The ERCC published 72 manuscripts during this past funding year (Aug 2020 – July 2021): 41 from ERCC2 (20 each from ERCC2 exRNA carrier and single EV grants; plus workshop review), and 32 from ERCC1 grants (some manuscript acknowledge ERCC1 and ECC2). There were several papers on acoustofluidic and microfluidic analysis of EVs from the Huang lab (Notre Dame) (1-12), developments in flow cytometry technology for studying EVs by the Jones (NCI) (13-17) and Chiu (U. Washington) (18-19) labs, and standardization of methods for separation and characterization of EVs from Witwer (Johns Hopkins) and colleagues (20-21). Tosar (Institut Pasteur de Montevideo) (22) as well as Tewari (U. Michigan) and colleagues (23) published methods for detecting exRNAs with 5’ or 3’ end biochemistry incompatible with the standard enzymes used for DNA amplification, including a method for amplifying long RNAs (mRNA, lncRNA) that have been under-studied (23). Enhanced bioinformatic analysis tools were highlighted in several papers (24-26). The Galas lab continued its development of methods to uncover multi-gene interactions in exRNA-seq and other datasets (24, 26). The Milosavljevic lab developed the Connect the Dots (CTD) method to characterize variations in metabolomic and transcriptomic data (25).
Several papers focused on the COVID-19 pandemic, including tools for viral monitoring (27, 28) and insights into the basic biology of SAR-CoV-2 (29-31). The Breakefield lab published a major review in Nature Reviews Molecular Cell Biology of RNA delivery by EVs in mammalian cells and its applications (32). The Charest (33) and Witwer (34) labs published papers on EV proteomics. Tosar and colleagues published studies that revealed new insights into the mechanisms of biogenesis of non-vesicular exRNAs (35-37).
Several studies described new exRNA and EV biomarkers of disease, including biomarkers tracking the effectiveness of PD-L1 cancer immunotherapy (38), differentiating different classes of heart disease (39, 40), and identifying Alzheimer’s disease (41) and pancreatic cancer (42). Similarly, a number of new studies shed new light into the basic biology and pathology of exRNAs and EVs, including the role of lncRNA in colorectal cancer therapy (43), EV-mediated resistance to anti-VEGF cancer therapy (44), the role of EVs in glioblastoma (45, 46), transfer of mutant p53 via EVs in cancer progression (47), oncogene-regulated release of EVs (48), and the role of EV miRNA in mitochondrial disease (49) and of EVs in multiple sclerosis (50-51) and heart function (52). Finally, several reviews discussed the use of EVs for therapeutics (53-55).
ERCC2 webinars continued to cover the biology of exRNAs and EVs and the development of exRNA / EV therapeutics. Pieter Mestdagh (Cancer Research Institute, Ghent) introduced the Human Biofluid RNA Atlas, and Gustavo Stolovitsky (IBM Research) presented on how to develop an exRNA data analysis challenge. These webinars were viewed 370 times on YouTube (36 hours viewing time). Apart from blogs covering the standard topics of exRNA and EV biology and development of exRNA and EV biomarkers and therapeutics, two blogs this year focused on efforts to improve testing for the SARS-CoV-2 virus.
The ERCC hosted a two-day online workshop (April 19-20, 2021) on the unique challenges of exRNA data analysis. The goal was to foster an open dialog about best practices and discuss open problems in the field, focusing initially on small exRNA sequencing data. There were 300 registrants for the workshop from around the world (Figure 1). All workshop lectures are available on YouTube, and have been viewed 900 times (75 hr viewing time so far). A manuscript authored by workshop lecturers is in press at Frontiers in Genetics (72).
The exRNA Atlas is a core ERCC resource for exRNA studies. To further extend it’s utility for scientific exploration, DMRR and consortium PIs plan to extend preliminary work that highlights the utility of the exRNA Atlas for examining RNA binding proteins (RBPs). There are hundreds of RNA binding proteins (RBPs) but the vast majority of them are poorly characterized, with much yet to be learned about their RNA binding specificity and functional role in exRNA cargo transport. For example, using current Atlas data files in conjunction with Bedgraph files, one may ask: where do RBPs bind across the transcriptome, or are there high occupancy sites, and do RBP levels change across physiological states? To enable this type of scientific exploration, DCC reprocessed the 7,000 FASTQ Atlas data files to generate Begraph files. Pilot studies using Bedgraph files and ENCODE eCLIP (enhanced crosslinking and immunosuppression) data files demonstrate how one may computationally explore questions related to RBP binding of exRNAs. During the coming year, these studies will be expanded to more fully expand the use of ERCC data to examine RBP biology.
