High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer

ABSTRACT

Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the “reference noise”). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.

KEYWORDS:

• Nanofacs
• sorting
• flow cytometry
• phenotyping
• extracellular vesicles

Acknowledgments

The authors would like to thank Mario Roederer (NIH), Dragan Maric (NIH), Bridget McLaughlin (UC Davis), Giacomo Vacca (Kinetic River Corporation), Tim Knaak (Stanford), Cathy Crumpton (Stanford), Penny Chisholm and Stephen Biller (MIT), John Daly (DFCI), Samuel Strober (Stanford), Ronald and Shoshana Levy (Stanford), Marca Wauben, Ger Arkesteijn and Sten Libgrets (University of Utrecht), and John Nolan (Scintillon Institute, San Diego) for helpful discussions during the development of the nanoFACS methods presented here. We also thank the Beckman Coulter Flow Cytometry and Life Sciences groups for their work with the NCI Vaccine Branch as part of a Collaborative Research and Development Agreement. We also thank Kunio Nagashima and Ulrich Baxa for performing the EM analysis of sorted virus. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: U373-MAGI-CCR5E and U373-MAGI-CXCR4_CEM, from Dr. Michael Emerman. This research was supported in part by the Intramural Research Programs of the National Cancer Institute and National Institute of Biomedical Imaging and Bioengineering, and by the NCI Assistant Clinical Investigator Program (JCJ), an Innovative Partnership Grant from the Beckman Foundation (MB and DP, Stanford FACS Core), a Radiological Society of North America Research & Education Foundation Resident Research Grant (JCJ), a Leukemia Lymphoma Society grant (SJK and JCJ), and NIH grants: 1ZIABC011502 (JCJ); S10RR025518-01 (MB); S10 RR12964 (JVD); S10 RR 026825 (JVD); P01AI054456 (GJF); U01-126497 (IG); U01-OD-019750 (IG); R01 CA218500 (IG and JCJ); R01 HL1266497 (IG); and R01AI089955 (GJF); C06-RR12088 (JVD); P30 CA0933730 (JVD).

Author contributions

JCJ, MB, EJF, CDR, JVD, JT, VT, DRP, KW, SP, KM, RO, IG, and WT designed and performed various instrument tests to improve nanoFACS configurations (improved instrument signal:noise performance and tested technical aspects of assay performance). TAM, AMK, TD, MRG, JAB, and JCJ designed the biological assays to test the nanoFACS method. JCJ, TM, TD, and AMK performed the assays for testing nanoFACS with biological materials. JCSW, SP, and JCJ developed the use of the LED-pulser to facilitate evaluation of instrument background noise. LP, JAB, MT, AMK, and JCJ designed and performed tumour and immune EV staining experiments. JCJ, JAW, AR, AHK, GJF, JBT, PG, AP, SJK, PC, BY, XC, MT, and HK provided input, materials, and assistance with experiments and interpretation of results. All coauthors wrote and/or edited the manuscript.

Supplementary materials

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Cancer Institute [BC011503]; National Cancer Institute [ZIA BC011502]; National Institutes of Health [R01AI089955];National Institutes of Health [C06-RR12088]; National Institutes of Health [S10 RR12964]; National Institutes of Health [P01AI054456]; National Institutes of Health [S10RR025518-01]; National Institutes of Health [1U01HL126497-01]; National Institutes of Health [P30 CA0933730].