Circulating Biomarkers, Exosomes & Liquid Biopsy Asia 2019 Agenda

Chairman's Welcome and Introduction to the Conference
Yong Song Gho, Professor, Pohang University of Science And Technology (POSTECH), Korea South

Chairperson's Welcome and Introduction to the Conference
Chulhee Choi, Professor and Chair, BioMedical Imaging Center, Korea Advanced Institute of Science and Technology (KAIST), President, ILIAS Biologics Incorporated, Korea South

Emerging Applications of Extracellular Vesicles in Medicine
Jan Lötvall, Professor, University of Gothenburg; Founding President of ISEV; Editor-in-Chief, Journal of Extracellular Vesicles, Sweden

Extracellular vesicles (EVs) are released by all cells in the human body, and can reflect the status and function of the releasing cell. Further, EVs can shuttle cargo molecules from one cell to another, including proteins and RNA. The human body is exceptionally rich in EVs, and it has over the last ten years been acknowledge that the phenotype of these vesicles change in disease, and that EVs indeed can be utilized for diagnostic purposes, for example in cancer. Further, the functionality of EVs can in multiple ways be harnessed for the development of future biological medications. This presentation will discuss the diversity of EVs isolated from human tissues, and will also touch upon the opportunities of developing EVs as powerful therapeutics.

Plasma Extracellular Vesicles as a Biomarker Source for Cardiovascular Disease
Dominique PV de Kleijn, Professor Experimental Vascular Surgery, Professor Netherlands Heart Institute, University Medical Center Utrecht, The Netherlands, Netherlands

Cardiovascular Disease (CVD) is with cardiovascular events of Ischemic Heart Disease (IHD) and Stroke, the number 1 and 2 cause of death in the world and expect to increase especially in Asia. We use protein signatures measured in 3 different subsets of plasma extracellular vesicle (EV) as an accurate source for early diagnosis and prediction of cardiovascular events like IHD and Stroke.

The diagnosis IHD is challenging, as many patients present with atypical symptoms. It is known that women have a different symptom sensation than men. Troponins are the main diagnostic tool for detection of MI. Blood biomarkers for stable angina (SA) and unstable angina (UA), however, are not available. These diagnoses frequently require hospital visits/admissions for time-consuming and costly (non)invasive tests. Using a simplified method that is suitable for automation and microfluidics to isolate the plasma EV subfractions in 25 ul plasma, we now validated our data in a large case control study (150 cases vs 300 controls) that showed that  EV subfractions can diagnose accurately SA in a drop of blood. Next to this, using another signature in plasma EV subfractions for patient that undergo a carotid endatherectomy (CEA) we could accurately determine which patient will get a second cardiovascular event (myocardial infarction, stroke or death) or not within 3 years after CEA.

Plasma EV subfractions  are a very valuable biomarker source for the diagnosis and prognosis of cardiovascular disease.

Immunomodulatory MSC Exosome and Small Extracellular Vesicles
Sai Kiang Lim, Research Director, Institute of Medical Biology, A*STAR, Singapore

Mesenchymal stromal cell (MSC) are the most clinically tested cell type for regenerative medicine.  Despite much setbacks, MSC is now gaining traction as a safe. viable regenerative medicine with recent market approval for use in GVHD and Crohn’s disease. However, it is increasingly clear that small extracellular vesicles (sEVs) of 50 to 200 nm diameter such as exosomes mediate the therapeutic potency of MSCs.  Since the first demonstration that MSC secretes cardioprotective sEVs in a mouse and pig models of myocardial ischemia/reperfusion injury, these sEVs were widely reported to therapeutically efficacious against at least 30 other indications.  A common observation in many MSC sEV-mediated efficacies is a significant immunomodulatory effect.  This talk will describe our studies on the immunomodulatory potential of MSC sEVs.

