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Papers by Daniela Osti

Journal of Cellular Physiology

Clinical Cancer Research

Purpose: Glioblastoma (GBM) is the most common primary brain tumor. The identification of blood b... more Purpose: Glioblastoma (GBM) is the most common primary brain tumor. The identification of blood biomarkers reflecting the tumor status represents a major unmet need for optimal clinical management of patients with GBM. Their high number in body fluids, their stability, and the presence of many tumor-associated proteins and RNAs make extracellular vesicles potentially optimal biomarkers. Here, we investigated the potential role of plasma extracellular vesicles from patients with GBM for diagnosis and follow-up after treatment and as a prognostic tool. Experimental Design: Plasma from healthy controls (n ¼ 33), patients with GBM (n ¼ 43), and patients with different central nervous system malignancies (n ¼ 25) were collected. Extracellular vesicles were isolated by ultracentrifugation and characterized in terms of morphology by transmission electron microscopy, concentration, and size by nanoparticle tracking analysis, and protein composition by mass spectrometry. An orthotopic mouse model of human GBM confirmed human plasma extracellular vesicle quantifications. Associations between plasma extracellular vesicle concentration and clinicopathologic features of patients with GBM were analyzed. All statistical tests were two-sided. Results: GBM releases heterogeneous extracellular vesicles detectable in plasma. Plasma extracellular vesicle concentration was higher in GBM compared with healthy controls (P < 0.001), brain metastases (P < 0.001), and extra-axial brain tumors (P < 0.001). After surgery, a significant drop in plasma extracellular vesicle concentration was measured (P < 0.001). Plasma extracellular vesicle concentration was also increased in GBM-bearing mice (P < 0.001). Proteomic profiling revealed a GBM-distinctive signature. Conclusions: Higher extracellular vesicle plasma levels may assist in GBM clinical diagnosis: their reduction after GBM resection, their rise at recurrence, and their protein cargo might provide indications about tumor, therapy response, and monitoring.

Nucleic acids research, Jan 4, 2018

Histone post-translational modifications (PTMs) generate a complex combinatorial code that regula... more Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are inste...

Oncotarget, 2014

Glioblastoma (GBM) is maintained by a small subpopulation of tumor-initiating cells (TICs). The a... more Glioblastoma (GBM) is maintained by a small subpopulation of tumor-initiating cells (TICs). The arduous assessment of TIC frequencies challenges the prognostic role of TICs in predicting the clinical outcome in GBM patients. We estimated the TIC frequency in human GBM injecting intracerebrally in mice dissociated cells without any passage in culture. All GBMs contained rare TICsand were tumorigenic in vivo but only 54% of them grew in vitro as neurospheres. We demonstrated that neurosphere formation in vitro did not foretell tumorigenic ability in vivo and frequencies calculated in vitro overestimated the TIC content. Our findings assert the pathological significance of GBM TICs. TIC number correlated positively with tumor incidence and inversely with survival of tumorbearing mice. Stratification of GBM patients according to TIC content revealed that patients with low TIC frequency experienced a trend towards a longer progression free survival. The expression of either putative stem-cell markers or markers associated with different GBM molecular subtypes did not associate with either TIC content or neurosphere formation underlying the limitations of TIC identification based on the expression of some putative stem cell-markers.

J Cell Mol Med, 2004

We have previously demonstrated that the delta isoform of Protein Kinase C (PKCδ) acts as a tumor... more We have previously demonstrated that the delta isoform of Protein Kinase C (PKCδ) acts as a tumor suppressor in HCT116 human colon cancer cells, and that p21 waf1/cip1 is an essential downstream effector of PKCδ. Our data suggested that p53 might also be involved in the suppression of the neoplastic phenotype induced by PKCδ. Here we show that homozygous knockout of p53 renders the HCT116 cell line unresponsive to PKCδ overexpression. Whereas reconstitution of p53 alone did not modify the morphology and growth properties of HCT116/p53null cells, overexpression of both p53 and PKCδ induced a number of alterations indicating suppression of the transformed phenotype. Interestingly, PKCδ was ineffective when overexpressed in HT29 cells, a human colon cancer line characterized by the Arg273His dominant-negative mutation of p53. Thus, our data indicate that wild-type p53 is an essential effector of PKCδ in human colon cancer cells.

