Circulating Biomarkers World Congress 2020 Agenda

Conference Registration, Materials Pick-Up, Morning Coffee and Pastries

Morning Coffee Break and Networking in the Exhibit Hall

Biofluid Biomarkers for the Brain
Kendall Van Keuren-Jensen, Professor and Deputy Director, Translational Genomics Research Institute, United States of America

We will describe some of our efforts in using exRNAs as diagnostics for central nervous system injury and disease.

Networking Lunch in the Exhibit Hall, Exhibits and Poster Viewing

Luncheon Technology Spotlight Presentation: Advancements in Exosome Isolation: Impacts on Research and Therapeutic Development
Jim West, CEO, Clara Biotech, United States of America
Mei He, Assistant Professor, University of Kansas and Chief Science Officer, Clara Biotech, United States of America

Don’t miss this informative lunch session!

The newest technologies laying the groundwork for future exosome research and commercialization will be discussed, highlighting advancements that will have a far-reaching impact on this burgeoning space.

Exosome isolation, purification, yield and viability are critical to research, biomarker identification and therapeutic development. As the industry learns more, being able to focus on specific exosome subtypes is becoming increasingly important to this growing field.

This seminar will review current and future exosome isolation technologies and platforms, while also discussing their respective impacts on the molecular components of exosomes.

*** Every attendee will get an exclusive conference gift for attending this seminar ***

Exosome Biomarkers: Multiparametric Characterization and Phenotypic Analysis
Clayton Deighan, North American Sales and Applications Manager, NanoFCM

Unlocking the full diagnostic potential of exosomes is dependent, in part, on the ability to accurately analyze these small particles, to acquire proteomic data on the single-exosome level, and to do both for rare events. ExoView is uniquely positioned to measure and characterize single exosomes of the smallest sizes. ExoView provides quantification, sizing, and protein expression information, including surface and luminal proteins, all without the need for sample purification. A custom panel can be built to assay up to 6 biomarkers of interest.

Phenotyping Extracellular Vesicles Using Tetraspanins and Fluorescence-NTA
Ross Jacobson, Technical Sales Leader, Particle Metrix, Inc.

Nanoparticle Tracking Analysis (NTA) measures size and concentration of nanoparticles in the size  range  from  10  nm  to  1  µm.  While  getting  a  particle  size  distribution,  the  user  typically cannot  discriminate  whether  the  particle  is  a  vesicle,  protein  aggregate,  cellular  trash  or  an inorganic  precipitate  from  scatter  data  alone.  The  fluorescent  detection  capability  of  NTA enables the user to gain biochemical information about the particle and increases the resolution and the discrimination power. NTA instruments traditionally are equipped with one laser wavelength in operation. This limits the detection of biomarkers to only a couple fluorescence dyes. To expand the number of excitation wavelengths, it was necessary to change of the laser  source,  a  time  and sample  consuming  step.  To  overcome  this  issue  Particle  Metrix developed a unique QUATT laser technology and now allows the rapid analysis of biomarker concentrations or ratios with four excitation lasers on the same sample.  Here we report the quick and easy EV Phenotyping via multi-wavelength f-NTA on the new Particle Metrix four laser system. Alignment-free switching   between excitation wavelengths and   measurement mode (scatter and fluorescence) allows quantification of biomarker ratios such as Tetraspanins (CD63, CD81, CD9 and CD41) on the same sample within minutes reducing measurement time and precious sample volumes.

Afternoon Coffee and Tea Break Plus Networking

Isolation and Characterization of Extracellular Vesicles; An Integrated, High-Precision and Standardizable Approach
Shayne Harrel, Chief Scientist-Americas, Izon Science US LTD

Tremendous interest exists towards the development and implementation of extracellular vesicles (EVs) in bio-fluids as rich sources of diagnostic and prognostic biomarkers.  EVs derived from bio-fluids exhibit extensive heterogeneity with regards to particle size, concentration, membrane composition and cargo resulting in unique challenges in terms of EVs isolation and characterization.  We present an approach to purify EVs from complex bio-fluids using Izon’s qEV line of size exclusion chromatography (SEC) based columns, resulting in a highly pure and unperturbed sample.  Reproducibility and standardization is enhanced by incorporating Izon’s Automated Fraction Collector (AFC) into the isolation.  Tunable Resistive Pulse Sensing (TRPS) is used to characterize the biophysical properties (size, concentration and Zeta Potential) of purified EVs.  TRPS measurements are conducted at the single particle level and results are compared against a known standard, ensuring the accuracy, repeatability and traceability of the data.

