Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
ReviewTumor interstitial fluid — A treasure trove of cancer biomarkers☆
Highlights
► Tumor interstitial fluid (TIF) is a novel source for cancer biomarker discovery. ► Methods for recovering TIF from fresh tissue specimens are critically discussed. ► Multiple proteomic technologies that profiled TIF are discussed. ► The main key studies of TIF proteomes for several types of cancers are overviewed.
Introduction
Interstitial fluid is an extracellular liquid that accumulates inside the tissue and is implicated in the regulation of the local tissue environment thus having a profound influence on the biological and physiological potential of a lesion. During the last two decades, significant advances have been made in our understanding of the role of interstitial fluid in tissue homeostasis, physiology, inflammation, immune regulation, and transport of metabolites between cells, lymphatic fluid and blood (extensively reviewed in Wiig and Swartz [1]). With our growing knowledge of the impact that changes in the microenvironment have in the development of cancer and furthering of tumor progression, interstitial fluid of tumors has become the subject of increasing attention.
The extracellular space is the bioactive site for the majority of growth factors and increased knowledge of protein activation in this compartment is of utmost importance for our comprehension of tumor biology ([2], [3], [4], [5], [6] and references therein). The term “secretome” was initially coined to classify the “proteome” complement that resides outside the cells, released by classical secretion pathways [7]. Today, the term has significantly expanded its scope to encompass the spectrum of secretory machineries by which proteins can be externalized by cells, tissues or organs, and includes classical/non-classical secretion, release by exosomes and cell membrane protein shedding. Tumor growth and progression, as well as the abnormal drainage of fluids into the tumor extracellular space, are associated with increased secretion and shedding of tumor antigens into the extracellular medium as well as with significant changes in cell adhesion and damage of both the tumor mass and the surrounding tissues. Such conditions may result in the leakage of interstitial components into the circulation and/or body cavities and may lead to the elevation of the levels of some relevant molecules in body fluids.
Blood is the most commonly analyzed clinical biospecimen and is recognized as presumably the most important source of disease-related biomarkers available, and as a consequence, the plasma proteome is perhaps the most extensively studied to date [8], [9], [10], [11]. However, the human plasma proteome is possibly also the most complex human proteome [12], [13], since in addition to proteins that do have a systemic function, it also presents proteins that were externalized by cells, because of locoregional functions, as well as proteins leaked to proximal fluids as a result of cell damage or death, or due to disease, all of which are drained by lymph and end up in the blood. As a result, researchers believe that the majority of proteins in the body, at some point in time, will end up in the blood. The complexity and dynamic range of protein abundance (> 10 orders of magnitude) in human plasma have greatly hampered protein biomarker discovery. Potential cancer-related proteins externalized from a specific tissue or cell type, because of a very high dilution ratio, may be present in plasma at very low levels, around 1–10 pg/ml or lower [14], which may not allow detection of these proteins with high accuracy and precision. The usefulness of blood as a source of disease biomarkers, stems from the fact that telltale proteins secreted or shed by diseased tissues into proximal fluids can be transported to the circulation by drainage through lymphatic or capillary system, depending on their native molecular mass. Accordingly, a comparison of human body fluid proteomes of various origins with plasma is expected to provide valuable insight into their physiological significance and an understanding of the unique and overlapping disease diagnostic potential that each fluid provides [15], [16]. As a consequence, body fluids have become an important target for proteomic biomarker research and a rapidly growing number of studies have generated a large amount of body fluid-related protein data ([17], [18], [19] and references therein). A broad variety of body fluids have been examined for their proteome composition, including various types of proximal fluids, such as urine [20], [21], [22], cerebrospinal fluid [23], saliva [24], [25], [26], bronchoalveolar lavage fluid [27], [28], synovial fluid [29], [30], nipple aspirate fluid [31], [32], tear fluid [33], [34], [35], amniotic fluid [36], and seminal plasma [37], [38]. Recently, a comprehensive web-based human body fluid proteome database, Sys-BodyFluid, was developed that contains eleven kinds of body fluid proteomes and more than 10,000 protein entries [39].
Given that the concentration of disease biomarkers in local tumor microenvironment is estimated to be 1000–1500 times higher than in blood [18], lesion-proximal sampling is one popular strategy to enrich for disease biomarkers. In this respect, tumor interstitial fluid (TIF) is a very promising source of biomaterial for marker discovery, since low abundance proteins are enriched in the local tumor space, and the protein profile of any given TIF can be directly compared to that of matched non-malignant interstitial fluid (NIF), harvested from the same patient. A relevant strategy adopted by our laboratory for the identification of biomarkers for early diagnostics is illustrated in Fig. 1. Interstitial fluids recovered from malignant and adjacent nonmalignant lesions are comparatively scrutinize by various proteomic tools, such as 2D PAGE, LC–MS/MS and array-based assays to reveal secreted proteins which are highly presented in TIFs. Finding a protein of potential interest directs onwards to the next challenging step: validation by a large-scale antibody screening of independent sets of tissue microarray. Confirmed targets are subsequently primary objectives for the detection of them in the corresponding proximal body liquids of patient, e.g., in blood. This step may require one to design and test a highly-sensitive assay, aimed specifically at the detection of a given putative biomarker in blood; eventually this should end in a prototype assay development.
Here, we provide an overview of the features of tumor-associated interstitial fluids (TIFs), mainly based on recent and updated information obtained from our studies of breast cancer. Several interesting applications for analysis of TIFs harvested from other types of cancer are also discussed.
Section snippets
Tumor interstitial fluid: origin and features
The importance of the tumor microenvironment in cancer growth and progression is widely accepted, yet the significance of signaling cross-talk between numerous components of tumor and tumor interstitium remains poorly understood. There is an overwhelming evidence that tumor growth and progression are determined not only by the malignant potential of the tumor cells but also by the multidirectional interactions of factors produced by all the cell types forming the local tumor milieu – including
Proteomic profiling of TIF
The high complexity of the proteome in interstitial fluids makes the discovery of biomarkers based on the analysis of this biological material a challenging endeavor at best, requiring the development of standard operating procedures for the preparation and handling of specimens, increased sensitivity for detection and bioinformatics tools for distribution of proteomic data into the public domain [18]. The proteome of TIF is not only complex, as there may be many thousands of proteins,
Breast cancer
Clinically useful biomarkers for early detection of breast cancer could have a significant impact on breast cancer mortality. With the aim to identify abundant cancer up-regulated proteins that were externalized by cells to the tumor microenvironment we have undertaken a detailed comparative proteomic analysis of TIF and normal interstitial fluid (NIF) recovered prospectively, during the last 6 years, from 69 breast cancer patients that underwent mastectomy at the Copenhagen University Hospital
Outlines and prospects
Tumor cell proteins that are secreted and/or shed into the proximal interstitial fluid represent a potentially valuable source of circulating cancer markers for diagnostics and targets for therapeutics. The rationale of this assumption is that TIF proteins readily enter the blood circulation through the lymphatic system; hence, the elevated levels of proteins in TIF also may be detected in peripheral blood and may serve as easily accessible biomarkers. The search for biomarkers within TIF may
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This article is part of a Special Issue entitled: An Updated Secretome.