1. TRIM28 and [beta]-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkersIvana Jovchevska, Neja Šamec, Nina Kočevar Britovšek, Daniela Cesselli, Neža Podergajs, Clara Limbaeck Stanic, Michael P. Myers, Serge Muyldermans, Gholamreza Hassanzadeh Ghassabeh, Helena Motaln, Maria Elisabetta Ruaro, Evgenia Bourkoula, Tamara Lah Turnšek, Radovan Komel, 2014, izvirni znanstveni članek Povzetek: Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.
Ključne besede: malignant gliomas, cancer stem cells, nanobodies
Objavljeno v DiRROS: 02.08.2024; Ogledov: 105; Prenosov: 73
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2. Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cellsNeža Podergajs, Helena Motaln, Uroš Rajčević, Urška Verbovšek, Marjan Koršič, Nina Obad, Heidi Espedal, Miloš Vittori, Christel Herold-Mende, Hrvoje Miletic, Rolf Bjerkvig, Tamara Lah Turnšek, 2016, izvirni znanstveni članek Povzetek: The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.
Ključne besede: biomarker, CD9, glioblastoma stem cells, neural stem cells, tetraspanin
Objavljeno v DiRROS: 26.07.2024; Ogledov: 116; Prenosov: 87
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3. Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGFNeža Podergajs, Narve Brekka, Bernhard Radlwimmer, Christel Herold-Mende, Krishna M. Talasila, Katja Tiemann, Uroš Rajčević, Tamara Lah Turnšek, Rolf Bjerkvig, Hrvoje Miletic, 2013, objavljeni znanstveni prispevek na konferenci Povzetek: Background. Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stemlike cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.Meterials and methods. To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.Results. Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).Conclusions. We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.
Ključne besede: glioblastoma, stem cell cultures, bFGF
Objavljeno v DiRROS: 03.04.2024; Ogledov: 246; Prenosov: 111
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