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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.