Review
Tumor interstitial fluid — A treasure trove of cancer biomarkers

https://doi.org/10.1016/j.bbapap.2013.01.013Get rights and content

Abstract

Tumor interstitial fluid (TIF) is a proximal fluid that, in addition to the set of blood soluble phase-borne proteins, holds a subset of aberrantly externalized components, mainly proteins, released by tumor cells and tumor microenvironment through various mechanisms, which include classical secretion, non-classical secretion, secretion via exosomes and membrane protein shedding. Consequently, the interstitial aqueous phase of solid tumors is a highly promising resource for the discovery of molecules associated with pathological changes in tissues. Firstly, it allows one to delve deeper into the regulatory mechanisms and functions of secretion-related processes in tumor development. Secondly, the anomalous secretion of molecules that is innate to tumors and the tumor microenvironment, being associated with cancer progression, offers a valuable source for biomarker discovery and possible targets for therapeutic intervention. Here we provide an overview of the features of tumor-associated interstitial fluids, based on recent and updated information obtained mainly from our studies of breast cancer. Data from the study of interstitial fluids recovered from several other types of cancer are also discussed. This article is a part of a Special Issue entitled: The Updated Secretome.

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

References (142)

  • J.E. Celis et al.

    Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery

    Mol. Cell. Proteomics

    (2004)
  • J.E. Celis et al.

    Identification of extracellular and intracellular signaling components of the mammary adipose tissue and its interstitial fluid in high risk breast cancer patients: toward dissecting the molecular circuitry of epithelial-adipocyte stromal cell interactions

    Mol. Cell. Proteomics

    (2005)
  • T.H. Wang et al.

    Stress-induced phosphoprotein 1 as a secreted biomarker for human ovarian cancer promotes cancer cell proliferation

    Mol. Cell. Proteomics

    (2010)
  • J.E. Celis et al.

    2D protein electrophoresis: can it be perfected?

    Curr. Opin. Biotechnol.

    (1999)
  • J.E. Celis et al.

    Proteomics in translational cancer research: toward an integrated approach

    Cancer Cell

    (2003)
  • P.H. O'Farrell

    High resolution two-dimensional electrophoresis of proteins

    J. Biol. Chem.

    (1975)
  • H. Wiig et al.

    Interstitial fluid and lymph formation and transport: physiological regulation and roles in inflammation and cancer

    Physiol. Rev.

    (2012)
  • N.E. Sounni et al.

    Targeting the tumor microenvironment for cancer therapy

    Clin. Chem.

    (2013)
  • A. Artacho-Cordón et al.

    Tumor microenvironment and breast cancer progression: a complex scenario

    Cancer Biol. Ther.

    (2012)
  • D. Boral et al.

    Cancer stem cells and niche mircoenvironments

    Front. Biosci.

    (2012)
  • H. Tjalsma et al.

    Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome

    Microbiol. Mol. Biol. Rev.

    (2000)
  • S.M. Hanash et al.

    Mining the plasma proteome for cancer biomarkers

    Nature

    (2008)
  • S. Surinova et al.

    On the development of plasma protein biomarkers

    J. Proteome Res.

    (2011)
  • P. Zhu et al.

    Mass spectrometry of peptides and proteins from human blood

    Mass Spectrom. Rev.

    (2010)
  • N.L. Anderson

    The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum

    Clin. Chem.

    (2010)
  • T. Farrah et al.

    A high-confidence human plasma proteome reference set with estimated concentrations in PeptideAtlas

    Mol. Cell. Proteomics

    (2011)
  • A. Schmidt et al.

    High-accuracy proteome maps of human body fluids

    Genome Biol.

    (2006)
  • W. Yan et al.

    Systematic comparison of the human saliva and plasma proteomes

    Proteomics Clin. Appl.

    (2009)
  • S. Hu et al.

    Human body fluid proteome analysis

    Proteomics

    (2006)
  • S.-M. Ahn et al.

    Body fluid proteomics: prospects for biomarker discovery

    Proteomics Clin. Appl.

    (2007)
  • P.N. Teng et al.

    Advances in proximal fluid proteomics for disease biomarker discovery

    J. Proteome Res.

    (2010)
  • J. Adachi et al.

    The human urinary proteome contains more than 1500 proteins including a large proportion of membrane proteins

    Genome Biol.

    (2006)
  • J. Casado-Vela et al.

    Human urine proteomics: building a list of human urine cancer biomarkers

    Expert. Rev. Proteomics

    (2011)
  • P.G. Zerefos et al.

    Analysis of the urine proteome via a combination of multi-dimensional approaches

    Proteomics

    (2012)
  • S. Roche et al.

    Clinical proteomics of the cerebrospinal fluid: towards the discovery of new biomarkers

    Proteomics Clin. Appl.

    (2008)
  • S. Hu et al.

    Human saliva proteome analysis

    Ann. N. Y. Acad. Sci.

    (2007)
  • J.A. Loo et al.

    Comparative human salivary and plasma proteomes

    J. Dent. Res.

    (2010)
  • A. Zhang et al.