ERCC seeks to share ERCC data more broadly with the scientific community. The ERCC DMRR is also funded by the Common Fund Data Ecosystem (CFDE) project that is focused on integrating data across twelve different CF projects. DMRR DCC worked with the CFDE Coordinating Center to integrate RNA-seq and qPCR data from 39 ERCC projects (3,300 biosamples) with the CFDE data portal. This data is now available in the CFDE portal and available for cross-project queries. Although early in the project, the successful integration of ERCC data provides greater access and visibility for ERCC data that can now be considered in a much broader context of biological and scientific questions.
To improve data submissions to the exRNA Atlas, DMRR developed a submission Validator that presents to the submitter “Validation Results” indicating whether they have properly prepared their templates and hence “Pass”, or have errors and “Fail’, with an appropriate error message to guide corrective action. Data security was also enhanced by implementing FTPS.
American Society of Extracellular and Microsvesicles (ASEMV) Conference. The ERCC Data Management and Resource Repository (DMRR) and a couple of ERCC2 PIs were part of the ASEMV2020 organizing committee that hosted an online conference in November, 2020.
ERCC accepted its first associate membership this year. Associate members are expected to be actively engaged in ERCC activities (i.e. participate in working groups as appropriate, may attend the ERCC biannual meetings at their own expense) and to abide by all policies approved by the consortium and any other pertinent NIH policies. Associate members must sign the ERCC2 Confidentiality Disclosure Agreement to participate in all ERCC activities.
The consortium benchmarking efforts remain a major focus. The DifFi cell line has been identified as source of EVs and is being produced under an established protocol and distributed to participating lab. In addition, several labs are also evaluating single EV analysis using commercial flow cytometers adapted specifically for EV analysis. A position paper that summarizes these and other efforts are described in a position paper (in press, iScience). The abstract of the paper is provided here:
NIH Common Fund Extracellular RNA Communication Consortium, Phase 2 (ERCC2): Next-Generation Approaches for Isolating and Characterizing exRNA and their Carriers.
Phase 2 of the Extracellular RNA (exRNA) Communication Consortium (ERCC) was launched at a Kick-Off Meeting on September 17-18, 2019, in Bethesda, Maryland. The ERCC2 Kick-Off Meeting was the first of a series of bi-annual consortium meetings to promote the development of new technologies, resources, and knowledge about exRNA and their carriers with the following goals: 1) generation and sharing of data, as well as tools for management and analysis of these data through the exRNA atlas resource; 2) improved separation techniques for separation of extracellular vesicles (EVs) and other exRNA carriers; 3) comprehensive characterization of the molecular cargo of EVs and other exRNA carriers; and 4) development and dissemination of tools for single EV isolation and analysis. To promote progress toward these goals, Working Groups were established for Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, and Scientific Outreach. Working collaboratively with each other and members of the scientific community at large, the ERCC2 investigators aim to fill critical gaps in knowledge and technology to enable development of rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs, which are needed to improve our understanding of exRNA biology and to develop accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.
The exRNA field continues to grow and the ERCC has a unique opportunity to educate the broader community about exRNAs and related topics, and how ERCC science and technology development is contributing to this emerging field. To engage the community, the Scientific Outreach Working Group (SOWG) within the DMRR is developing an exRNA-themed online course that is set to launch in early 2022. The course will consist of twenty lecturers who have committed to participating and the SOWG continues to meet with them to review content and coordinate between shared topics.
DMRR Scientific Outreach is supporting the October 2021 conference of the newly organized American Society for Intercellular Communication (ASIC) to be held at the Bolger center in Bethesda, MD. The SOWG also worked closely with the ASIC2021 organizing committee and Roger Alexander (SOWG) presented at ASIC2021 on open problems in exRNA data analysis and the development of an exRNA data analysis challenge.
Several webinars covering a variety of exRNA related topics are planned. We have a webinar planned for September 2021 by Juan Pablo Tosar of the Universidad de la Republica, Uruguay, who will discuss his important insights into the metabolism of non-vesicular exRNAs.