Microfluidics Technologies for Cancer Diagnosis - From Cell Capture to Single Cell Analysis
Chwee Teck Lim, NUS Society Chair Professor, Department of Biomedical Engineering, Institute for Health Innovation & Technology (iHealthtech), Mechanobiology Institute, National University of Singapore, Singapore

The presence of Circulating Tumor Cells (CTCs) in bloodstream of patients is an important intermediate step in cancer metastasis and can provide valuable insights into disease detection and personalized treatment. As compared to obtaining a tissue biopsy which is invasive and painful, ‘‘liquid biopsy’’ for CTCs detection can be easily performed via a routine blood draw.  The presence and number of CTCs in peripheral blood has been associated with the severity of the disease and have potential uses for early detection, diagnosis, prognosis and treatment monitoring. However, the presence of the large number of blood cells complicates as well as makes detection of rare CTCs in blood of cancer patients extremely challenging. Here, we address these issues and demonstrate that physical biomarkers such as the unique size and deformability of CTCs can be effectively used for their detection and separation from blood via microfluidics.   We do this by leveraging on the many inherent advantages of microfluidics such as high sensitivity and spatial resolution, short processing time and low device cost. We developed a suite of microfluidic biochips that exploit the principles of cell size/deformability based separation as well as inertial focusing to perform high throughput continuous detection and separation of diseased cells.  These simple, efficient and cost effective microfluidic platforms will be invaluable in extracting viable CTCs for many downstream clinical applications including single cell analysis as well as personalized treatment.

Innovative Microfluidic Isolation of Circulating Biomarkers: cfDNA and Exosomes
Sehyun Shin, Professor & Director, Nano-Biofluignostic Engineering Research Center, Korea University and Anam/Guro Hospital of Korea University, Korea South

Liquid biopsy, non-invasively analyzing biomarkers circulating in various body fluids, has been emphasized as a promising technology to accelerate the realization of personalized treatment of cancer. Among the circulating biomarkers, cell-free DNA (cfDNA) and exosome have been implicated as important biomarkers in cancer management. Efficient techniques for isolation of such biomarkers from blood are prerequisites for precision liquid biopsy. Also, a sample-to-answer system for such biomarker extraction is highly required for clinical application. Thus, in the present study, two of technologies (PIBEX and PLL clustering) to isolate main circulating biomarkers (cfDNA and exosome) of liquid biopsy are presented. First, the PIBEX system is a centrifugation-free cfDNA extraction microfluidic chip capable of extracting cfDNA from plasma samples through microfluidic circuits within 15 min under vacuum pressure using an immiscible solvent. Second, the PLL clustering method is the microfluidic chip capable of isolating exosomes from plasma samples within an hour under vacuum pressure using a cationic polymer solution. These chips effectively eliminates the repetitive centrifugation processes and dramatically shortened the sample preparation time. The proposed microfluidic platform could facilitate the development of a sample-to-answer system for use in liquid biopsy of cancers.

Identification of Different Subpopulations of Circulating Tumor Cells in the Blood of Localized and Metastatic Cancer Patients Using Microfluidics
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

Liquid biopsies are becoming an attractive source of biomarkers that can be used to manage a variety of cancer-related diseases. One of the principle biomarkers for oncology-related diseases found in blood is circulating tumor cells (CTCs). The challenge associated with using CTCs as biomarkers for many cancer-related diseases has been the modest clinical sensitivity demonstrated using the FDA-approved platform. CTCs expressing invasive phenotypes down-regulate epithelial antigens, such as the epithelial cell adhesion molecule – EpCAM, which is typically used for the affinity selection of CTCs. As such, CTCs may have a continuum of phenotypes and thus, a single selection marker may not address all cells comprising the tumor microenvironment. We have developed a CTC selection strategy that employs two polymeric microfluidic chips modified with antibodies and connected in series with each selecting a distinct CTC subpopulation from a single blood sample. In addition to the common marker used for CTC positive selection (EpCAM), Fibroblast Activation Protein alpha (FAPa) expressing CTCs can also be selected. Using the dual selection strategy, both CTC types were detected from patients with clinical sensitivity that showed significant improvement compared to selection in which only EpCAM was used. Approximately 90% of the selected CTCs were found not to co-express both antigens. Due to the high purity (>80%) and clinical yields (>95% recovery) of the dual selection strategy, molecular analysis of both EpCAM and FAP-alpha CTCs could be carried out including next generation sequencing, and droplet digital PCR. Our results suggest FAP-alpha and EpCAM CTCs can be used in concert to guide therapeutic decisions for cancer patients. In this presentation, I will discuss the microfluidic chips we use for the selection of CTCs, the clinical results secured using these chips, and the molecular profiling of both CTC subpopulations.

