heterogeneity in HNC patient tumor cultures and allows relative quantification of PD-L1 levels in cells in a streamlined and robust manner. Furthermore, this imaging-based assay can capture additional spatial or subcellular localization information of PD-L1 expression in patient cultures and has the potential to be combined with other image-based assays for future drug development purposes. Importantly, we demonstrated that this assay was robust enough to evaluate dose-dependent PD-L1-modulatory effects of drugs in patient-derived tumor cultures and demonstrated patient-to-patient variability of drug responses for PD-L1 modulation. This assay has the potential to be adopted for high-throughput drug screening for identifying small molecules modulators of PD-L1 using individual patient tumor cultures of various cancer types.