We plan to highlight new technologies as much as possible in the ERCC blog series. An exciting new development in the field is the engineering of the human PEG10 protein to carry RNA cargoes to target cells of interest. An August 2021 blog post will explore the issue (https://exRNA.org/blog-PEG10/), and Michael Segel from MIT, first author of the PEG10 publication in Science, will give an ERCC webinar on the topic in November 2021.
A major focus for the coming year is to accept and process into the Atlas ERCC data generated during the year. This data will consist primarily of RNA-seq and qPCR data, with a new data type(s) to be determined in consultation with ERCC2 PIs. We will also continue to expand the preliminary RNA binding protein (RBP) studies initiated this past year as described above. DMRR DCC will also be facilitating the integration of new ERCC2 RNA-seq and qPCR data into the CFDE Data Portal as it becomes available.
DMRR plans to expand the exRNA Atlas ecosystem by adding an EV Flow Repository module. This module will accommodate flow cytometric data pertaining to EV surface and internal cargo which will contain metadata relating to EV purification and basic EV subset analytical processes. The plan is to make the EV Flow Repository available to consortium members to facilitate their ongoing EV Flow benchmarking studies and for internal validation, prior to releasing to the broader scientific community.
The EV Antibody Database (https://exRNA.org/EVAbdb/) has been live since November 2020 and currently contains 42 entries on antibodies for use in Western blots. There is ongoing discussion in the ERCC2 Joint working group about adding catalogs of antibodies with applications in flow cytometry and immunoprecipitation.
1. Liu P et al. Acoustofluidic multi-well plates for enrichment of micro/nano particles and cells. Lab Chip (2020) 20: 3399-3409.
2. Zhong Z et al. Hardware design and fault-tolerant synthesis for digital acoustofluidic biochips. IEEE Trans Biomed Circuits Syst (2020) 14: 1065-1078.
3. Tian Z et al. Generating multifunctional acoustic tweezers in Petri dishes for contactless, precise manipulation of bioparticles. Sci Adv (2020) 6: eabb0494.
4. Hao N et al. Acoustofluidics-assisted fluorescence-SERS bimodal biosensors. Small (2020) 16: e2005179.
5. Zhang P et al. Deterministic droplet coding via acoustofluidics. Lab Chip (2020) 20: 4466-4473.
6. Xie Y et al. Microfluidic isolation and enrichment of nanoparticles. ACS Nano (2020) 14: 16220–16240.
7. Zhang P et al. Acoustoelectronic nanotweezers enable dynamic and large-scale control of nanomaterials. Nat Commun (2021) 12: 3844.
8. Li J et al. Acoustic tweezer with complex boundary-free trapping and transport channel controlled by shadow waveguides. Sci Adv (2021) 7: eabi5502.
9. Gu Y et al. Acoustofluidic centrifuge for nanoparticle enrichment and separation. Sci Adv (2021) 7: eabc0467.
10. Zhu H et al. Acoustohydrodynamic tweezers via spatial arrangement of streaming vortices. Sci Adv (2021) 7: eabc7885.
11. Chen C et al. Acoustofluidic rotational tweezing enables high-speed contactless morphological phenotyping of zebrafish larvae. Nat Commun (2021) 12: 1118.
12. Zhao S et al. Fabrication of tunable, high-molecular-weight polymeric nanoparticles via ultrafast acoustofluidic micromixing. Lab Chip (2021) 21: 2453-2463.
13. Welsh JA & Jones JC. Small particle fluorescence and light scatter calibration using FCMPASS software. Curr Protoc Cytom (2020) 94: e79.
14. Welsh JA et al. Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration. J Extracell Vesicles (2020) 9: 1816641.
15. Morales-Kastresana A, Welsh JA & Jones JC. Detection and sorting of extracellular vesicles and viruses using nanoFACS. Curr Protoc Cytom (2020) 95: e81.
16. Burnie J et al. Flow virometry quantification of host proteins on the surface of HIV-1 pseudovirus particles. Viruses (2020) 12: 1296.
17. Welsh JA et al. A simple, high-throughput method of protein and label removal from extracellular vesicle samples. Nanoscale (2021) 13: 3737-3745.
18. Andronico LA et al. Sizing extracellular vesicles using membrane dyes and a single molecule-sensitive flow analyzer. Anal Chem (2021) 93: 5897-5905.
19. Jiang Y et al. High-throughput counting and superresolution mapping of tetraspanins on exosomes using a single-molecule sensitive flow technique and transistor-like semiconducting polymer dots. Angew Chem Int Ed Engl (2021) 60: 13470-13475.
