Anna Paola Carreca

Lung cancer represents the leading cause of cancer-related mortality worldwide, with around 1.8 million deaths in 2020. For this reason, there is an enormous interest in finding early diagnostic tools and novel therapeutic approaches, one of which is extracellular vesicles (EVs). EVs are nanoscale membranous particles that can carry proteins, lipids, and nucleic acids (DNA and RNA), mediating various biological processes, especially in cell–cell communication. As such, they represent an interesting biomarker for diagnostic analysis that can be performed easily by liquid biopsy. Moreover, their growing dataset shows promising results as drug delivery cargo. The aim of our work is to summarize the recent advances in and possible implications of EVs for early diagnosis and innovative therapies for lung cancer.

Novel DNA binding half Salphen cobalt(iii)complexes with promising anticancer activity on 3D cultured models.

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

Bone is a highly vascularized tissue and relies on the angiogenesis and response of cells in the immediate environmental niche at the defect site for regeneration. Hence, the ability to control angiogenesis and cellular responses during osteogenesis has important implications in tissue-engineered strategies. Self-assembling ionic-complementary peptides have received much interest as they mimic the natural extracellular matrix. Three-dimensional (3D)-printed biphasic calcium phosphate (BCP) scaffolds coated with self-assembling DAR 16-II peptide provide a support template with the ability to recruit and enhance the adhesion of cells. In vitro studies demonstrated prompt the adhesion of both human umbilical vein endothelial cells (HUVEC) and human mesenchymal stem cells (hMSC), favoring endothelial cell activation toward an angiogenic phenotype. The SEM-EDS and protein micro bicinchoninic acid (BCA) assays demonstrated the efficacy of the coating. Whole proteomic analysis of DAR 16-II-treated HUVECs demonstrated the upregulation of proteins involved in cell adhesion (HABP2), migration (AMOTL1), cytoskeletal re-arrangement (SHC1, TMOD2), immuno-modulation (AMBP, MIF), and morphogenesis (COL4A1). In vivo studies using DAR-16-II-coated scaffolds provided an architectural template, promoting cell colonization, osteogenesis, and angiogenesis. In conclusion, DAR 16-II acts as a proactive angiogenic factor when adsorbed onto BCP scaffolds and provides a simple and effective functionalization step to facilitate the translation of tailored 3D-printed BCP scaffolds for clinical applications.

Chondrosarcoma is the second most common bone tumor, accounting for 20% of all cases. Little is known about the pathology and molecular mechanisms involved in the development and in the metastatic process of chondrosarcoma. As a consequence, there are no approved therapies for this tumor and surgical resection is the only treatment currently available. Moreover, there are no available biomarkers for this type of tumor, and chondrosarcoma classification relies on operator-dependent histopathological assessment. Reliable biomarkers of chondrosarcoma are urgently needed, as well as greater understanding of the molecular mechanisms of its development for translational purposes. Hypoxia is a central feature of chondrosarcoma progression. The hypoxic tumor microenvironment of chondrosarcoma triggers a number of cellular events, culminating in increased invasiveness and migratory capability. Herein, we analyzed the effects of chemically-induced hypoxia on the secretome of SW 1353, a human chondrosarcoma cell line, using high-resolution quantitative proteomics. We found that hypoxia induced unconventional protein secretion and the release of proteins associated to exosomes. Among these proteins, which may be used to monitor chondrosarcoma development, we validated the increased secretion in response to hypoxia of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme well-known for its different functional roles in a wide range of tumors. In conclusion, by analyzing the changes induced by hypoxia in the secretome of chondrosarcoma cells, we identified molecular mechanisms that can play a role in chondrosarcoma progression and pinpointed proteins, including GAPDH, that may be developed as potential biomarkers for the diagnosis and therapeutic management of chondrosarcoma.

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.

Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium.

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.