| Abstract: | Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.Cholangiocarcinoma (CCA),1 which arises from the hepatic bile ducts, is the primary cancer accounting for ∼10–15% of all hepatobiliary malignancies. CCA is categorized by the International Classification of Diseases for Oncology into intrahepatic and extrahepatic forms, the latter including perihilar, hilar, and distal bile duct CCA. The global incidence rate of intrahepatic cholangiocarcinoma (ICC) has increased by 2–6% during recent decades, whereas the annual incidence rate of the more common, extrahepatic form has remained relatively stable (1, 2). The diagnosis of ICC remains particularly challenging because the disease can mimic metastasis to the liver or hepatocellular carcinoma (HCC). The only potentially curative treatment options available at present are surgical. Unfortunately, the majority of patients are diagnosed at an advanced, unresectable stage because of the initially silent clinical characteristics of this malignancy. The prognosis of ICC is therefore devastating with survival of less than 24 months following diagnosis. Although several risk factors have been reported as contributing to the development of the disease, most cases of ICC occur in the absence of known etiological factors (3–5). As a consequence, considerable efforts have been made to identify reliable markers to enable the early detection of biliary cancers and provide new insights into the pathogenesis of this deadly disease (6). Recent studies have focused on the cytokines and growth factors (7, 8) produced by CCA cells as well as on the proteomics analysis of serum and bile (9, 10). Follow-up studies are ongoing to determine the sensitivity and specificity of the markers that have emerged from these investigations.Tumor tissue is undeniably the most appropriate resource to investigate tumor-specific signals. Indeed, the determination of alterations to the protein profiles of ICC may offer opportunities to identify tumor-specific markers. To date, proteomics studies performed using tumor tissues have mainly tried to identify proteins with a differential expression between HCC and ICC to prevent the misclassification of these pathologies (11, 12). Furthermore, these studies, performed on total liver homogenates, were not appropriate to detect proteins with altered expression in tumorous cholangiocytes. Indeed, ICC tumor cells are essentially embedded in an abundant stroma containing inflammatory cells and fibroblasts, which may impair the detection of proteins displaying tumor-specific expression patterns (13). In summary, because no proteomics studies have so far been performed on isolated cholangiocytes, any proteome alterations in the setting of ICC remain a matter of speculation.Laser microdissection (LM) has emerged as a suitable tool to selectively extract cells of interest from their natural environment. This technology has been used extensively to profile global gene expression in purified tumor cells (14–16). It has also been used successfully in experiments designed to discover protein biomarkers through mass spectrometry-based proteomics studies (17–19). However, working with such small samples challenges conventional proteomics techniques in terms of sensitivity and the precision of quantitation.One particularly efficient method for proteome analysis is the so-called accurate mass and time (AMT) tag approach where high performance liquid chromatography (HPLC) and high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS or FTMS) work in synergy to achieve a broad proteome coverage even for small sample amounts (20, 21). A recent report demonstrated the significant advantage of the AMT tag method over more conventional proteomics technologies in enabling significantly broader proteome coverage using the minute protein quantities available from tissue microdissection (22).In the present study, we used an AMT tag strategy to determine the differential protein profile between tumorous and non-tumorous microdissected cholangiocytes to define specific proteome alterations of human ICC. Among proteins with altered expression in ICC, we detected a panel of proteins conforming to potential tumorigenic pathways that could be candidates as therapeutic targets and tumor markers for this lethal disease. |
