Intrahepatic cholangiocarcinoma (iCCA) is malignant tumor arising from the bile duct and peribiliary glands of the liver. This cancer is aggressive and with a very poor prognosis: about 50% of patients with untreated disease die within 4 months of presentation. The only curative treatment option is surgery, unfortunately limited to early stage disease. Thus, the early detection of iCCA is crucial to counteract the disease. To date, the diagnosis of iCCA requires multidisciplinary approaches and is still a challenge because of its paucicellular nature, anatomic location, and silent clinical character. Therefore, there is a need for accurate and sensitive circulating markers to distinguish the iCCA in respect to other liver malignancies and to increase the chance of having curative treatment interventions.
iCCA is characterized by a peculiar, dense and reactive desmoplastic stroma that provides the anatomical supporting structure for tumor mass and promotes tumor progression. The iCCA stromal microenvironment is mainly represented by an altered and excessive accumulation of extracellular matrix (ECM) components that arises from the fibrogenic activity of cancer-associated myofibroblasts (CAFs). Particularly, the iCCA-associated desmoplastic reaction requires a dynamic ECM remodeling with continuous degradation and new synthesis and secretion of ECM components by CAFs. Thus, ECM leakage proteins released into the circulation may reflect the state of the iCCA onset and progression and may be the most likely candidates as reliable, specific and non-invasive biomarkers. Supported by preliminary proteomic data showing many iCCA-specific ECM proteins released in the liquid tumor microenvironment, the present proposal aims to develop a panel of circulating biomarkers for the early and accurate tumor detection.
Serum contains a highly complex mixture of low abundant proteins secreted from tissues and organs which systemically reflects the physiological or pathological states of a living organism. Quantitative analysis of these low abundant proteins in serum samples might unveil an unexplored archive of histological information and might provide useful biomarkers for disease detection. In light of this, many scientists have recently developed mass spectrometry-based strategy for the circulating low abundant proteome characterization and for the discovery of tumor biomarkers from small serum samples. However, a reliable, specific and non-invasive biomarkers of iCCA is still not yet available. Therefore, the diagnosis of iCCA is still a challenge that requires multidisciplinary approaches, invasive procedures for patients and with high costs for the national public health system.
It is well established that iCCA leads to changes of the hepatic ECM composition. In fact, during cholangiocarcinogenesis CAFs release abundant amount of new ECM components, thus leading to the formation of a peculiar scaffold characterized by high turnover of ECM proteins released in the tumor liquid microenvironment. Consequently, since the desmoplastic reaction is the most peculiar hallmarks of iCCA, we believe that these proteins resulting from the ECM turnover, if detected in circulation, may be the most likely candidates as biomarkers for iCCA detection. Therefore, driven from robust experimental reports, the feasibility of the presented proposal is further encouraged by two our preliminary results:
i) Our recent proteomic report of the iCCA ECM scaffold molecular composition compared to the adjacent non-tumorous ECM unveiled a peculiar protein expression signature of the tumor desmoplastic stroma. In particular, iCCA tissues were found with high levels of collagen fibers and low abundance of reticular and elastic fibers suggested stiffness and loss of polarity. By proteomics analysis, 50 ECM proteins differently regulated in respect to surround noncancerous tissues indicated the basement membrane dismantling, reduced tumor angiogenesis, and downregulation of the oncosuppressive activity. Overall, this study contributed to the understanding of the molecular bases underlying desmoplasia in iCCA and prompted us to unveil the molecular composition of tumor liquid microenvironment during tumor-induced ECM remodelling.
ii) Recently, we therefore performed a comparative proteomic analysis of soluble proteins flushed out from both the iCCA and NCT specimens obtained from radical surgery of patients. Among the identifications, 12 proteins (11 of them found to be ECM components displaying a functional interaction network involved in the frame of ECM organization) were found highly enriched or exclusively present in the iCCA samples. Therefore, we believe that these proteins, if detected in circulation, may be the most likely candidates as reliable, specific and non-invasive biomarkers of iCCA.
The feasibility of this proposal is further guaranteed by the Principal Investigator, manager of the Proteomics Laboratory of the Sapienza University of Rome. He established the necessary lab space and proteomics equipment of his own independent research group and provides 18 years old experience in the field of proteomics, particularly in nanoLC-MS/MS quantitative applications such as (SILAC, Spike-in SILAC, iTRAQ, 18O labeling), applied to the liver biology. We believe that this proposal is innovative and will improve significantly the management of iCCA. This aspect is particularly relevant for the Italian National System. In fact, in the last ten years, incidence and mortality rate of tumors of the biliary tract show an increase of about 3% per year (data from Associazione Italiana per lo Studio del Fegato). This results in a high impact for the national public health system, particularly for the high cost of current diagnostic tests for the assessment of iCCA. In fact, clinical, laboratory, endoscopic, and radiographic analyses, in addition to be an invasive procedure, require high cost of hospitalizations. In light of all these considerations, we believe that a diagnostic test based on circulating iCCA ECM proteins could offer several advantages in terms of economy, tolerability and monitoring. We believe therefore, that our study will draw a new interest of the scientific community in this research area that may allow defining new innovative strategies for the early detection of diseases, drug susceptibility, and the evaluation of prognosis.