    Salivary proteomics in biomedical research

    Clin. Chim. Acta

    (2012)
  • B. Magi et al.

    Proteome analysis of bronchoalveolar lavage in lung diseases

    Proteomics

    (2006)
  • A.Y. Hui et al.

    A systems biology approach to synovial joint lubrication in health, injury, and disease

    Wiley Interdiscip. Rev. Syst. Biol. Med.

    (2012)
  • Q. Wang et al.

    Identification of a central role for complement in osteoarthritis

    Nat. Med.

    (2011)
  • M.P. Pavlou et al.

    Nipple aspirate fluid proteome of healthy females and patients with breast cancer

    Clin. Chem.

    (2010)
  • H. Alexander et al.

    Proteomic analysis to identify breast cancer biomarkers in nipple aspirate fluid

    Clin. Cancer Res.

    (2004)
  • N. Li et al.

    Characterization of human tear proteome using multiple proteomic analysis techniques

    J. Proteome Res.

    (2005)
  • B.S. Koo et al.

    Comparative analysis of the tear protein expression in blepharitis patients using two-dimensional electrophoresis

    J. Proteome Res.

    (2005)
  • G.A. De Souza et al.

    Identification of 491 proteins in the tear fluid proteome reveals a large number of pro-teases and protease inhibitors

    Genome Biol.

    (2006)
  • A. Kolialexi et al.

    Proteomic analysis of amniotic fluid for the diagnosis of fetal aneuploidies

    Expert. Rev. Proteomics

    (2011)
  • B. Pilch et al.

    Large-scale and high-confidence proteomic analysis of human seminal plasma

    Genome Biol.

    (2006)
  • T. Zhao et al.

    Relative quantitation of proteins in expressed prostatic secretion with a stable isotope labeled secretome standard

    J. Proteome Res.

    (2012)
  • S.J. Li et al.

    Sys-BodyFluid: a systematical database for human body fluid proteome research

    Nucleic Acids Res.

    (2008)
  • Cited by (66)

    • Three-dimensional photonic nitrocellulose for minimally invasive detection of biomarker in tumor interstitial fluid

      2022, Chemical Engineering Journal
      Citation Excerpt :

      Tumor interstitial fluid (TIF), which is the extracellular fluid bathing the tumor and stroma cells, is now considered as a crucial source for identification of biomarkers associated with cancer development and progression [1–3]. As a proximal fluid, TIF contains a subset of highly concentrated components released by tumor cells and their microenvironment via various mechanisms [4]. It is estimated that concentration of cancer biomarkers in TIF can be 1000–1500 times higher than that in blood [5].

    • Beyond liquid biopsy: Toward non-invasive assays for distanced cancer diagnostics in pandemics

      2022, Biosensors and Bioelectronics
      Citation Excerpt :

      Among these, factors which can affect the quality of specimen are: the presence low-concentrated proteins in situ or diluted during the process of fluid recovering; the handling process could damage cells causing a release and contamination by structural or other non-secreted proteins into the extracellular space; an uncontrolled proteolytic activity may occur during isolation procedure. In a detailed review paper, Gromov and co-workers describe methods to obtain TIF from biopsies and they focus their attention on tissue centrifugation at low G-forces and passive elution from fresh tumor specimen (Celis et al., 2004; Gromov et al., 2013). In vivo sampling of interstitial fluid without recurring to surgical biopsy could represent a very invasive method, above all if the tissue under investigation is not easily accessible.

    • Multifunctional hydrogel microsphere with reflection in near-infrared region for in vivo pH monitoring and drug release in tumor microenvironment

      2021, Chemical Engineering Journal
      Citation Excerpt :

      A blue shift of the reflection peak wavelength was observed when the pH of the injected solution changed from 7.4 to 4.5, akin to the trend for the in vitro testing. However, the sensitivity for the in vivo tests was smaller than that for the in vitro tests, which can be probably attributed to dilution and pH buffering capacity of skin interstitial fluid (ISF) [36]. To attenuate the influence of matrix effects and improve accuracy for the in vivo testing, hydrogel microspheres synthesized without the MAA monomer (Fig. S3 in the Supporting Information), therefore having negligible pH response, were used as the control (Fig. S4 in the Supporting Information).

    • A practical guide to the development of microneedle systems – In clinical trials or on the market

      2020, International Journal of Pharmaceutics
      Citation Excerpt :

      It has been shown that 50% of the proteins in the interstitial fluid are not present in the serum, whereas the interstitial fluid contains 83% of the proteins found in the serum (Samant and Prausnitz, 2018). This suggests that interstitial fluid can be a unique source of new biomarkers and an alternative source for blood biomarkers (Gromov et al., 2013; Kool et al., 2007; Muller et al., 2012). A recent study has shown that extracted interstitial fluid is sufficient for transcriptome and proteome analyses and has a profile similar to those of blood and serum (Miller et al., 2018).

    View all citing articles on Scopus

    This article is part of a Special Issue entitled: An Updated Secretome.

    View full text