Novel Methods For EV Isolation and Their Use in a Pilot Liver Disease Biomarker Discovery Study
Shannon Pendergrast, Chief Scientific Officer, Ymir Genomics

We will describe several novel products and methods for the rapid and effective isolation of biofluid extracellular vesicles (EVs). Also, the use of one such method to discover novel EV-associated urine biomarkers for liver disease will be presented.

Stem Cell Extracellular Vesicles as a Novel Therapy for Age-related Tendinopathy
John Ludlow, Executive Director, Regenerative Medicine, Zen-Bio, Inc.

Aging is the principal risk factor associated with tendinopathy and also contributes to a significant decrease in the ability to efficiently heal chronic tendon injury by altering the complex and highly coordinated processes required for normal resolution. In addition to increases in lifespan and a growing aging population that is remaining active, there is a concomitant rise in the prevalence of comorbidities and tendinopathy risk factors such as obesity and diabetes. These factors have led to a very high prevalence of tendon injuries in those over 60 years of age, reaching 50% of this population, resulting in decreased quality of life and an economic burden in the billions of dollars. The use of biological and stem cell therapies for tendon healing and repair have been widely investigated and have demonstrated some potential for soft tissue regeneration. Secreted extracellular vesicles (EVs), such as exosomes, are packed with potent pro-repair proteins and RNA cargos that are both cell type-specific, as well as differentially produced and secreted according to the cellular environment. We have demonstrated that mild heat shock improves the repair activity of stem cell-derived EVs in vitro and in vivo. By manipulating the cellular environment, we have found that we can alter stem cell EV production and secretion. Our results have validated an approach of manipulating the cellular environment in a closed-system bioreactor to modify the bioactive cargo of secreted EVs for improved tendon healing. This presentation will describe the further development of our pro-healing EV therapeutic approach to treat tendon injuries and tendinopathy specifically afflicting the elderly.

Extracellular Vesicles Spread Neurotoxicity in Amyotrophic Lateral Sclerosis
Davide Trotti, Professor, Scientific Director, Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, United States of America

A Multi-Dimensional Approach to EV Flow Cytometry
Andries Zijlstra, Associate Professor of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center and Vanderbilt Ingram Cancer Center, United States of America

Flow cytometry has proven to be very promising method for Single-EV analysis. Unfortunately, the heterogeneity of EV populations prevents the application of the single-particle classification strategy derived from conventional flow cytometry of intact cells fails in our attempts to resolve distinct classes of EVs. In simple terms, the small size of most EVs prevents the incorporation of all protein markers that define a uniform class of EVs. Consequently, EVs generated through the same biogenesis pathway from the same donor cells, could contain vastly different cargo. Conversely, restricting EV classification only to a size range is equally limiting. To improve EV sub-classification and enable the identification of biologically-significant alterations in EV populations in response to alterations in physiological or pathological states of the donor cell/tissue, we have developed a multi-dimensional approach to EV flow cytometry that attempts to cluster EVs into subpopulations on the basis of many, rather than single parameters.

Networking Reception with Beer, Wine and a Buffet Dinner in the Exhibit Hall

Close of Day 1 of the Main Conference

Exosomes-EV Training Course in Ballroom A&B [Separate Registration Required to Attend this Training Course]

Morning Coffee, Tea and Pastries in the Exhibit Hall

Applying Phage-Displayed Random Peptide Libraries to Identify Exosomes as Biomarkers in Patients with Central Nervous System Disorders
Xiaoli Yu, Assistant Professor, Department of Neurosurgery, University of Colorado Anschutz Medical Campus, United States of America

