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Proteomic Profiling Identifies Markers for Inflammation-Related Tumor

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Proteomic Profiling Identifies Markers for Inflammation-Related Tumor Drev et al. Clin Proteom (2017) 14:33 DOI 10.1186/s12014-017-9168-7 Clinical Proteomics RESEARCH Open Access Proteomic profling identifes markers for infammation‑related tumor–fbroblast interaction Daniel Drev1†, Andrea Bileck2†, Zeynep N. Erdem1, Thomas Mohr1, Gerald Timelthaler1, Andrea Beer3, Christopher Gerner2*‡ and Brigitte Marian1*‡ Abstract Background: Cancer associated fbroblasts are activated in the tumor microenvironment and contribute to tumor progression, angiogenesis, extracellular matrix remodeling, and infammation. Methods: To identify proteins characteristic for fbroblasts in colorectal cancer we used liquid chromatography-tan- dem mass spectrometry to derive protein abundance from whole-tissue homogenates of human colorectal cancer/ normal mucosa pairs. Alterations of protein levels were determined by two-sided t test with greater than threefold dif- ference and an FDR of < 0.05. Public available datasets were used to predict proteins of stromal origin and link protein with mRNA regulation. Immunohistochemistry confrmed the localization of selected proteins. Results: We identifed a set of 24 proteins associated with infammation, matrix organization, TGFβ receptor signal- ing and angiogenesis mainly originating from the stroma. Most prominent were increased abundance of SerpinB5 in the parenchyme and latent transforming growth factor β-binding protein, thrombospondin-B2, and secreted protein acidic-and-cysteine-rich in the stroma. Extracellular matrix remodeling involved collagens type VIII, XII, XIV, and VI as well as lysyl-oxidase-2. In silico analysis of mRNA levels demonstrated altered expression in the tumor and the adja- cent normal tissue as compared to mucosa of healthy individuals indicating that infammatory activation afected the surrounding tissue. Immunohistochemistry of 26 tumor specimen confrmed upregulation of SerpinB5, thrombos- pondin B2 and secreted protein acidic-and-cysteine-rich. Conclusions: This study demonstrates the feasibility of detecting tumor- and compartment-specifc protein-signa- tures that are functionally meaningful by proteomic profling of whole-tissue extracts together with mining of RNA expression datasets. The results provide the basis for further exploration of infammation-related stromal markers in larger patient cohorts and experimental models. Keywords: Infammation signature, Colorectal cancer, Cancer associated fbroblasts, SPARC, THBS2, Extracellular matrix organization, Proteomic profling *Correspondence: [email protected]; brigitte.marian@ meduniwien.ac.at †Daniel Drev and Andrea Bileck have contributed equally to this work ‡Christopher Gerner and Brigitte Marian have contributed equally and are co-corresponding authors 1 Department of Medicine 1, Institute of Cancer Research and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria 2 Institute of Analytical Chemistry, University of Vienna, Vienna, Austria Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Drev et al. Clin Proteom (2017) 14:33 Page 2 of 16 Background Methods Accumulating evidence powerfully demonstrates that Tissue acquisition reactive fbroblasts are central players in physiologi- Te study was approved by the Ethics Commission of the cal and pathological processes [21, 42, 50, 52]. Activa- Medical University of Vienna (EK 1659/2012). Patients tion patterns consist of alterations in the cytoskeleton, sufering from colorectal cancer who underwent surgery production of growth factors as well as cytokines, mod- at the General Hospital of Vienna were asked for their ulation of the extracellular matrix (ECM) and a migra- informed consent to use part of the resected tissue for tory phenotype. Cancer associated fbroblasts (CAF) analysis of tumor protein markers. Tumor tissue and nor- are activated by growth factors derived from the can- mal mucosa 15 cm or as far as possible from the tumor cer cells (e.g. transforming growth factor β—TGFβ) (control tissue) were excised by the pathologist and and develop a wound healing phenotype [58]. Tey stored at − 80 °C. For protein extraction and LC–MS/MS are also stimulated by the infammatory environment analysis 6 tissue pairs were used, 3 of them from stage II which is a hallmark of cancer [38]. Especially cells of tumors without any sign of invasion (low-stage) and 3 the innate immune system may be pro-tumorigenic, from tumors that had