IDENTIFICATION OF NEW POTENTIALLY ACCESSIBLE BIOMARKERS SUITABLE FOR THE DEVELOPMENT OF THE TARGETED THERAPY OF GLIOBLASTOMA MULTIFORME

Background: Glioblastoma multiforme (GBM) is the most aggressive, highly invasive and neurologically destructive among malignant brain tumors. None of the treatments currently used is effective, making GBM lethal within 12 months from diagnosis. Despite major evolution in the understanding of the molecular mechanisms involved during GBM development and progression, patients are yet in need of a successful treatment. Nowadays, one of the most promising approaches aimed to cure cancer consists on the antibody-based therapy. The systemic delivery of specific antibodies, coupled with highly cytotoxic drugs, directed towards the tumor site is considered as a promising way of treatment. Unfortunately, the bottleneck of such approach consists in the identification of antigens accessible by the blood stream. Aim of the work: This work aims to identify and validate in-vivo new biomarkers produced either by the tumor itself or by the surrounding stroma in response to tumoral demand, which are reachable by a systemic administered compound and could be used as therapeutic targets in human GBM. Material and methods: Human GBM specimens, two different human glioblastoma cell lines (U373 and T98G) grafted in nude mice and U87-derived tumors grown in egg choriallantoic membrane (CAM) were biotinilated ex-vivo. Labeled proteins were successively isolated using affinity chromatography based on streptavidin beads. Proteins were eluted and digested with trypsin and the resulting peptides were analyzed using the 2D nano-LC MS/MS technique. Protein identification was carried out using the Mascot® search engine (Matrix Sciences, Boston, MA, USA) and the Swisprot® protein database (Swiss Institute for Bioinformatics, Basel, Switzerland). For the purpose of validation U87-derived tumors were grown on CAM. Intravenous administration of specific monoclonal antibodies into the CAM vessels was carried out in order to validate in-vivo the accessibility of the target. Results and conclusion: expression profiles for each experimental model were determined using the Mud-PIT technique. About 30 to 35% of the total proteome were found to be accessible (membrane associated, extracellular and secreted proteins). Among the proteins identified, the study highlighted the hyaluronan receptor CD44 and tenascin-C (onco-fetal Tenascin) already known to be overexpressed in human GBM along with other new potential targets such as sparc-like 1, prosaposin and collagen 6 α1. The validation phase carried out using immunohistochemical analysis confirmed the overexpression of the proteins in high-grade gliomas. Additionally, in-vivo experiment with the systemic administration of the monoclonal anti-human CD44 and COL6α1 antibodies into CAM vessels resulted in a site-specific tumor accumulation of the antibodies suggesting these proteins as readily accessible target for the treatment of GBM. Taken together, these results demonstrate the potential of the biotinilation technique in searching for potential accessible biomarkers. The study pointed at the usefulness of the CAM system as an alternative model of biomarker validation in comparison to the more cost and labor intense mouse model. Further investigations focusing on the development of antibody-based treatment of tumor bearing animals are the next step to envision before proposing these targets for clinical trials on humans

Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma

Pancreas ductal adenocarcinoma (PDAC) remains a deadly malignancy with poor early diagnostic and no effective therapy. Although several proteomic studies have performed comparative analysis between normal and malignant tissues, there is a lack of clear characterization of proteins that could be of clinical value. Systemically reachable ("potentially accessible") proteins, suitable for imaging technologies and targeted therapies represent a major group of interest. The current study explores potentially accessible proteins overexpressed in PDAC, employing innovative proteomics technologies. In the discovery phase, potentially accessible proteins from fresh human normal and PDAC tissues were ex vivo biotinylated, isolated and identified using 2D-nano-HPLC-MS/MS method. The analysis revealed 422 up-regulated proteins in the tumor, of which 83 (including protein isoforms) were evaluated as potentially accessible. Eleven selected candidates were further confirmed as up-regulated using Western blot and multiple reaction monitoring protein quantification. Of these, transforming growth factor beta-induced (TGFBI), latent transforming growth factor beta binding 2 (LTBP2), and asporin (ASPN) were further investigated by employing large scale immunohistochemistry-based validations. They were found to be significantly expressed in a large group of clinical PDAC samples compared to corresponding normal and inflammatory tissues. In conclusion, TGFBI, LTBP2, and ASPN are novel, overexpressed, and potentially accessible proteins in human PDAC. They bear the potential to be of clinical value for diagnostic and therapeutic applications and merit further studies using in vivo models. Peer reviewe

Proteomic analysis of human pancreas cancers for the identification of targetable biomarkers

peer reviewe

Restauro della chiesa di san Giovanni Battista - Disvetro di Cavezzo (Mo)