Like most other cells, brain tumor cells shed extracellular vesicles (EV) into their external environment and are considered potentially high-value biomarker reservoirs. We hypothesize that phage-displayed random peptide libraries can be applied to identify EV of CNS origin, and high affinity phage peptides can be used for rapid isolation and characterization of extracellular vesicles from blood of patients with brain tumors. Phage display has been successfully applied to many different areas of research, including immunology, cancer, drug discovery, epitope mapping, protein-protein interactions, and infectious diseases, targeting a broad cross section of protein families. Phage display technology is based on the construction of a polypeptide library fused to a bacteriophage coat protein. Phage are screened (“panned”) on surfaces coated with potential targets. We have applied two Phage-display peptide libraries (7mer &12 mer) and identified high affinity peptides specific to exosomes derived from brain tumor cell-lines and plasma of patients with other CNS disorders. The peptides shared high sequence homologies within the ones selected by the same panning targets as well as with others panned by exosomes from different sources. Our data suggest that phage peptide technologies can be used for enrichment, isolation, and characterization of EVs derived from different CNS cell types from blood of brain tumor patients and other CNS disorders.

Highly Sensitive Direct Quantification of cf-microRNAs for Biomarker Profiling by Next Generation Sequencing (NGS)
Sergio Barberan-Soler, CEO, RealSeq Biosciences

Cell-free circulating microRNAs (cf-miRNAs) are promising diagnostic and prognostic biomarkers for cancer and other diseases. cf-miRNAs are highly stable in biofluids and are therefore attractive as potential biomarkers via NGS-based detection compared to circulating DNA and protein markers. However, NGS detection of cf-miRNAs suffers from a high level of incorporation bias when sequencing libraries are prepared using the most widely used commercial kits. SomaGenics RealSeq library prep platform addresses this issue, with proven best-in-class accuracy of detection. The RealSeq platform includes a kit designed to profile miRNAs from biofluids (RealSeq-Biofluids) that typically detects four times as many cf-miRNAs as commercially available technologies. The latest kit in the RealSeq platform is RealSeq-T, the first targeted sequencing approach to detect panels of cf-miRNAs (up to 1,200) directly from plasma, with no organic extraction needed. Nearly 95% of sequenced reads from RealSeq-T libraries mapped to the miRNA panel. With these new capabilities, the RealSeq platform should advance the prospects of cf-miRNAs as clinical biomarkers.

Sequencing of cfDNA to Monitor Treatment Response in Cancer Patients
Taylor Jensen, Director, Research and Development, Sequenom (a LabCorp Company), United States of America

Networking Coffee Break in the Exhibit Hall -- Visit Exhibitors and View Posters

CSF Based New Generation Molecular Diagnostics: Improving Sensitivity and Actionability
Santosh Kesari, Founder, Pacific Neuroscience Institute, United States of America

Isolation of Human Urine Exosomes: Potential Markers For Disease Conditions
Antonio De Maio, Professor, University of California-San Diego, United States of America

The role of exosomes or extracellular vesicles as mediators of intercellular communication has gained a great deal of attention, particularly within pathological conditions. In addition, interest in them has emerged as potential biomarkers of pathological conditions. Exosomes have been detected in a variety of bodily fluids, such as blood, saliva, and urine. The study of exosomes in urine samples is ideal for the detection of disease markers because they can be easily obtained in abundant quantities in a non-invasive and painless fashion. We have systematically optimized the collection of human urine samples for the isolation of exosomes by differential centrifugation, including the time of collection, reproducibility, and conditions for storage. We have observed that glycoproteins within the membrane of exosomes provide a specific pattern in healthy individuals that opens the possibility that alteration in their presence may result in a disease signature. Based on this investigation, we are in conditions to evaluate the use of urine exosomes as markers for a variety of diseases.

The Use of Vesicles from Cow Milk for Oral Drug Delivery
Tom Anchordoquy, Professor, University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences, United States of America

Many pharmaceuticals must be administered intravenously due to their poor oral bioavailability.  In addition to issues associated with sterility and inconvenience, the cost of repeated infusion over a six-week course of therapy costs the healthcare system tens of billions of dollars per year.  Attempts to improve oral bioavailability have traditionally focused on enhancing drug solubility and membrane permeability, and the use of synthetic nanoparticles has also been investigated.  As an alternative strategy, some reports have clearly demonstrated that exosomes from cow milk are absorbed from the gastrointestinal tract in humans, and could potentially be used for oral delivery of drugs that are traditionally administered intravenously.  Our results demonstrate that milk exosomes are absorbed from the gut as intact particles that can be modified with ligands to promote retention in target tissues.