Oncotarget, 2015

Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and ... more Little progresses have been made in the treatment of glioblastoma (GBM), the most aggressive and lethal among brain tumors. Recently we have demonstrated that Chloride Intracellular Channel-1 (CLIC1) is overexpressed in GBM compared to 2 normal tissues, with highest expression in patients with poor prognosis. Moreover, CLIC1-silencing in cancer stem cells (CSCs) isolated from human GBM patients negatively influences proliferative capacity and self-renewal properties in vitro and impairs the in vivo tumorigenic potential. Here we show that CLIC1 exists also as a circulating protein, secreted via extracellular vesicles (EVs) released by either cell lines or GBM-derived CSCs. Extracellular vesicles (EVs), comprising exosomes and microvesicles based on their composition and biophysical properties, have been shown to sustain tumor growth in a variety of model systems, including GBM. Interestingly, treatment of GBM cells with CLIC1-containing EVs stimulates cell growth both in vitro and in vivo in a CLIC1-dose dependent manner. EVs derived from CLIC1-overexpressing GBM cells are strong inducers of proliferation in vitro and tumor engraftment in vivo. These stimulations are significantly attenuated by treatment of GBM cells with EVs derived from CLIC1-silenced cells. However, CLIC1 modulation appears to have no direct role in EV structure, biogenesis and secretion. These findings reveal that, apart from the function of CLIC1 cellular reservoir, CLIC1 contained in EVs is a novel regulator of GBM growth.

STEM CELLS, 2012

The invasive nature of glioblastoma (GBM) is one important reason for treatment failure. GBM stem... more The invasive nature of glioblastoma (GBM) is one important reason for treatment failure. GBM stem/progenitor cells retain the migratory ability of normal neural stem/ progenitor cells and infiltrate the brain parenchyma. Here, we identify Rai (ShcC/N-Shc), a member of the family of Shc-like adaptor proteins, as a new regulator of migration of normal and cancer stem/progenitor cells. Rai is expressed in neurogenic areas of the brain and its knockdown impairs progenitor migration to the olfactory bulb. Its expression is retained in GBM stem/progenitor cells where it exerts the same promigratory activity. Rai silencing in cancer stem/progenitor cells isolated from different patients causes significant decrease in cell migration and invasion, both in vitro and in vivo, providing survival benefit. Rai depletion is associated with alteration of multiple-signaling pathways, yet it always leads to reduced expression of proinvasive genes. STEM CELLS 2012;30:817-832

