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In-Depth Proteomic Analysis of Tissue Interstitial Fluid for Hepatocellular Carcinoma Serum Biomarker Discovery

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In-Depth Proteomic Analysis of Tissue Interstitial Fluid for Hepatocellular Carcinoma Serum Biomarker Discovery FULL PAPER British Journal of Cancer (2017) 117, 1676–1684 | doi: 10.1038/bjc.2017.344 Keywords: biomarker; tissue interstitial fluid; hepatocellular carcinoma; proteomics; prognosis In-depth proteomic analysis of tissue interstitial fluid for hepatocellular carcinoma serum biomarker discovery Jian Zhang1, Ning Hao1, Wei Liu2, Min Lu3, Longqin Sun1, Ning Chen1, Miantao Wu4, Xiaohang Zhao5, Baocai Xing2,7, Wei Sun*,1,7 and Fuchu He*,1,6,7 1State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206, China; 2Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Hepato-Pancreato-Biliary Surgery I, Peking University Cancer Hospital and Institute, Beijing 100036, China; 3Department of Pathology, School of Basic Medical Sciences, Peking University, Beijing 100191, China; 4State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China; 5State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China and 6Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China Background: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. New serum biomarkers for HCC screening are needed, especially for alpha-fetoprotein (AFP) negative patients. As a proximal fluid between body fluids and intracellular fluid, tissue interstitial fluid (TIF) is a suitable source for serum biomarker discovery. Methods: Sixteen paired TIF samples from HCC tumour and adjacent non-tumour tissues were analysed by isobaric tags for relative and absolute quantitation (iTRAQ) method. Two proteins were selected for ELISA validation in serum samples. Results: Totally, 3629 proteins were identified and 3357 proteins were quantified in TIF samples. Among them, 232 proteins were significantly upregulated in HCC-TIF and 257 proteins down-regulated. Two overexpressed extracellular matrix proteins, SPARC and thrombospondin-2 (THBS2) were selected for further validation. ELISA result showed that the serum levels of SPARC and THBS2 in HCC patients were both significantly higher than those in healthy controls. The combination of serum SPARC and THBS2 could distinguish HCC (AUC ¼ 0.97, sensitivity ¼ 86%, specificity ¼ 100%) or AFP-negative HCC (AUC ¼ 0.95, sensitivity ¼ 91%, specificity ¼ 93%) from healthy controls. And the combination of serum SPARC and THBS2 could also distinguish HCC patients from benign liver disease patients (AUC ¼ 0.93, sensitivity ¼ 80%, specificity ¼ 94%). In addition, serum THBS2 was found to be a novel independent indicator for poor prognosis of HCC. Conclusions: Novel HCC candidate serum markers were found through in-depth proteomic analysis of TIF, which demonstrated the successful utility of TIF in cancer serum biomarker discovery. Hepatocellular carcinoma (HCC) is one of the most common regions such as Asia (Mittal and El-Serag, 2013). Alpha-fetoprotein malignancies worldwide. The mortality of HCC is high mainly due (AFP) is recommended for HCC screening and surveillance by to the late diagnosis. Screening programs have improved the early many guidelines from Asia such as the Asian Pacific Association detection of HCC (Sherman, 2005), especially in high-incidence for the Study of the Liver (APASL) (Omata et al, 2010) and the *Correspondence: Dr W Sun; E-mail: [email protected] or Professor F He; E-mail: [email protected] 7These authors share co-last authorship. Received 1 March 2017; revised 25 July 2017; accepted 1 September 2017; published online 12 October 2017 r 2017 Cancer Research UK. All rights reserved 0007 – 0920/17 1676 www.bjcancer.com | DOI:10.1038/bjc.2017.344 Hepatocellular carcinoma biomarker discovery BRITISH JOURNAL OF CANCER Japan Society of Hepatology (JSH) (2010). The elevation of AFP is Table 1. Clinical characteristics of HCC patients involved in a risk factor for HCC and can be used to help define patients at iTRAQ experiment risk. However, the sensitivity of AFP is insufficient (Daniele et al, Clinical characteristics HCC group (N ¼ 16) n (%) 2004). Thus, new biomarkers are urgently needed, especially for AFP-negative HCC patients. Age (year) As a kind of body fluid commonly used in the clinic, blood is an o60 13 (81.2%) X ideal source for biomarker discovery. However, identification of 60 3 (18.8%) low-abundance proteins in the blood remains a challenge due to Gender the complexity of the blood proteome. Tissue-based proteomic Male 14 (87.5%) studies have also been carried out for biomarker discovery Female 2 (12.5%) (Kikuchi et al, 2012). But only secreted proteins with dysregulated HBV abundance in blood have the potential for clinical application. How Positive 8 (50.0%) to get the tumour-originated proteins which could be released into