title: TRIM28 and β-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.
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 β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.
authors.affiliation: Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Ivana
authors.initials: I
authors.lastname: Jovčevska
authors.affiliation: Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Neja
authors.initials: N
authors.lastname: Zupanec
authors.affiliation: Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Nina
authors.initials: N
authors.lastname: Kočevar
authors.affiliation: Department of Medical and Biological Sciences (DSMB), University of Udine, Udine, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Cesselli
authors.affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology (NIB), Ljubljana, Slovenia.
authors.forename: Neža
authors.initials: N
authors.lastname: Podergajs
authors.affiliation: Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Clara Limbaeck
authors.initials: CL
authors.lastname: Stokin
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Myers
authors.affiliation: Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Structural Biology Research Center, VIB, Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Brussels, Belgium; Nanobody Service Facility, VIB, Brussels, Belgium.
authors.forename: Gholamreza Hassanzadeh
authors.initials: GH
authors.lastname: Ghassabeh
authors.affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology (NIB), Ljubljana, Slovenia.
authors.forename: Helena
authors.initials: H
authors.lastname: Motaln
authors.affiliation: Department of Medical and Biological Sciences (DSMB), University of Udine, Udine, Italy.
authors.forename: Maria Elisabetta
authors.initials: ME
authors.lastname: Ruaro
authors.affiliation: Department of Medical and Biological Sciences (DSMB), University of Udine, Udine, Italy.
authors.forename: Evgenia
authors.initials: E
authors.lastname: Bourkoula
authors.affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology (NIB), Ljubljana, Slovenia.
authors.forename: Tamara Lah
authors.initials: TL
authors.lastname: Turnšek
authors.affiliation: Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Radovan
authors.initials: R
authors.lastname: Komel
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D002161:Camelids, New World; D045744:Cell Line, Tumor; D005909:Glioblastoma / Q000175:diagnosis / Q000378:metabolism*; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D014411:Neoplastic Stem Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D019151:Peptide Library; D040901:Proteomics / Q000379:methods; D012097:Repressor Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000076183:Tripartite Motif-Containing Protein 28
chemical_list: D000199:Actins; D014408:Biomarkers, Tumor; D019151:Peptide Library; D012097:Repressor Proteins; D061905:Single-Domain Antibodies; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28