A New Method for Isolation of Interstitial Fluid from Human Solid Tumors Applied to Proteomic Analysis of Ovarian Carcinoma Tissue Hanne Haslene-Hox 1 , Eystein Oveland 1 , Kaja C. Berg 1 , Odd Kolmannskog 1 , Kathrine Woie 2 , Helga B. Salvesen 2,3 , Olav Tenstad 1. , Helge Wiig 1. * 1 Department of Biomedicine, University of Bergen, Bergen, Norway, 2 Department of Obstetrics and Gynecology, Haukeland University Hospital, Bergen, Norway, 3 Department of Clinical Medicine, University of Bergen, Bergen, Norway Abstract Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample’s origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na + , two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods. Citation: Haslene-Hox H, Oveland E, Berg KC, Kolmannskog O, Woie K, et al. (2011) A New Method for Isolation of Interstitial Fluid from Human Solid Tumors Applied to Proteomic Analysis of Ovarian Carcinoma Tissue. PLoS ONE 6(4): e19217. doi:10.1371/journal.pone.0019217 Editor: Yihai Cao, Karolinska Institutet, Sweden Received October 26, 2010; Accepted March 30, 2011; Published April 26, 2011 Copyright: ß 2011 Haslene-Hox et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors gratefully acknowledge grants from The Research Council of Norway, The Norwegian Cancer Association and The Western Norway Regional Health Authority. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: wiig@biomed.uib.no . These authors contributed equally to this work. Introduction Significant efforts have been invested in the search for biological disease indicators in plasma, for diagnostic and prognostic purposes [1]. However, the high range of protein concentrations in plasma is a major obstacle in biomarker discovery using proteomics [2]. Many proteins suggested as biomarkers are relatively abundant and related to non-specific global reactions to the disease resulting in low sensitivity and specificity, thus most candidates are never translated into clinical use [3,4]. Furthermore, proteins secreted from tumor cells and shed membrane proteins will have several orders of magnitude higher concentration in the tumor extracellular or interstitial microenvironment compared to plasma [5]. In the search for tumor specific biomarkers the focus should accord- ingly be on the tumor interstitial environment and the secretome and thus in the fluid phase bathing the tumor cells and the extracellular matrix elements, i.e. in the tumor interstitial fluid (TIF) [6]. To our knowledge there is, however, no technique available whereby native interstitial fluid can be isolated from solid human tumors. Microdialysis is a technique frequently used to access the interstitial space in experimental animals and humans in vivo, but because of low recovery of macromolecules, fluid isolated with this technique will not reflect the protein composition of native interstitial fluid [7,8,9]. Attempts have been made to isolate TIF ex vivo and to apply this fluid as substrate for proteomic analysis [6,10,11,12]. Little evidence has, however, been presented that such fluid originates solely from the interstitial fluid phase. Admixture of intracellular proteins in the isolated TIF will result in identification of proteins that will not be secreted and may thus be erroneously identified as biomarker candidates. By isolating native or undisturbed TIF without causing cellular damage, the sample will be a pre-sorted selection of proteins with character- istics that are required for proteins to be used as biomarkers (i.e. produced in the tumor), making detection of clinically relevant biomarker candidates more likely. As pointed out in a recent review [9], access to native fluid will enable us to address a wide range of questions regarding the tumor microenvironment and tumor biology in general. As an example, we may measure interstitial fluid colloid osmotic pressure (COP), one of the determinants of transcapillary fluid exchange that also PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19217