already spread to the lymph-nodes pro-angiogenic and pro-metastatic [23, 32, 60]. Tis or the peritoneum (high-stage). Each tissue was analysed resulted in the description of cancer as a wound that individually and the abundance data pooled for statistical does not heal [25]. In this chronic infammatory envi- analysis. Formalin-fxed parafn-embedded tissue sec- ronment, endothelial cells and fbroblasts have been tions were analyzed by IHC. shown to express cell type-specifc pro-infammatory For immunohistochemistry formalin-fxed parafn- signatures [33, 76] that can be modeled by exposure of embedded tissue sections were obtained from the origi- cells to interleukin 1β (IL1β) in vitro [75]. Comparison nal 6 patients as well as additional 20 tumors (stages II, of fbroblasts obtained from the skin, the lung and the III and IV) and 10 normal mucosa resection margins. bone marrow demonstrated that these signatures are tissue-specifc and that tissue-specifc characteristics Extraction and digestion of proteins are retained in cancer-associated fbroblasts [76]. Frozen tissue samples were incubated in sample bufer In colorectal cancer (CRC) such activated fbroblasts (7.5 M urea, 1.5 M thiourea, 4% CHAPS, 0.05% SDS, have been identifed as a driving force of tumor devel- 100 mM dithiothreitol) for 10 min on ice. Subsequently, opment [14, 41, 52] and both TGFβ and infammation proteins were extracted by means of an ultrasonic stick. are activating factors. High expression of TGFβ is a Protein concentrations were determined using a Brad- well-established characteristic in CRC [18, 64, 83] and ford assay (Bio-Rad-Laboratories, Germany). Tereafter, has been identifed as a marker of poor prognosis [64]. in-solution digestion of proteins was performed with Infammation is already apparent in premalignant lesions trypsin (Roche Diagnostics, Germany), as described derived either from chronic infammatory bowel disease previously [9, 74]. Briefy, 20 µg of protein were concen- giving rise to typically infammation-driven tumors [65] trated on a pre-washed 10 kDa molecular weight cut-of or from upregulation of cyclooxygenase-2 in colonic pol- flter (Pall Austria Filter GmbH, Vienna, Austria). Upon yps [26, 36]. Te infammatory environment has been reduction with dithiothreitol (5 mg/ml dissolved in 8 M shown to support expansion of tumor-initiating cells guanidinium hydrochloride in 50 mM ammonium bicar- by providing eicosanoids and interleukins [7, 40, 88]. bonate bufer, pH 8) and alkylation with iodoacetamide Tese small subpopulations of tumor-initiating cells have (10 mg/ml in 8 M guanidinium hydrochloride in 50 mM been identifed in CRC [28, 63] and were reported to be ammonium bicarbonate bufer), proteins were digested involved in therapy resistance and metastasis [24]. enzymatically overnight at 37 °C using trypsin (Roche Tis paper aims to determine whether a deeper insight Diagnostics, Germany). After digestion of proteins, pep- into the connective tissue processes in CRC and the tide samples were cleaned up using C-18 spin columns contribution of cancer-associated fbroblasts can be (Pierce, Termo Fisher Scientifc), dried and stored until gained by using tissue-proteomics. For this purpose, we further LC–MS/MS analyses. have undertaken a liquid chromatography-tandem mass spectrometry (LC–MS/MS) based proteome analy- LC–MS/MS analysis sis of human CRC tissue specimens and paired normal Samples were reconstituted in 5 µl 30% formic acid con- intestine. We focused on stromal, infammation related taining 10 fmol each of 4 synthetic standard peptides and proteins, verifed selected markers using immunohis- then immediately diluted with 40 µl mobile phase A (98% tochemistry (IHC) and linked those candidates to the H2O, 2% acetonitrile, and 0.1% formic acid). Te synthetic Consensus Molecular Subtype signatures by screening peptides [Glu1-Fribrinopeptide B, EGVNDNEEGFFSAR; publicly available RNA expression datasets. M28, TTPAVLDSDGSYFLYSK; HK0, VLETKSLYVR; Drev et al. Clin Proteom (2017) 14:33 Page 3 of 16 HK1, VLETK(ε-AC)SLYVR] were spiked into each sam- Additionally, raw fles were analyzed using Proteome ple as an internal quality control for monitoring LC–MS Discoverer 1.4 (Termo Fisher Scientifc, Austria) utiliz- instrument stability. Five microliters of the solution were ing Mascot 2.5 (Matrix Science, UK) in order to enable injected into the nano HPLC-system (Dionex Ultimate upload of mass spectrometric data to a publicly avail- 3000) loading peptides on a 2 cm × 75 µm C18 Pep- able repository. Terefore, protein
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