Single Extracellular Vesicle Protein Analysis Using Immuno‐Droplet Digital Polymerase Chain Reaction Amplification

There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno‐droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody–DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read‐out by droplet imaging. In these proof‐of‐principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.

[1]  Jun Wang,et al.  Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies , 2019, Proteomics.

[2]  A. Karlström,et al.  Fast and efficient Fc-specific photoaffinity labelling to produce antibody-DNA-conjugates. , 2019, Bioconjugate chemistry.

[3]  C. Genoud,et al.  Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging , 2019, Journal of extracellular vesicles.

[4]  A. Cashikar,et al.  A cell-based assay for CD63-containing extracellular vesicles , 2019, PloS one.

[5]  Yu Wang,et al.  Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues , 2019, Nature Biotechnology.

[6]  Hakho Lee,et al.  Physical and Molecular Landscapes of Mouse Glioma Extracellular Vesicles Define Heterogeneity , 2019, Cell reports.

[7]  T. D. de Greef,et al.  Efficient Small-Scale Conjugation of DNA to Primary Antibodies for Multiplexed Cellular Targeting , 2019, bioRxiv.

[8]  Virginia Savova,et al.  Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species. , 2019, Immunity.

[9]  L. Martínez-Piñeiro,et al.  High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry , 2019, Scientific Reports.

[10]  Hakho Lee,et al.  Characterization of single microvesicles in plasma from glioblastoma patients. , 2018, Neuro-oncology.

[11]  Paul J. Hoover,et al.  Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma , 2018, Cell.

[12]  R. Weissleder,et al.  Single-cell barcode analysis provides a rapid readout of cellular signaling pathways in clinical specimens , 2018, Nature Communications.

[13]  Lei Zheng,et al.  Single-Exosome-Counting Immunoassays for Cancer Diagnostics. , 2018, Nano letters.

[14]  R. Tang,et al.  Nanoparticle Counting by Microscopic Digital Detection: Selective Quantitative Analysis of Exosomes via Surface-Anchored Nucleic Acid Amplification. , 2018, Analytical chemistry.

[15]  Imre Mäger,et al.  Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression , 2018, Front. Immunol..

[16]  Hakho Lee,et al.  Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles , 2018, Science Advances.

[17]  Hakho Lee,et al.  Multiplexed Profiling of Single Extracellular Vesicles. , 2018, ACS nano.

[18]  J. Nolan,et al.  Analysis of Individual Extracellular Vesicles by Flow Cytometry. , 2018, Methods in molecular biology.

[19]  Omar K. Yaghi,et al.  Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils , 2017, Science.

[20]  James S. Wilkinson,et al.  Extracellular Vesicle Flow Cytometry Analysis and Standardization , 2017, Front. Cell Dev. Biol..

[21]  Jennifer C. Jones,et al.  Flow Cytometric Analysis of Extracellular Vesicles. , 2017, Methods in molecular biology.

[22]  Melissa M. Sprachman,et al.  SCS macrophages suppress melanoma by restricting tumor-derived vesicle–B cell interactions , 2016, Science.

[23]  A. deMello,et al.  The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. , 2015, Lab on a chip.

[24]  Razelle Kurzrock,et al.  PD-L1 Expression as a Predictive Biomarker in Cancer Immunotherapy , 2015, Molecular Cancer Therapeutics.

[25]  Hakho Lee,et al.  Sensitive and direct detection of circulating tumor cells by multimarker µ-nuclear magnetic resonance. , 2012, Neoplasia.

[26]  Yoshitaka Narita,et al.  Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma. , 2010, Genes & development.

[27]  Christoph A. Merten,et al.  Drop-based microfluidic devices for encapsulation of single cells. , 2008, Lab on a chip.

[28]  A. deMello,et al.  Quantitative detection of protein expression in single cells using droplet microfluidics. , 2007, Chemical communications.

[29]  O. Bogler,et al.  A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. , 1996, Cancer research.

[30]  W. Cavenee,et al.  A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. , 1994, Proceedings of the National Academy of Sciences of the United States of America.