Circulating EV-miRNAs For a Novel Diagnosis and Early Detection of Cancer
Takahiro Ochiya, Professor, Tokyo Medical University, Japan

Currently, it was discovered that extracellular miRNAs circulate in the blood of both healthy and diseased patients. Most of the circulating miRNAs are included in protein, lipid or lipoprotein complexes, such as RNA-binding proteins, apoptotic bodies, extracellular vesicles (EVs) named as exosomes, and are, therefore, highly stable. The existence of circulating miRNAs and exosomes in the blood of cancer patients has raised the possibility that disease-specific miRNAs and exosomes may serve as a novel diagnostic marker for early detection of cancer and monitoring for cancer development. Here we present our current progress on our project for miRNA-based novel cancer diagnosis in Japan.

Lab-on-a-Disc for Liquid Biopsy
Yoon-Kyoung Cho, Professor, Biomedical Engineering, Ulsan National Institute of Science & Technology; Group leader, IBS; FRSC, Fellow of Royal Society of Chemistry, Korea South

The lab-on-a-disc systems to isolate and detect liquid biopsy markers such as circulating tumor cells (CTCs) and cell-free DNA(cfDNA) or Extracellular vesicles (EVs) are developed and tested with clinical samples such as whole blood or urine from cancer patients. First, we will introduce the fluid-assisted separation technology (FAST), which enables ultrafast, uniform, clog-free, and highly efficient isolation of CTCs from whole blood with pressure drop much less than in conventional filtration. In addition, we demonstrate a fully automated cfDNA enrichment device integrating the plasma separation, sample lysis, DNA binding and elution on a single device. We used the lab-on-a-disc to isolate cfDNA from patients with non-small cell lung cancer and successfully detected epidermal growth factor receptor gene mutations (L858R, T790M) during targeted drug therapy. Next, we will present a rapid, label-free, and highly sensitive method for EVs isolation and quantification using a lab-on-a-disc integrated with nanofilters (Exodisc). Urinary EVs from prostate cancer patients could be automatically enriched within 30 min using a tabletop-sized centrifugal microfluidic system followed by molecular analysis or on-chip ELISA. As a proof of concept study, we analyzed androgen receptor splice variant 7 (AR-V7) which is associated with castration-resistant prostate cancer (CRPC) and resistance to anti-androgen therapy in the RNA isolated from the urine-derived extracellular vesicles (EVs) without the need for blood withdrawal. Further, the device also facilitates temporal monitoring of tumor progression within live mouse xenograft models over a period of 13 weeks whilst using minimal volume of weekly collected blood samples. Taken together, Exodisc is a powerful tool to provide a rapid, sensitive, and point-of-care-type method for extracting intact EVs from urine or small volumes of blood samples for disease diagnosis and monitoring. We believe that this revolutionary method can contribute to accelerate the acceptance of liquid biopsy-based cancer diagnostics as a standard practice in clinical settings.

An Integrated Microfluidic Device for Selective Isolation and Detection of Cancer-specific Exosomes
Hyo-Il Jung, Professor, Yonsei University, Korea South

Intercellular communications can be accomplished by means of extracellular vesicles (EVs) responsible for transferring biological cargo such as lipids, proteins, functional messenger RNAs, microRNAs, and double-stranded DNA. One of the EVs, exosome, is the size of 30-150 nm membrane vesicles released from both normal and cancer cells. Therefore the isolation and detection of exosomes are of great importance in the liquid biopsy-based cancer diagnosis and monitoring of treatment response as the functionality of the exosomes reflects the cancer progression and metastasis. There have been a myriad of researches on the microfluidic devices for the probing tool and some of them demonstrated a breakthrough to replace the time-consuming and tedious steps of ultracentrifugation in purifying exosomes. Here I will describe the microfluidic devices for the isolation and detection of cancer-specific exosomes and then discuss the critical concerns and future directions in the field of exosome work.

Protein Content of Extracellular Vesicles as Biomarker in Cancer
Andreas Möller, Associate Professor, Group Leader and Head, Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Australia

Despite significant therapeutic advances, cancer remains a leading cause of death worldwide. A significant clinical problem is the generally late discovery of a cancer and the uncertainty of choosing the most effective therapy for the individual patient. Several novel biomarkers are proposed, ranging from genetic and genomic evaluations of the cancer or the cancer material in circulation to assessing nucleic acids, proteins or lipids. Accurate cancer biomarkers, in particular non-invasive liquid biomarkers based on blood samples or other body fluids, will allow clinicians to identify cancer patients early, triage them to the most appropriate intervention and follow the response of the cancer in real time over the course of the therapy.

