Ivana Jovchevska (Author), Neja Zupanec (Author), Nina Kočevar (Author), Daniela Cesselli (Author), Neža Podergajs (Author), Clara Limbäck-Stokin (Author), Tamara Lah Turnšek (Author), Radovan Komel (Author), Michael P. Myers (Author), Serge Muyldermans (Author), Gholamreza Hassanzadeh Ghassabeh (Author), Helena Motaln (Author), Maria Elisabetta Ruaro (Author), Evgenia Bourkoula (Author)

Abstract

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 beta-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

Keywords

malignant gliomas;cancer stem cells;nanobodies;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Organization: NIB - National Institute of Biology
UDC: 620.3
COBISS: 3251791 Link will open in a new window
ISSN: 1932-6203
Views: 38
Downloads: 30
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Other data

Secondary language: Slovenian
Secondary keywords: maligni gliomi;izvorne rakave celice;nano telesa;
Source comment: Nasl. z nasl. zaslona; Opis vira z dne 25. 11. 2014; Članek v pdf obsega 22 str.;
Volume: ǂVol. ǂ9
Issue: ǂiss. ǂ11
Chronology: Nov. 2014
DOI: 10.1371/journal.pone.0113688
ID: 24644280