CD200-Expresson on Vascular Endothelial cells is Mediated Through VEGF
Michael Olin, Associate Professor, University of Minnesota Masonic Cancer Center, United States of America

Central nervous system tumors are the number one killer of children with cancer. Among those, glioblastoma multiforme (GBM) is an incurable primary brain tumor. The standard of care consists of resection followed by radiation and chemotherapy using temozolomide and is associated with a median overall survival of 14.6 months. To overcome this dismal outcome, clinicians are turning to immunotherapy approaches, which have demonstrated promising results in other solid tumors. However, immunosuppression by the tumor microenvironment prohibits a durable anti-tumor response and the optimization of immunotherapy, especially in GBM. Tumors have capitalized on immune regulatory mechanisms that facilitate the inhibition of an immune response. The CD200 checkpoint includes the tumor-bound protein CD200 and its inhibitory receptor. We recently reported that CD200 is also expressed on tumor vascular endothelial cells and that this expression is upregulated by VEGF, creating an immunological barrier around the tumor microenvironment. CD200 is also shed by tumors, and it has been reported that this soluble CD200 interacts with its inhibitory receptor restricted to immune cells to create an immunosuppressive environment. However, as extracellular proteases are abundant in the sera, it is highly probable for the soluble CD200 to be degraded. We hypothesis is that tumor-derived extracellular vesicles modulate CD200-induced immunosuppression. There is abundant evidence in the literature that extracellular vesicles derived from tumors suppress antigen-specific and non-specific anti-tumor responses, mediating a broad array of detrimental effects on the immune system. We now know that tumor-derived extracellular vesicles contain CD200 and VEGF. In addition, others have reported that miR-150, which plays a role in tumorigenesis, interacts with TAMs to induce VEGF, suggesting a link between tumor-derived extracellular vesicles, and the upregulation of C200. We propose i) CD200-expressing tumor-derived extracellular vesicles induce immune suppression, ii) VEGFpos-tEVs upregulates CD200 on vascular endothelial cells and/or iii) miR150 interaction with tumor associated macrophage upregulate CD200 on vascular endothelial cells maintain an immune suppressive tumor microenvironment.

Networking Lunch in the Exhibit Hall

Single EV Analysis Workshop -- Principles of EV Analysis
John Nolan, CEO, Cellarcus Biosciences, Inc., United States of America

Attend an on-site workshop covering principles of EV analysis. This 60 minute session will describe guidelines for rigorous EV characterization including an overview of current methods, considerations and controls for generating interpretable data, and a discussion of published guidelines including the new MIFlowCyt-EV manuscript. The workshop will conclude with a hands-on EV analysis lab demonstrating methods for standardized, reproducible measurement of EV size, concentration and cargo by flow cytometry.

Exosomal Carboxypeptidase E Confers and CPE-shRNA Loaded Exosomes Inhibit Tumorigenesis
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

Carboxypeptidase E (CPE) has many extracellular non-enzymatic functions including its ability to promote cancer growth and metastasis. Exosomes carry biomolecules (proteins, DNA, mRNA and miRNA) unique to their cell of origin and deliver them to recipient target cells thereby mediating cell-cell communication. We have explored the ability of exosomes from high metastatic HCCH (liver cancer) cells to confer growth and metastatic properties to HCCL (low metastatic). Exosomes were isolated from the supernatant culture media of cancer cells and a correlation was found between elevated CPE mRNA levels in exosomes from high vs low metastatic cell lines across various cancer types. Content analysis of exosomes derived from highly metastatic HCC97H cells revealed CPE-WT mRNA and protein. We showed that exosomes released from HCC97H cells were able to enhance invasion of HCC cells with poor metastatic ability (HCC97L) in Matrigel invasion assay and proliferation in MTT assay. However, when CPE expression was suppressed in the HCC97H cells before exosome isolation, the exosomes had no effect on proliferation and invasion. These data demonstrate the ability of exosomes to confer metastasis in cancer cells and the role of exosomal CPE in driving the process. We then utilized the inherent property of exosomes to act as efficient delivery tools to carry a therapeutic agent such as shRNA. Previously it was shown that down-regulation of CPE expression by shRNA can reverse tumor growth and metastasis in an HCC mouse model. We therefore loaded CPE-shRNA into exosomes by infecting HEK293 (Human Embryonic Kidney) cells with adenovirus carrying CPE shRNA-GFP. These modified exosomes were harvested from the cell medium, purified and then used to transfer CPE-shRNA to HCC97H cells. The exosomes taken up by the recipient cells resulted in significant reduction of CPE mRNA levels and decrease in colony formation of these cells. Thus, these studies demonstrate the ability of exosomal CPE to enhance invasion in low metastatic HCC cells and the potential to use shRNA loaded exosomes to target CPE as a therapeutic strategy to treat liver and other cancers.