In this presentation, I will share data on a novel biomarker for Non-Small-Cell Lung Cancer (NSCLC). We have developed a blood-based multi-protein signature capable of accurately prognosticating clinical outcome in NSCLC patient cohorts. This signature is contained in small circulating nano-vesicles termed exosomes. Overall, this work describes a novel prognostic biomarker signature to identify early stage NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalized adjuvant treatment decisions.

Extracellular Vesicles (EVs)-derived exRNA as Liquid biopsy Using NGS
Hidetoshi Tahara, Deputy Executive Director, Industry-Academia Collaboration; Professor, Department of Cellular and Molecular Biology; Head, The Research Center for Drug development and Biomarker Discovery, Hiroshima University, Japan

MicroRNAs are small non-coding genes that regulate numerous pathways through targeting the number of mRNAs. Dysregulation of miRNAs including genomic alteration, DNA methylation, transcriptional regulation and abnormal miRNA biogenesis are reported in most cancers. miRNA also found in body fluids including blood, salvia and urea, as important sources for early detection of cancer. Therefore, circulating microRNA may serve as novel diagnostic tools to detect early cancer as well as other diseases. We examined the expression of microRNA using serum/plasma samples obtained from pancreatic cancer patients, head and neck cancer patients hepatic carcinoma cell patients compared with healthy controls. To identify significant biomarker from body fluids, proper normalization strategy is of critical importance. We used microRNA array, qRT-PCR and next generation sequencing (NGS) platform to analyze microRNA, and found that appropriate normalized microRNAs in each platform. We identified a number of biomarker candidates such as mature miRNAs, isomiR, tRNA-derived fragments (tRFs), and other ncRNA, are known to regulate expression of genes involved in cell metabolism and are released into body fluid from various cells with extracellular vesicles. To test the possibilities that these candidate small RNAs are secreted from cancer cells, we purified EVs from serum in health volunteer and cancer patients, and analyzed small RNAs using NGS, and found that some of candidate small RNAs can be found in EVs. Interestingly, these small RNAs are also found in culture media in breast cancer cell lines. Based on our alogism, we identified significant biomarker secreted from cancer cells for early detection of cancer.

Extracellular Vesicles and Mimetic Technologies for Theranostics
Yong Song Gho, Professor, Pohang University of Science And Technology (POSTECH), Korea South

Communication between cells and environment is an essential process in living organisms. The secretion of extracellular vesicles is a universal cellular process occurring from simple organisms to complex multicellular organisms, including humans. Throughout evolution, both prokaryotic and eukaryotic cells have adapted to manipulate extracellular vesicles for intercellular communication via outer membrane vesicles in the case of Gram-negative bacteria and ectosomes (also known as microvesicles) or exosomes in eukaryotic cells. Recent progress in this area has revealed that extracellular vesicles play multiple roles in intercellular and interspecies communication, suggesting that extracellular vesicles are NanoCosmos, i.e., extracellular organelles that play diverse roles in intercellular communication (http://evpedia.info). This presentation will briefly introduce the state-of-art extracellular vesicle research and focuses on our recent progress in novel extracellular vesicle-mimetic technologies for targeted drug delivery, theranostics, and epigenetic reprogramming as well as for adjuvant-free, non-toxic vaccine delivery system against bacterial infection. Future research directions on our extracellular vesicle isolation technology ‘ExoLutE’ and bacterial extracellular vesicles-based cancer immunotherapy for basic researches and clinical application will be briefly introduced.

Exosome-based Delivery of Therapeutic Proteins: Principles and Applications
Chulhee Choi, Professor and Chair, BioMedical Imaging Center, Korea Advanced Institute of Science and Technology (KAIST), President, ILIAS Biologics Incorporated, Korea South

Our group has recently developed an opto-genetically engineered exosome system, named ‘exosomes for protein loading via optically reversible protein–protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol of target cells via controlled, reversible protein–protein interactions (PPI). By integrating a reversible PPI module controlled by specific wavelength of light with the endogenous process of exosome biogenesis, cargo proteins of our interest can be loaded into the exosomes. Engineered exosomes were shown to significantly increase intracellular levels of cargo proteins and their function in the recipient cells in both a time- and dose-dependent manner. In this presentation, I will introduce the basic principles of EXPLOR technology and follow-up studies for therapeutic applications focusing on novel anti-inflammatory exosomes in various disease models.