Matrix Matters: Biomarker Potentials of Phagocytes, Exosomes, and Cytokines

Abstract

Overview: The Applied Biosciences Professional Science Masters (ABS-PSM) program at the University of Arizona prepares students in the fields of biological science to enter areas of business and scientific competition. This interdisciplinary course of study involves the completion of an internship wherein students demonstrate scientific inquiry in the context of the goals and economic pursuits of the hosting agency. Students are required to convey how their projects contribute to the ambitions of the company or academic institution as well as to the larger scientific field. In this report, two internship projects completed to fulfill this requirement for the ABS-PSM degree will be described. Biomedical research and diagnostics rely heavily on the use of biomarkers for drug discovery and disease management. Biomarkers are “characteristics that are objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to therapeutic interventions,” as defined by the National Institutes of Health Biomarkers Definitions Working Group (2001). Drug development and many facets of clinical diagnostics involve the measurement of a combination of biomarkers to evaluate the status of disease in an individual or their physiological changes following some treatment. In contrast to symptoms, biomarkers are not perceived by the patient but rather are observed from outside the patient (Strimbu & Tavel, 2010). The most well-studied biomarkers, such as troponin for the assessment of cardiac injury (Babuin & Jaffe, 2005), are applied regularly in predicting the incidence or outcome of a disease. This use of biomarkers as clinically meaningful surrogate endpoints is entirely justifiable, but only when such a characteristic has extensively and repeatedly proven predictive of outcome (Strimbu & Tavel, 2010). Additionally, the most logical biomarkers are those directly involved in the pathophysiology of a certain pathway, enabling more accurate interpretations of an individual’s disease status to be made. The evaluation of a biomarker’s potential is challenged by the way in which it is measured. Biological samples are wide in variety, and, aside from ensuring the marker is present in the medium at all, determining what sample type is most compatible with existing instruments and what is most associated with a disease or anatomic site is a complex task. Cardiac troponin, for example, is measured in a peripheral blood sample to assess heart damage. The correlation of this enzyme’s concentration in the blood with heart muscle damage makes it a valuable disease indicator because the enzyme is produced in cardiac tissue and immediately released into the circulation (Antman et al., 1996). Neurodegenerative disease markers are markedly complicated because their presence in conventional fluid samples may not be accurately representative of concentrations in the brain. MSDx, Inc. (Tucson, AZ), a company that develops diagnostic solutions for neurodegenerative diseases, has identified phagocytes as a suitable source of biomarkers for this purpose. These cells naturally concentrate and carry remnants of disease pathology through phagocytosis, thereby preserving intact markers of neurodegeneration from the potentially degradative extracellular environment that can be quantified in a blood sample. In comparing this approach to conventional fluid analyses, the first chapter in this report describes a literature review performed on various potential neurodegenerative disease biomarkers and their alterations relative to controls by sample matrix. A second aspect of this project that will be discussed was an exploration of entities involved in the research of exosomes, a type of extracellular microvesicle capable of carrying proteins between cells.

Cytokine concentrations in the blood also change during various disease processes such as inflammation, making them attractive potential biomarkers. Their use, though, is hindered by variation in normal levels from person to person and an uncertainty as to the optimal sample matrix in which to quantify them. For biomarkers to be identified among cytokines, it is essential to first determine what constitutes “normal” levels of a selection of pathologically relevant cytokines, whether serum or plasma samples should be used, and what collection and processing practices such as anticoagulant should be employed. This is the overarching goal of a group at the University of Arizona BIO5 Institute whose small pilot study will be the focus of the second chapter of this report. The study involved recruiting a cohort of self-identified healthy adult volunteers at specific time intervals and obtaining blood samples for quantifying their cytokines using a commercial multiplex Luminex-based assay. An abbreviated preliminary analysis of the results was then conducted, which concerned the levels of cytokines obtained at different time points and in different sample matrices, supporting a brief evaluation of the benefits and drawbacks of using certain anticoagulants and preparation strategies over others.

Both projects produced exciting findings related to biomarker discovery and measurement. Based on my review, phagocytes seem to be largely ignored in neurodegeneration research, yet represent a promising medium in that they avoid many of the drawbacks associated with measuring central nervous system components outside the brain; as well, they did not appear to be studied elsewhere beyond MSDx nor their potential to carry important biomarkers dismissed. On the other hand, exosomes have been shown to be increasingly studied in neurology with broad potential, but important challenges mostly related to their small size remain. Cytokines, in contrast to such biomarker cargo carriers, are information couriers that may also have biomarker potential. These intercellular messengers mediate inflammatory processes but are in constant production at different degrees depending on one’s immunologic and overall health status. Determining what is normal must first be established and is a major challenge since cytokine levels vary by individual, even in an uninfected state. To advance this process, plasma collected with EDTA as an anticoagulant may be optimal, though serum is attractive due to its widespread use and would help make comparisons with the majority of other cytokine studies possible. Despite its invaluable capability to simultaneously measure multiple cytokines in a single sample, the bead-based assay also has its weaknesses that may be related to the matrix type or antibodies being used. Thus, determining the most appropriate method for sample retrieval, reliable measurement of the marker of interest, and establishment of reference values for novel biomarkers appear to be the most pressing challenges associated with the goals described here.