20. Royo F et al. Methods for separation and characterization of extracellular vesicles: Results of a worldwide survey performed by the ISEV Rigor and Standardization subcommittee. Cells (2020) 9: 1955.
21. Arab T et al. Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms. J Extracell Vesicles (2021) 10: e12079.
22. Tosar JP et al & . RI-SEC-seq: Comprehensive profiling of nonvesicular extracellular RNAs with different stabilities. Bio Protoc (2021) 11: e3918.
23. Giraldez MD & Tewari M. Phospho-RNAseq profiling of extracellular mRNAs and lncRNAs. Methods Mol Biol (2021) 2348: 257-271.
24. Kunert-Graf J, Sakhanenko N & Galas D. Partial information decomposition and the information delta: A geometric unification disentangling non-pairwise information. Entropy (Basel) (2020) 22: 1333.
25. Thistlethwaite LR et al. CTD: An information-theoretic algorithm to interpret sets of metabolomic and transcriptomic perturbations in the context of graphical models. PLoS Comput Biol (2021) 17: e1008550.
26. Kunert-Graf JM, Sakhanenko NA & Galas DJ. Optimized permutation testing for information theoretic measures of multi-gene interactions. BMC Bioinformatics (2021) 22: 180.
27. Suea-Ngam A et al. Enzyme-assisted nucleic acid detection for infectious disease diagnostics: Moving toward the point-of-care. ACS Sens (2020) 5: 2701-2723.
28. Chen L et al. Elliptical pipette generated large microdroplets for POC visual ddPCR quantification of low viral load. Anal Chem (2021) 93: 6456-6462.
29. Guo C et al. The D614G mutation enhances the lysosomal trafficking of SARS-CoV-2 spike. bioRxiv AOP 2020-12-09.
30. Su Y et al. Multi-omics resolves a sharp disease-state shift between mild and moderate COVID-19. Cell (2020) 183: 1479-1495.e20.
31. Zhang Q et al. Angiotensin-converting Enzyme 2-containing small extracellular vesicles and exomeres bind the Severe Acute Respiratory Syndrome Coronavirus 2 spike protein. Gastroenterology (2021) 160: 958-961.e3.
32. O’Brien K et al. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat Rev Mol Cell Biol (2020) 21: 585-606.
33. Charest A. Experimental and biological insights from proteomic analyses of extracellular vesicle cargos in normalcy and disease. Adv Biosyst (2020) 4: e20
34. Martin-Jaular L et al. Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection. EMBO J AOP 2021-03-11.
35. Tosar JP et al. Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome. Nucleic Acids Res (2020) 48: 12874-12888.
36. Tosar JP, Garcia-Silva MR & Cayota A. Circulating SNORD57 rather than piR-54265 is a promising biomarker for colorectal cancer: Common pitfalls in the study of somatic piRNAs in cancer. RNA (2021) 27: 403-410.
37. Tosar JP, Witwer K & Cayota A. Revisiting extracellular RNA release, processing, and function. Trends Biochem Sci (2021) 46: 438-445.
38. Shukuya T et al. Circulating microRNAs and extracellular vesicle-containing microRNAs as response biomarkers of Anti-programmed Cell Death Protein 1 or Programmed Death-Ligand 1 Therapy in NSCLC. J Thorac Oncol (2020) 15: 1773-1781.
39. Vaze A et al. Relations between plasma microRNAs, echocardiographic markers of atrial remodeling, and atrial fibrillation: Data from the Framingham Offspring study. PLoS ONE (2020) 15: e0236960.
40. Crouser ED et al. Circulating exosomal microRNA expression patterns distinguish cardiac sarcoidosis from myocardial ischemia. PLoS ONE (2021) 16: e0246083.
41. Sandau US et al. Performance of validated microRNA biomarkers for Alzheimer’s disease in mild cognitive impairment. J Alzheimers Dis (2020) 78: 245-263.
42. Kaczor-Urbanowicz KE et al. Reviews on current liquid biopsy for detection and management of pancreatic cancers. Pancreas (2020) 49: 1141-1152.
43. Pichler M et al. Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer. Gut (2020) 69: 1818-1831.
44. Ma S et al. CD63-mediated cloaking of VEGF in small extracellular vesicles contributes to anti-VEGF therapy resistance. Cell Rep (2021) 36: 109549.
45. Nieland L et al. Extracellular vesicle-mediated bilateral communication between glioblastoma and astrocytes. Trends Neurosci (2021) 44: 215-226.
46. Zeng A et al. Glioblastoma-derived extracellular vesicles facilitate transformation of astrocytes via reprogramming oncogenic metabolism. iScience (2020) 23: 101420.
47. Ma S et al. Gain-of-function p53 protein transferred via small extracellular vesicles promotes conversion of fibroblasts to a cancer-associated phenotype. Cell Rep (2021) 34: 108726.
48. Kilinc S et al. Oncogene-regulated release of extracellular vesicles. Dev Cell (2021) 56: 1989-2006.e6.
49. Ahn JY et al. Release of extracellular vesicle miR-494-3p by ARPE-19 cells with impaired mitochondria. Biochim Biophys Acta Gen Subj (2021) 1865: 129598.
50. Pusic KM et al & . Environmental enrichment and its benefits for migraine: Dendritic cell extracellular vesicles as an effective mimetic. J Cell Immunol (2021) 3: 215-225.
51. Pusic KM, Kraig RP & Pusic AD. IFNgamma-stimulated dendritic cell extracellular vesicles can be nasally administered to the brain and enter oligodendrocytes. PLoS ONE (2021) 16: e0255778.
52. Saheera S et al & . Extracellular vesicle interplay in cardiovascular pathophysiology. Am J Physiol Heart Circ Physiol AOP 2021-03-05.
53. Noren Hooten N et al. Hitting the bullseye: Are extracellular vesicles on target?J Extracell Vesicles (2020) 10: e12032.
54. Nguyen VVT et al. Functional assays to assess the therapeutic potential of extracellular vesicles. J Extracell Vesicles (2020) 10: e12033.
55. Witwer KW. On your MARCKS, get set, deliver: Engineering extracellular vesicles. Mol Ther (2021) 29: 1664-1665.
56. Meabon JS et al. Chronic elevation of plasma vascular endothelial growth factor-A (VEGF-A) is associated with a history of blast exposure. J Neurol Sci (2020) 417: 117049.
57. Chen B et al. The long noncoding RNA CCAT2 induces chromosomal instability through BOP1 – AURKB signaling. Gastroenterology (2020) 159: 2146-2162.e33. 00069.
58. Li J et al. miR-30d regulates cardiac remodeling by intracellular and paracrine signaling. Circ Res (2020) 128: e1-e23.
59. Zhao Z et al. miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages. FEBS Open Bio (2020) 10: 2021-2039.
60. Palace SG et al. Yersinia pestis escapes entrapment in thrombi by targeting platelet function. J Thromb Haemost (2020) 18: 3236-3248.
61. Chiu LS et al. The association of non-alcoholic fatty liver disease and cardiac structure and function-Framingham Heart Study. Liver Int (2020) 40: 2445-2454.
62. Muralidharan K et al. TERT promoter mutation analysis for blood-based diagnosis and monitoring of gliomas. Clin Cancer Res (2021) 27: 169-178.
63. Gorur A et al. ncRNA therapy with miRNA-22-3p suppresses the growth of triple-negative breast cancer. Mol Ther Nucleic Acids (2021) 23: 930-943.
64. Guo C et al. Choice of selectable marker affects recombinant protein expression in cells and exosomes. J Biol Chem (2021) 297: 100838.
65. Fazzalari A et al. A translational model for venous thromboembolism: microRNA expression in hibernating black bears. J Surg Res (2021) 257: 203-212.
66. Ezzaty Mirhashemi M et al. The dynamic platelet transcriptome in obesity and weight loss. Arterioscler Thromb Vasc Biol (2021) 41: 854-864.
67. Silverman DA et al. Cancer-associated neurogenesis and nerve-cancer cross-talk. Cancer Res (2021) 81: 1431-1440.
68. Li J et al. Angiotensin II-induced muscle atrophy via PPARgamma suppression is mediated by miR-29b. Mol Ther Nucleic Acids (2021) 23: 743-756.
69. Tulpule A et al. Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules. Cell (2021) 184: 2649-2664.e18.
70. Webster NJ et al. Testicular germ cell tumors arise in the absence of sex-specific differentiation. Development (2021) 148.
71. Thompson L et al. Quantification of cellular densities and antigenic properties using magnetic levitation. J Vis Exp AOP 2021-05-17.
72. Alexander RP et al. Open problems in extracellular RNA data analysis: Insights from an online workshop. Front Genet in press.