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Cancer immunotherapy has made recent breakthrough, including immune checkpoint blockade (ICB) that inhibits immunosuppressive checkpoints such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1). However, most cancer patients do not durably respond to ICB. To predict ICB responses for patient stratification, conventional immunostaining has been used to analyze the PD-L1 expression level on biopsied tumor tissues but has limitations of invasiveness and tumor heterogeneity. Recently, PD-L1 levels on tumor cell exosomes showed the potential to predict ICB response. Here, we developed a non-invasive, sensitive, and fast assay, termed as exosome-hybridization chain reaction (ExoHCR), to analyze tumor cell exosomal PD-L1 levels. First, using αCD63-conjugated magnetic beads, we isolated exosomes from B16F10 melanoma and CT26 colorectal cancer cells that were immunostimulated to generate PD-L1-positive exosomes. Exosomes were then incubated with a conjugate of PD-L1 antibody with an HCR trigger DNA (T), in which one αPD-L1-T conjugate carried multiple copies of T. Next, a pair of metastable fluorophore-labeled hairpin DNA (H1 and H2) were added, allowing T on αPD-L1-T to initiate HCR in situ on bead-conjugated exosome surfaces. By flow cytometric analysis of the resulting beads, relative to αPD-L1-fluorophore conjugates, ExoHCR amplified the fluorescence signal intensities for exosome detection by 3-7 times in B16F10 cells and CT26 cells. Moreover, we validated the biostability of ExoHCR in culture medium supplemented with 50% FBS. These results suggest the potential of ExoHCR for non-invasive, sensitive, and fast PD-L1 exosomal profiling in patient stratification of cancer immunotherapy.

ExoHCR: a sensitive assay to profile PD-L1 level on tumor exosomes for immunotherapeutic prognosis