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

Lujun Hu; Wenjie Chen; Shurong Zhou; Guizhi Zhu

doi:10.1007/s41048-020-00122-x

Volume 6 Issue 6

Mar. 2021

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Lujun Hu, Wenjie Chen, Shurong Zhou, Guizhi Zhu. ExoHCR: a sensitive assay to profile PD-L1 level on tumor exosomes for immunotherapeutic prognosis[J]. Biophysics Reports, 2020, 6(6): 290-298. doi: 10.1007/s41048-020-00122-x

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Lujun Hu, Wenjie Chen, Shurong Zhou, Guizhi Zhu. ExoHCR: a sensitive assay to profile PD-L1 level on tumor exosomes for immunotherapeutic prognosis[J]. Biophysics Reports, 2020, 6(6): 290-298. doi: 10.1007/s41048-020-00122-x

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

doi: 10.1007/s41048-020-00122-x

Lujun Hu^1,2,
Wenjie Chen²,
Shurong Zhou²,
Guizhi Zhu^{2,3,4
,
,}

1 College of Bioengineering, Sichuan University of Science and Engineering, Zigong 643000, Sichuan, China
2 Center for Pharmaceutical Engineering and Sciences, Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA
3 The Developmental Therapeutics Program, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA
4 Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA

Funds: We thank Drs. da Rocha, Hawkridge, McClay, and McRae for facility sharing. L. Hu was supported with the China Scholarship Council scholarship. G. Zhu acknowledges partial support from the Center for Pharmaceutical Engineering and Sciences-VCU School of Pharmacy, NIH KL2 Scholarship and Endowment Fund from VCU C. Kenneth and Dianne Wright Center for Clinical and Translational Research (UL1TR002649 and KL2TR002648), American Cancer Society Institutional Research Grants (IRG-18-159-43), and VCU Presidential Research Quest Fund. Microscopy was performed at the VCU Microscopy Facility, supported in part by funding from NIH-NINDS Center (5 P30 NS047463) and, in part, by funding from the NIH-NCI Cancer Center (P30 CA016059). Flow cytometry was performed at the VCU Massey Cancer Center Flow Cytometry Shared Resource, which is supported, in part, with funding from NIH-NCI Cancer Center (P30 CA016059).

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Corresponding author: Guizhi Zhu
Received Date: 04 July 2020
Rev Recd Date: 14 September 2020
Publish Date: 10 March 2021

Abstract

Abstract

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-L1-positive exosomes. Exosomes were then incubated with a conjugate of PD-L1 antibody with an HCR trigger DNA (T), in which one aPD-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 aPD-L1-T to initiate HCR in situ on bead-conjugated exosome surfaces. By flow cytometric analysis of the resulting beads, relative to aPD-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.
- Exosome,
- Hybridization chain reaction,
- Immune checkpoint,
- PD-L1 analysis,
- Immunotherapy

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References(34)

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