Stem Cell Research & Therapy, 2013

Cancer stem cells and neural stem cells: common features with diff erent purposes Parallels betwe... more Cancer stem cells and neural stem cells: common features with diff erent purposes Parallels between neurogenesis and the processes contributing to brain tumor formation exist. Neural stem cells (NSCs) are quiescent cells able to self-renew and generate partially committed, highly proliferative progenitors that subsequently undergo complete diff erentiation into one of the three lineages composing the brain. A recognized hallmark of neural stem/progenitor cells is their ability to migrate, an essential process for recovery after brain injury [1]. Th e same role exerted by NSCs in the physiological context has been proposed to be played in glioblastoma (GBM) by a rare fraction of self-renewing, multipotent tumor-initiating cells called cancer stem cells (CSCs), responsible for tumor progression, maintenance, and recurrence [2,3]. Th is subpopulation has shown intrinsic resistance to therapy, being able to repopulate the tumor after treatment [4]. Recently, many studies have ascribed to CSCs the infi ltrative property of GBM. Cancer stem cells and invasive cells: two sides of the same coin? Th e clinically distinct feature of GBM lies within its infi ltrative potential, rendering complete tumor resection nearly impossible. Tumor infi ltration is an extremely com plex program that requires the steady supply of extra cellular cues, abrogation of cell-cell interactions, and extracellular matrix (ECM) remodeling. Invading GBM cells are particularly resistant to current therapies and are often localized within the neurovascular niche, two features in common with CSCs [5]. Recent experimental data started to suggest that CSCs are responsible for GBM invasiveness. Cells enriched for the putative stem cell marker CD133 display greater migratory and invasive potential in vitro and in vivo when compared with matched CD133-negative tumor cells derived from human primary GBMs, GBM xenografts [4], and brain tumor cell lines [6-9]. We and others reported a marked upregulation of proteins involved in the processes of migration and invasion in GBM CSCs, such as diff erent types of matrix metalloproteinases, or diff erent members of both ADAMs (a disintegrin and metallo proteinases) and ADAMTS (ADAM with thrombo spondin motifs) families [4,6-8,10]. Th erefore, the highly migrating and invasive ability of GBM CSCs may be due to increased expression of proinvasive genes. Based on the findings that the GBM CSCs are more infiltrative than their diff rentiated descendants, a novel strategy has also been proposed to isolate and enrich CSCs from the whole tumor population by exploiting the tumor cell heterogeneity of invasiveness [11]. Cells at the leading edge of the tumor have been found to be positive for putative stem cell markers such as Abstract Glioblastoma (GBM) is the most aggressive and lethal brain tumor in adults. Its invasive nature currently represents the most challenging hurdle to surgical resection. The mechanism adopted by GBM cells to carry out their invasive strategy is an intricate program that recalls what takes place in embryonic cells during development and in carcinoma cells during metastasis formation, the so-called epithelial-to-mesenchymal transition. GBM cells undergo a series of molecular and conformational changes shifting the tumor toward mesenchymal traits, including extracellular matrix remodeling, cytoskeletal re-patterning, and stemlike trait acquisition. A deeper understanding of the mechanisms driving the whole infi ltrative process represents the fi rst step toward successful treatment of this pathology. Here, we review recent fi ndings demonstrating the invasive nature of GBM cancer stem cells, together with novel candidate molecules associated with both cancer stem cell biology and GBM invasion, like doublecortin and microRNAs. These fi ndings may aff ect the design of eff ective therapies currently not considered for GBM invasive progression.

Journal of Virological Methods, 2006

Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral ve... more Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3) of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5 HS4 chromatin insulator of the chicken ␤-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored-in transfection experiments-the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.

The Journal of Immunology, 2009

Journal of Cellular and Molecular Medicine, 2004

International Journal of Cancer, 2005

We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importan... more We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.

European Journal of Cancer, 2010

Human gliomas represent an unmet clinical challenge as nearly two-thirds of them are highly malig... more Human gliomas represent an unmet clinical challenge as nearly two-thirds of them are highly malignant lesions with fast progression, resistance to treatment and poor prognosis. The most severe form, the glioblastoma multiforme, is characterised by a marked and diffuse infiltration through the normal brain parenchyma. Given the multiple effects of chemokines on tumour progression, aim of this study was to analyse the expression of the chemokine CX3CL1 and of its specific receptor CX3CR1 in 36 human surgical glioma samples, with different degrees of histological malignancy and in glioblastoma-derived neurospheres. Herein we show that both ligand and receptor are expressed at the mRNA and protein levels in most specimens (31/36). While receptor expression was similarly detected in low or high grade tumours, the uppermost scores of CX3CL1 were found in grades III-IV tumours: oligodendrogliomas, anaplastic astrocytomas and glioblastomas. Accordingly, the expression of CX3CL1 was inversely correlated with patient overall survival (p = 0.01). Glioblastoma-derived neurospheres, containing a mixed population of stem and progenitor cells, were positive for both CX3CR1 and for the membrane-bound chemokine, which was further up-regulated and secreted after TNF-IFNγ stimulation. Confocal microscopy of 3D neurospheres showed that the ligand was primarily expressed in the outer layer cells, with points of co-localisation with CX3CR1, indicating that this ligand-receptor pair may have important intercellular adhesive functions. The high expression of CXC3L1 in the most severe forms of gliomas suggests the involvement of this chemokine and its receptor in the malignant behaviour of these tumours.

JNCI Journal of the National Cancer Institute, 2013

Background Chloride channels are physiologically involved in cell division and motility. Chloride... more Background Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis. Methods We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane's multiple comparison test. Kaplan-Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided. Results CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1 low vs CLIC1 high survival: χ 2 = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient-derived neurospheres. Conclusions Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

Biophysical Journal, 2012

Chloride channels are physiologically involved in cell division and motility. Chloride intracellu... more Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis.