Extracellular Vesicles (EVs: Exosomes and Microvesicles): Research, Diagnostics and Therapeutics Applications Agenda

Aspirations For and the Current Status of Blood-based Cancer Diagnostics
Walter Koch, Vice President, Roche Molecular Systems, United States of America

Dozens of published research studies have reported that quantitative analysis of somatic mutations in blood is associated with dynamic responses of tumors to therapy, development of drug resistance during therapy and relapse after treatment, as well as genomic evolution of primary tumors and metastases.  To date, only one “liquid biopsy” test has gained FDA approval; the cobas® EGFR Mutation Test v2 is used to genotype NSCLC for dozens of EGFR activating and resistance mutations used for selection of tyrosine kinase inhibitors Erlotinib or Osimertinib.  Beyond genotyping tumors for therapy selection or clinical trial enrollment, there is an unmet medical need for better noninvasive biomarkers to monitor solid tumor therapy response, recurrence, and residual disease after surgical removal of early stage tumors.  This talk will explore the clinical development needed for liquid biopsy tests to be broadly included in cancer diagnosis and treatment guidelines, and to be used in routine clinical care with reimbursement.

Subgroups of Extracellular Vesicles from Tumor Tissues for Biomarker Discovery and Liquid Biopsy Development in Malignant Disease
Jan Lötvall, Professor Krefting Research Centre, University of Gothenburg, Chief Scientist, Codiak BioSciences; Founding President of ISEV, United States of America

Extracellular vesicles (EVs) have the capacity to shuttle both proteins, lipids and nucleotides such as RNA between cells, leading to an array of functional changes in a recipient cell. Importantly, the EV secretome changes significantly in disease, especially in cancer. We have recently developed a process to isolate EVs specifically from tumor tissues, and have utilized this technology to identify an array of biomarker candidates in malignant melanoma, breast cancer and colon cancer. Specifically, the technique identifies EV surface molecules from tumor tissue EVs, which are not present on plasma EVs from healthy individuals. Using this information, we have been able to develop assays to specifically identify cancer-EVs in the circulation in cancer patients compared to healthy individuals. This presentation will discuss EV diversity, and will give several examples of circulating cancer EV biomarkers that can function as liquid biopsies in cancer subgroup identification, cancer monitoring and putatively cancer screening.

Carboxypeptidase E: A Biomarker for Multiple Cancers in Circulating Exosomes
Y. Peng Loh, Chief and Senior Investigator, Section on Cellular Neurobiology, Eunice Kennedy Shriver National Institute of Child health and Human Development, National Institutes of Health (NIH), United States of America

Extracellular Vesicles and Neuro-Oncologic Cancer
Bob Carter, Professor and Chief of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, United States of America

Will discuss EVs as biomarkers in neuro-oncologic cancer.

Exosomal Integrins: Biomarkers for Liquid Biopsy
Lucia Languino, Professor of Cancer Biology, Thomas Jefferson University, United States of America

Cancer Extracellular Vesicles (EVs) found in blood are heterogeneous and show unique protein compositions. Our analysis shows that a prostate cancer EV subset, designated exosomes, carries a unique repertoire of integrins, which are surface receptors for extracellular matrix.  Using iodixanol gradients, immunoblotting and size measurements we show that integrins are differentially expressed in cancer exosomes. Our recent studies demonstrate that exosomes from prostate cancer cells contribute to horizontal propagation of unique integrin-associated invasive phenotypes.  We also show that exosomal integrins are transferred to cells in the tumor microenvironment and mediate response to therapy. This presentation will discuss the role of exosomal integrins in cancer progression and therapy response, and their potential use as clinical biomarkers for non-invasive diagnosis of cancer.

Extracellular Vesicles (EVs) and Cell Free DNA (cfDNA) as Blood-based Biomarkers: Plastic-based Microfluidics for their Enrichment and Analysis
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

While there are a plethora of different blood-based markers, EVs are generating significant interests due to their relatively high abundance (~1013 particles per mL of blood) and the information they carry. EVs contain a diverse array of nucleic acids, such as mRNA, lncRNA, and miRNA that can be used for disease management. In addition to EVs, cfDNA also are biomarkers that can be used to help manage different disease states using the mutations they possess that can have high diagnostic value. In spite of the relatively high abundance of cfDNA in diseased patients (~160 ng/mL), the extraction and enrichment of cfDNA has been inefficient, even by commercial kits, due to the low abundance of the tumor bearing DNA fragments (<0.01%) and the short nature of these fragments, especially cancer-related cfDNA (as small as 50 bp). In this presentation, we will discuss the design, fabrication and analytical figures-of-merit of a microfluidic device that can serve the dual purpose for the affinity-based selection of EVs and the solid phase extraction of cfDNA directly from plasma using the same device. The microfluidic is made from a plastic that can be injection molded to produce high quality devices at low cost. For EVs, the device is made cyclic olefin copolymer (COC) is UV/O3 activated to allow for the efficient immobilization of affinity agents to the surface of the device. In the case of cfDNA, the device is made from COC as well, but is only UV/O3 activated (i.e., no affinity agents used). Information will be provided as to the ability to molecularly profile the cargo contained within the affinity-selected EVs, in particular mRNA expression profiling. We will also discuss the use of this microfluidic to isolate with high recovery cfDNA from plasma samples with size selection capabilities. The isolated cfDNA could be queried for mutations using an allele-specific ligation detection reaction at a mutant to wild-type ratio <0.1%.

New Tools for Liquid Biopsy: Rare Cells, Exosomes, and Circulating DNA
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America

This presentation will describe new technologies we developed for liquid biopsy and precision medicine. The three new tools include a rare-cell isolation instrument we call eDAR (ensemble decision aliquot ranking), a nanofluidic technology for the high-sensitivity and high-purity enrichment of exosomes, and a digital-nucleic-acid detection and analysis platform based on our SD (self-digitization) chip. I will outline the workings of these new tools, describe their performance, and discuss the clinical questions we are addressing with these next-generation technologies.

Utility and Challenges of ctDNA
J. Carl Barrett, Vice President, Translational Sciences Oncology, AstraZeneca, United States of America

ctDNA is rapidly becoming a major clinical tool for identifying patients for therapy, monitoring response of therapy and exploring mechanisms of resistance to therapies. Examples of all these applications will be presented. It is imperative that ctDNA assay are sensitive and specific. Data on the performance of different assays will be presented as well the basis for lack of concordance in certain situations.

Mt-SEA Pipeline: High Throughput Flow Cytometric Analysis of Exosomes in Clinical Biofluids
Jennifer Jones, NIH Stadtman Investigator, Head of Transnational Nanobiology, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, United States of America

Because Extracellular Vesicles (EVs) carry surface receptors that are characteristic of their cells of origin, EVs have tremendous potential as non-invasive biomarkers for diagnosis, risk-stratification, treatment selection, and treatment monitoring. We developed a first-in-class pipeline to characterize EV heterogeneity and provide high-sensitivity quantification of informative EVs in biofluids before, during, and after treatment. By combining multiplex assays with high-resolution, single EV flow cytometric methods together into a Mutiplex-to-Single EV Analysis (Mt-SEA) pipeline, we are able to characterize a broad range of relevant EV subsets, while also accurately measuring the concentration of specific EV populations.  Detection of tumor-associated EVs and detection of EV repertoire changes during treatment paves the way to future evaluation of EVs as as biomarkers for use in personalized, adaptive therapies.

EFIRM Liquid Biopsy (eLB)
David Wong, Felix and Mildred Yip Endowed Chair in Dentistry; Director for UCLA Center for Oral/Head & Neck Oncology Research, University of California-Los Angeles, United States of America

The advent of personalized medicine employing molecular targeted therapies has markedly changed the treatment of cancer in the past decade. Although tumor tissue biopsy-based genotyping is the current clinical practice for guiding clinical management, biopsy procedures can result in significant morbidity, limiting sampling to static snapshots which are further limited in scope by the inherent sampling bias of the analysis itself. To overcome these issues, technologies are needed for rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease. Liquid biopsy is a rapidly emerging field to address this unmet clinical need as diagnostics based on cell-free circulating tumor DNA (ctDNA) can be a surrogate for the entire tumor genome. The use of ctDNA via liquid biopsy will facilitate analysis of tumor genomics that is urgently needed for molecular targeted therapy. Currently, most targeted approaches are based on PCR and/or next generation sequencing (NGS) for liquid biopsy applications with performance concordance in the 60-80% range with biopsy-based genotyping. We have developed a liquid biopsy technology “Electric Field Induced Release and Measurement (EFIRM)- Liquid Biopsy (eLB)” provides the most accurate targeted detection that can assist clinical treatment decisions for the most common subtype of lung cancer, non-small cell lung cancer (NSCLC), where tyrosine kinase inhibitors (TKI) that can extend the disease progress free survival period of these patients. eLB requires only 40 µl of sample volume, no sample processing, reaction time is 15min and can be performed at the point-of-care or high throughput reference lab using plasma or saliva. eLB detects actionable EGFR mutations in NSCLC patients with >95% concordance with biopsy-based genotyping. eLB is minimally (plasma)/ non-invasive (saliva) detecting the most common EGFR gene mutations that are treatable with TKI such as Gefitinib or Erlotinib to effectively extend the progression free survival of lung cancer patients.

Microfluidics for the Isolation of Biomarkers From Glioblastoma Patients
Shannon Stott, Assistant Professor, Massachusetts General Hospital & Harvard Medical School, United States of America

Glioblastoma is a highly fatal disease with few treatment options. Due to the location of the tumor, it is challenging to get dynamic, real-time information about the cancer. To address this need, we have developed microfluidic technologies to obtain information about these tumors by isolating rare cells (circulating tumor cells or “CTCs”) and tiny lipid particles, referred to as extracellular vesicles (EVs), from a simple blood draw. In this talk, data will be presented on our technological approach as well as our effort to interrogate their molecular content using next generation RNA sequencing and ddPCR. While the first application of this ‘liquid biopsy’ technology is in monitoring glioblastoma, it can be readily expanded to many different cancers and used to explore the underlying biology of metastasis.

Extracellular Vesicles as Mediators of Tissue Repair and Disease Reversal
Peter Quesenberry, Professor of Medicine, The Warren Alpert Medical School of Brown University, United States of America

Extracellular vesicle from marrow derived mesenchymal stem cells (MSCs) have been shown to have tissue repair characteristics.  We have studied several models examining the capacity of MSC vesicles to promote tissue repair or disease reversal.  In a murine model of monocrotaline induced pulmonary hypertension we have demonstrated that MSC derived vesicles will reverse or prevent the development of pulmonary hypertension as measured by vascular remodeling and right ventricular hypertrophy.  In a similar vein we have demonstrated that MSC-derived vesicles mitigates radiation damage to murine bone marrow stem cells both in vitro and in vivo.  This latter appears to be mediated by miRNA species. Lastly, MSC-derived vesicles have been shown to reverse the malignant phenotype of both colorectal and prostate cancer cells in vitro and in vivo.

The potential therapeutic applications of possible vesicle therapy are apparent and await appropriate progress in scale up approaches for harvest of vesicle types.

About Noam Chomsky, DNA Motifs, Non-coding RNAs and Cancer Patients
George Calin, Professor and The Alan M. Gewirtz Leukemia & Lymphoma Society Scholar, University of Texas MD Anderson Cancer Center, United States of America

The newly discovered differential expression in numerous tissues, key cellular processes and multiple diseases for several families of long and short non-codingRNAs (ncRNAs, RNAs that do not codify for proteins but for RNAs with regulatory functions), including the already famous class of microRNAs (miRNAs) strongly suggest that the scientific and medical communities have significantly underestimated the spectrum of ncRNAs whose altered expression has significant consequences in diseases. MicroRNA and other short or long non-codingRNAs alterations are involved in the initiation, progression and metastases of human cancer. The main molecular alterations are represented by variations in gene expression, usually mild and with consequences for a vast number of target protein coding genes. The causes of the widespread differential expression of non-codingRNAs in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the processing machinery. MicroRNA and other short or long non-codingRNAs expression profiling of human tumors has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify non-codingRNAs that may represent downstream targets of activated oncogenic pathways or that are targeting protein coding genes involved in cancer. Recent studies proved that miRNAs and non-coding ultraconserved genes are main candidates for the elusive class of cancer predisposing genes and that other types of non-codingRNAs participate in the genetic puzzle giving rise to the malignant phenotype. Last, but not least, the shown expression correlations of these new ncRNAs with cancer metastatic potential and overall survival rates suggest that at least some member of these novel classes of molecules could potentially find use as biomarkers or novel therapeutics in cancers and other diseases.

Extracellular Vesicles (EVs) as Therapeutics for Extending Healthspan
Paul Robbins, Professor, Department of Biochemistry, Molecular Biology, and Biophysics, and the Institute on the Biology of Aging and Metabolism, University of Minnesota Medical School, United States of America

With aging, there is an inevitable and progressive loss of the ability of tissues to recover from stress, in part through loss of stem cell function. As a consequence, the incidence of chronic degenerative diseases increases exponentially starting at the age of 65. This includes neurodegeneration, cardiovascular disease, diabetes, osteoarthritis, cancers, and osteoporosis.  More than 90% of people over 65 years of age have at least one chronic disease, while 75% have at least two. Thus, it is imperative to find a way to target therapeutically the process of aging to compress the period of functional decline in old age. Such a therapeutic approach would simultaneously prevent, delay or alleviate multiple diseases of old age. We are using both naturally aged mice and the ERCC1-deficient mouse model of accelerated aging mice as a model of accelerated aging to identify therapeutic strategies for extending healthy aging. Previously we demonstrated that intraperitoneal (IP) administration muscle-derived stem/progenitor cells (MDSPCs) isolated from young wild-type mice into ERCC1-deficient mice conferred significant lifespan and healthspan extension through a paracrine/endocrine mechanism. More recently, we demonstrated that BM-MSCs from young, but not old mice, also prolonged lifespan and healthspan in ERCC1-deficient mice, similar to MDSPCs. Taken together, these results suggest that at least two types of adult stem cell populations, BM-MSCs and MDSPCs, isolated from young mice extend lifespan and healthspan following IP injection in a mouse model of accelerated aging. We also have been characterizing and identifying the factors released by young, functional stem cells important for this extension of lifespan and healthspan. Conditioned media (CM) from young, but not old MDSPCs and BM-MSCs, rescued the function of aged, dysfunctional stem cells as well as senescent fibroblasts in culture.  Moreover, this activity in the CM co-purifies with extracellular vesicles (EVs) released by young, but not old stem cells. Treatment of ERCC1-deficient mice with EVs from young stem cells is able to extend healthspan. Interestingly, EVs from the serum of young, but not old mice also can rescue cellular senescence suggesting that the effect of young serum on aging could be mediated in part by EVs. In addition, senescent cells appear to take up stem cell derived EVs more efficiently.  Progress towards developing clinically relevant approaches using stem cell-derived extracellular vesicles to treat autoimmune and age-related pathologies will be presented.

Circulating Extracellular Vesicles are Biomarkers and Mediators of Alcoholic Liver Disease
Gyongyi Szabo, Professor & Vice Chair for Research, Department of Medicine, University of Massachusetts Medical School, United States of America

A salient feature of alcoholic liver disease (ALD) is Kupffer cell (KC) activation and recruitment of inflammatory monocytes and macrophages (MØs). We studied whether these key cellular events were mediated by extracellular vesicles (EVs) in the pathogenesis of ALD. EVs transfer biomaterials, including proteins and microRNAs, and have recently emerged as important effectors of intercellular communication. We also hypothesized that circulating EVs from mice with ALD have a characteristic protein cargo that may serve as biomarker of disease. The total number of circulating EVs was increased in mice with ALD compared to pair-fed controls. Mass spectrometric analysis of circulating EVs revealed a distinct signature of proteins involved in inflammatory responses, cellular development, and cellular movement between ALD EVs and control EVs. We also identified uniquely important proteins in ALD EVs that were not present in control EVs. When ALD EVs were injected intravenously into alcohol naive mice, we found evidence of uptake of ALD EVs in recipient livers in hepatocytes and MØs. Hepatocytes isolated from mice after transfer of ALD EVs, but not control EVs, showed increased monocyte chemoattractant protein 1 mRNA and protein expression, suggesting a biological effect of ALD EVs. Compared to control EV recipient mice, ALD EV recipient mice had increased numbers of F4/80hi cluster of differentiation 11b (CD11b) lo KCs and increased percentages of tumor necrosis factor alpha–positive/interleukin 12/23– positive (inflammatory/M1) KCs and infiltrating monocytes (F4/80intCD11bhi), while the percentage of CD2061CD1631 (anti-inflammatory/M2) KCs was decreased. In vitro, ALD EVs increased tumor necrosis factor alpha and interleukin-1b production in MØs and reduced CD163 and CD206 expression. We identified heat shock protein 90 in ALD EVs as the mediator of ALD-EV- induced MØ activation. In conclusion these  results indicate a specific protein signature of ALD EVs and demonstrate a functional role of circulating EVs in mediating KC/MØ activation in the liver.

Acoustic Tweezers: Separating Exosomes, Circulating Tumor Cells, and Other Tiny Objects Using Sound Waves
Tony Jun Huang, William Bevan Distinguished Professor of Mechanical Engineering and Materials Science, Duke University, United States of America

The ability to isolate exosomes, circulating tumor cells (CTCs), and other circulating factors from biological fluids in a precise, biocompatible, and convenient manner is critical for many biomedical studies and applications. Here we summarize our recent progress on an “acoustic tweezers” technology that utilizes sound waves to manipulate exosomes, CTCs, and other tiny particles. For example, we developed an acoustic separation method to isolate exosomes directly from undiluted whole blood in a label-free and contact-free manner. This device consists of two modules. Micro-scale blood components are first removed by the cell-removal module, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110 nm particles from a mixture of micro- and nano-sized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. The acoustic tweezers technology is capable of delivering high-precision, high-throughput, high-efficiency cell/particle/fluid manipulation in a simple, inexpensive, cell-phone-sized device. More importantly, the acoustic power intensity and frequency used in the acoustic tweezers technology are in a similar range as those used in ultrasonic imaging, which has proven to be extremely safe for health monitoring, even during various stages of pregnancy. As a result, the acoustic tweezers technology is extremely biocompatible; i.e., cells can maintain their natural states and highest integrity during the acoustic cell-manipulation process.