La chiesa ad oggi si presenta senza coperture: per il terremoto sono crollati il tetto, la volta a botte della navata, i muri del claristorio, parte della facciata e il campanile. L’intervento prevede l’ancoraggio alle murature superstiti riconsolidate di una struttura in legno lamellare composta da capriate controventate a cui si agganciano il tetto, i nuovi pannelli del claristorio, e una sottostruttura composta da archi in legno che vanno a ricreare l’invaso spaziale prodotto dalla vecchia volta a botte, ma lasciano passare la luce e intravedere la struttura oltre gli archi. Le nuove pareti del claristorio sono pensate in pannelli lignei all’interno e lamine longitudinali di zinco-titanio all’esterno. Le aperture verticali sono poste in corrispondenza delle vecchie lesene. La facciata della chiesa deve essere ricostruita e reintegrata cromaticamente in modo che sia distinguibile l’intervento. Il campanile è stato progettato con una struttura in legno alta come l’originale, impernata nella muratura residua. Possiede la stessa finitura del claristorio, ad eccezione della sommità che è interamente realizzata in rete metallica, riproducendo la forma originale. Questo intervento si presenta come un layer che si sovrappone e coesiste ai resti della chiesa, dichiarandosi come altro e andando a ricostituire la fruibilità e l’originale geometria degli spazi

Novel Relative ICPL Based Quantitative Phospho- and Glycoproteome Analysis Method

Large scale proteomic analysis remains challenging partially because proteins are inhomogeneous and often influenced by a variety of structural modifications. In particular, these specific chemical modifications called posttranslational modifications (PTM) are crucial determinants for the protein function and biological role. Up to now there have been a growing number of studies describing the enrichment and identification of PTM. However, a significant dearth of data offering a reliable methodology for PTM quantification does exist. The present work aims at developing a label based protein PTM quantification strategy and demonstrating its value on comparative analysis of cells originating from two distinct prostate metastasis sites. PC3 and LNCaP cells isolated from bone and lymph node prostate cancer metastasis sites respectively, were lysed and spiked with three non-human proteins serving as internal standards. Following this, the samples were reduced and alkylated, digested with trypsin and subjected to peptide ICPL (isotope coded protein label) labeling. The two peptide containing samples were joined together followed by the affinity isolation of phospho- (using TiO2 metal affinity chromatography) and glycopeptides (oxidized glycans were bound on hydrazide resin). The enriched fraction as well as the flow-through were analyzed on a 2D-(SCX and C18-RP)-nano-HPLC system. The peptide identification and quantification was conducted using electrospray ion-trap mass spectrometer (Bruker, HCT-ultra). Validation of the differentially modulated proteins was conducted in several biological and technical replicates using the label free MSe based quantification strategy. This PTM based, novel relative protein quantification using post-digest ICPL has detected over 598 individual proteins. Of these more than 95 % have been successfully quantified. PTM enrichment methodologies allowed an isolation rate of 91 % and 50 % for phosphorylated and glycosylated proteins respectively. The detailed comparison of PC3 and LNCaP cells has shown specific overexpression of selected proteins indicating differences between these two prostate metastatic cell lines. Several of these modulated proteins have been previously described to be related to prostate cancer (e.g. annexin A2 and vimentin) while others could be considered as potentially novel. These proteins might be implicated in the fundamental process related to metastasis dissemination. However, because of the known discrepancy between cell systems and clinical material, the present study can be regarded only as a step towards elucidation of these complex interactions

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO(2), whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC-MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼600 individual proteins) and quantified proteins (>95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins

Sparc-Like Protein 1 Is a New Marker of Human Glioma Progression

High-grade gliomas (glioblastomas) are the most common and deadly brain tumors in adults, currently with no satisfactory treatment available. Apart from <i>de novo</i> glioblastoma, it is currently accepted that these malignancies mainly progress from lower grade glial tumors. However, the molecular entities governing the progression of gliomas are poorly understood. Extracellular and membrane proteins are key biomolecules found at the cell-to-cell communication interface and hence are a promising proteome subpopulation that could help understand the development of glioma. Accordingly, the current study aims at identifying new protein markers of human glioma progression. For this purpose, we used glial tumors generated orthotopically with T98G and U373 human glioma cells in nude mice. This setup allowed also to discriminate the protein origin, namely, human (tumor) or mouse (host). Extracellular and membrane proteins were selectively purified using biotinylation followed by streptavidin affinity chromatography. Isolated proteins were digested and then identified and quantified employing 2D-nano-HPLC–MS/MS analysis. A total of 23 and 27 up-regulated extracellular and membrane proteins were identified in the T98G and U373 models, respectively. Approximately two-thirds of these were predominantly produced by the tumor, whereas the remaining proteins appeared to be mainly overexpressed by the host tissue. Following extensive validation, we have focused our attention on sparc-like protein 1. This protein was further investigated using immunohistochemistry in a large collection of human glioma samples of different grades. The results showed that sparc-like protein 1 expression correlates with glioma grade, suggesting the possible role for this protein in the progression of this malignancy