Negative 8 (50.0%) the blood is an important issue in cancer biomarker discovery. Tumour size (cm) In this regard, proximal fluids have emerged as a novel source. o5 6 (37.5%) Tissue interstitial fluid (TIF), as the proximal liquid surrounding X5 10 (62.5%) the tissue cells, could reflect the microenvironment alterations Microvascular invasion during tumour development. Compared with serum, TIF contains Positive 1 (7.7%) less high abundant proteins and more suitable for mass spectro- Negative 13 (92.3%) metry analysis (Sun et al, 2010). Besides, the concentration of À 1 proteins secreted or shed from the tumour cells is higher in TIF AFP (ng ml ) than that in serum. Those characteristics make TIF a good source o20 12 (75.0%) 20–100 4 (25.0%) for biomarker discovery. In fact, many groups have proved that TIF could be used for biomarker discovery since Celis’ group (Celis TNM stage et al, 2004) established tissue elution method for TIF preparation. I 7 (43.8%) For example, Teng et al (2011) analysed the TIF of renal cell II 4 (25.0%) III 5 (31.2%) carcinoma (RCC) and validated the elevation of enolase 2 in the serum of RCC patients. Xie et al (2016) identified six serine Abbreviation: HCC ¼ hepatocellular carcinoma. proteases in colon cancer TIF and four of them were validated in serum. Our group (Sun et al, 2010) has established the extraction (Supplementary Table S2). Blood samples were centrifuged at method of liver TIF in previous work. In this study, in order to 2000 g for 5 min at 4 1C within 1 h of collection. The supernatant identify serum markers for AFP-negative HCC patients, paired was further centrifuged at 13 500 g for 15 min. The serum was tumour and adjacent non-tumour TIFs from 16 HCC patients with aliquot and stored at À 80 1C until use. HCC samples were from low serum AFP levels were analysed with four batches of 8-plex Peking University Cancer Hospital. Benign liver disease samples iTRAQ. Bioinformatics analysis showed that proteins involved in were from Peking University Cancer Hospital and Sun Yat-sen extracellular matrix (ECM) organisation were significantly University Cancer Center. Healthy samples were from Cancer enriched in HCC TIF and in one subtype of HCC patients with Hospital of Chinese Academy of Medical Sciences. All patients poor prognosis. The ECM is a collection of extracellular molecules, provided informed consents. The use of human samples was which composed primarily of collagens, laminins and various approved by the Ethics Committees of Peking University Cancer glycoproteins. The ECM proteins, which could regulate inter- Hospital, Sun Yat-sen University Cancer Center and Cancer cellular communication, may play an important role in promoting Hospital of Chinese Academy of Medical Sciences. tumour growth and metastasis. Two ECM-associated proteins, SPARC and thrombospondin-2 (THBS2), were selected for further TIF collection. The TIF was collected according to the protocol validation. SPARC is a secreted protein which may support described before (Sun et al, 2010). In brief, fresh tissues were cut tumour-stroma interactions and contribute to cancer progression into 1–3 mm3 pieces and washed carefully with PBS. After in some certain cancers such as lung cancer (Wong and Sukkar, incubation in a CO2 incubator at 37 1C for 2 h, the samples were 2017). THBS2 is a secreted glycoprotein which may have an centrifuged at 1000 g for 3 min to remove cells and the supernatant oncogenic function in lung adenocarcinoma and breast cancer but was transferred to a new tube for further centrifugation at 2000 g may have an anti-angiogenic role in gastric cancer (Weng et al, for 8 min and then 20 000 g for 30 min at 4 1C. TIF samples were 2016). Our study found that SPARC and THBS2 could be aliquot and stored at À 80 1C. The protein concentration of TIF candidate serum screening markers for HCC, including AFP- was measured using the Bradford assay (Bio-Rad Laboratories, negative HCC. THBS2 was also identified to be a candidate serum Hercules, CA, USA). prognostic marker for HCC. iTRAQ analysis of TIF samples. For each TIF sample, 50 mgof proteins were mixed with 200 ml of UA buffer (8 mol l À 1 urea in 0.1 mol l À 1 Tris/HCl pH 8.5) in Amicon Ultra-0.5 Centrifugal MATERIALS AND METHODS Filter (10 kDa; Millipore Corp, Billerica, MA, USA) and centri- fuged at 14 000 g for 20 min. Then samples were treated with Sample preparation. Paired tumour and non-tumour liver tissues 20 mmol l À 1 dithiothreitol at 37 1C for 4 h. After centrifugation, from 16 HCC patients who underwent hepatectomy were collected 50 mmol l À 1 iodoacetamide was added and incubated for 30 min at for TIF extraction. All the patients were with serum room temperature in the dark. After washing with UA buffer, AFPo100 ng ml À 1, including 12 patients with serum triethylammonium bicarbonate buffer (TEAB) was added and the AFPo20 ng ml À 1.
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