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

• 作者: Guizhi Zhu Lujun Hu Shurong Zhou Wenjie Chen
• 来源期刊: 生物物理学报:英文版
• 学科: 医学
• 关键词: EXOSOME Hybridization chain reaction Immune checkpoint PD-L1 analysis IMMUNOTHERAPY
• 年，卷（期）: swwlxbywb_2020,（6）
• 页码范围: 290-298
• 页数: 9页
• 分类号: R73

摘要

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 aCD63-conjugated magnetic beads,we isolated exosomes from B16F10 melanoma and CT26 colorectal cancer cells that were immunostimulated to generate PD-Ll-positive exosomes.Exosomes were then incubated with a conjugate of PD-L1 antibody with an HCR trigger DNA(T],in which one aPD-Ll-T conjugate carried multiple copies of T.Next,a pair of metastable fluorophore-labeled hairpin DNA(HI and H2)were added,allowing T on〇(PD-Ll-T to initiate HCR in situ on bead-conjugated exosome surfaces.By flow cytometric analysis of the resulting beads,relative to aPD-Ll-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.