EV Engineering For Biomedical Applications
Samir EL-Andaloussi, Associate Professor, Karolinska Institutet, Sweden
Joel Nordin, Research Fellow, Karolinska Institutet, Sweden

The presentation will describe work-flows for scalable purification of bioengineered EVs, down-stream vesicular analytics and application of such EVs in therapeutic contexts.

Capturing Heterogeneous Circulating Tumor Cells in Breast and Lung Cancer Using Nanotube-CTC-Chip
Balaji Panchapakesan, Professor, Department of Mechanical Engineering, Worcester Polytechnic Institute (WPI), United States of America

The ability to detect cancer and its heterogeneity non-invasively in the earliest of stages can make a significant clinical impact. Therefore, biopsy of blood and other bodily fluids also called “liquid biopsy” is an emerging concept for non-invasive detection and capture of circulating tumor cells. Circulating biomarkers is a rapidly growing field of research. Circulating biomarkers include, circulating tumor cells (CTCs), exosomes, circulating tumor DNA (ctDNA), cell-free DNA and RNA (mRNA, miRNA, non-coding RNA, and other RNAs), circulating proteins and metabolites. However, the clinical impacts of liquid biopsy are yet to be realized. We describe the nanotube-CTC-chip for capture and analysis of heterogeneous circulating tumor cells. Using a micro-array technology, well established in the clinical domain, and utilizing nanosurfaces, we demonstrate the capture and isolation of invasive CTCs and CTCs of multiple phenotypes in breast and lung cancer patients.

Integrated Microfluidic Systems for the Efficient Isolation of Circulating Leukemia Cells and Circulating Plasma Cells
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America
Malgorzata Witek, Associate Research Professor, The University of Kansas, United States of America

Liquid biopsies are generating great interest within the biomedical community due to the simplicity for securing important biomarkers to manage complex diseases. We are developing a suite of microfluidic devices that can process whole blood directly and engineered to efficiently search for a variety of disease-associated liquid biopsy markers and their subsequent molecular analysis. One microfluidic device can isolate targets with recovery >90% and high purity (>80%) to enable downstream analysis of the particular biomarker without requiring single cell picking. We have also developed a microfluidic device for imaging single cells. The aforementioned microfluidic devices can be interfaced to a fluidic motherboard to allow for full process automation, including cell selection from blood and then, immunophenotyping and/or FISH of the isolated cells. In this presentation, information will be shared on the operational parameters of these devices for the selection of liquid biopsy markers, and the downstream molecular information that can be garnered from the isolated markers in acute myeloid / acute lymphoblastic leukemia (circulating leukemia cells) and multiple myeloma (circulating plasma cells). The attractive nature of using liquid biopsy markers for these two diseases is that it circumvents the need for a patient to undergo a painful bone marrow biopsy. Information will be provided as to the use of these liquid biopsy markers to monitor relapse from minimum residual disease, and stage patients for directing therapy.

Linking Pain-Regulated Exosomal-miRNAs to Target Expression in the Brain
Natasha Sosanya, Staff Scientist, USAISR, United States of America

Nerve injury in male rats results in differential expression (DE) of circulating exosomal-miRNAs (ex-miRNAs). The DE ex-miRNAs affect target expression and signaling pathways that contribute to the development of neuropathic pain.

A Platform for Detecting Tumor-Derived Extracellular Vesicles
Lydia Sohn, Almy C. Maynard and Agnes Offield Maynard Chair in Mechanical Engineering, University of California-Berkeley, United States of America
Thomas Carey, Graduate Student Researcher, UC Berkeley, United States of America

Close of Day 2 of the Conference