Human GBM cell-line preparation

A GBM cell line from patient tumour tissue was isolated and grown on laminin under conditions for enrichment in GBM stem-like cells following the protocols of Bourkoula et al. [18].

Library construction

An adult male alpaca (23 months old) was repeatedly immunized with whole glioma cells enriched in GBM stem-like cells. Each immunization used 10⁶ whole cells, which were subcutaneously injected in the animal. In total, seven immunizations were performed, at 1 week interval. After the last immunization, 100 mL of blood was drawn from the immunized alpaca and used for the extraction of lymphocytes.

The nanobody library was constructed at the Vrije Universiteit Brussel using in-house protocols of the Nanobody Service Facility, as published by Hassanzadeh Ghassabeh et al. [19]. The library construction was started immediately after receiving the blood. Peripheral blood lymphocytes were isolated and their mRNA was extracted and used as template for cDNA synthesis. Two PCRs were carried out. The first PCR used the primers CALL001 (5′→3′, GTC CTG GCT GCT CTT CTA CAA GG) and CALL002 (5′→3′, GGT ACG TGC TGT TGA ACT GTT CC) to differentiate between classical Abs (ca. 900 bp) and HCAbs (ca. 600 bp). The HCAb fragments from the first PCR reaction were excised from the electrophoretic gel, purified using gel extraction kits (Qiagen), and amplified by nested PCR using the specifically designed primers A6E (5′→3′, GAT GTG CAG CTG CAG GAG TCT GGR GGA GG) and 38 (5′→3′, GGA CTA GTG CGG CCG CTG GAG ACG GTG ACC TGG GT), to amplify the variable domains of the HCAbs only (i.e., VHH or nanobodies). The VHH amplicons were purified using Qiaquick PCR purification kits (Qiagen) and ligated into a pHEN4 phagemid vector that was already digested with PstI and NotI. This pHEN4 is a modified pHEN1 vector that was created by substituting the c-myc with an HA tag [16], and its structure is schematically represented in Hassanzadeh Ghassabeh et al. [19]. The nanobody library was created by transformation of the phagemids obtained into electro-competent TG1 E. coli cells (Lucigen).

Sample preparation for selection

Library panning was performed on whole protein extracts from GBM tissues of eight patients (five males, three females; 40–76 years old) that underwent surgery at the University Hospital of Udine. The proteins were extracted following the procedure described by Alhamdani et al. [20]. Briefly, the frozen tissue (ca. 60 mg pooled sample) was homogenized in 300 µL extraction buffer (20 mM HEPES, pH 7.9, 10 mM glycerol, 0.5% Triton X-100, 0.5% Na-cholate, 0.5% NP-40, 0.5% CHAPS), and incubated on ice for 30 min. Upon centrifugation at 20,000×g for 20 min at 4°C, the supernatant was transferred into a clean microcentrifuge tube (Eppendorf, Germany) and deep frozen until used for panning.

Bio-panning was also performed on fractions enriched in membrane proteins that were extracted from the same human GBM tissue samples, and on membrane protein-enriched fractions extracted from commercially available GBM stem-like cell lines (NCH644 and NCH421K cells; Cell Line Service GmbH, Eppelheim, Germany), initially prepared and described by Campos et al. [21]. The NCH644 and NCH421K cells were cultivated in complete neurobasal medium: neurobasal medium (Gibco)supplemented with 20 µM/mL L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 2% B-27 supplement, 20 ng/mL bFGF, 20 ng/mL EGF and 1 U/mL heparin [22].

Membrane protein-enriched fractions were obtained using two commercially available kits: ProteoExtract Transmembrane Protein Extraction kits (Novagen) and ProteoExtract Native Membrane Protein Extraction kits (Calbiochem), according to the manufacturer guidelines. The extracted fractions were analyzed by Western blot, using 1∶1500 Anti-pan Cadherin [CH-19] (abcam) and 1∶3000 monoclonal anti-GAPDH antibody produced in mouse (Sigma Aldrich) for detecting the membrane protein-enriched and cytosolic fractions, respectively, and 1∶5000 goat anti-mouse IgG (H+L) horse radish peroxidase-conjugated antibody (Jackson ImmunoResearch) as a secondary antibody. The membrane was treated with SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific) and the bands were visualised using chemiluminescence and a LAS-4000 CCD camera (Fujifilm; Tokyo, Japan). The bands were analysed with the Multi Gauge version 3.2 software (for detailed Western blot procedure see Antigen validation).

The normal post-mortem brain samples that were used as the negative control in the enrichment of the immobilized protein extracts and for screening were provided by the Institute of Pathology, Faculty of Medicine, University of Ljubljana (Slovenia). The used brain samples originated from the hippocampus, and subventricular and periventricular areas of the brain. The membrane protein-enriched fractions from the human brains were extracted using the above-mentioned kits (from Novagen and Calbiochem). The whole protein fraction was extracted following the same protocol as for the GBM tissue samples. The concentrations of the extracted proteins from the biological samples were determined according to the Bradford method [23], using the commercial Bradford reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Phage production

An aliquot of the TG1 library that contained a representative selection of the cloned VHHs was grown in 2x Yeast extract and Tryptone (2× TY) medium containing 100 µg/mL ampicillin and 2% glucose, until the TG1 E. coli were growing exponentially (3–4 h at 37°C, shaking). The TG1 E. coli cells were then infected with an excess of M13K07 helper phages (10¹² phages/mL) and incubated for 20 min at 37°C, without shaking. The culture was centrifuged at 1467×g for 10 min, and the supernatant was removed. The pellet was resuspended in 1 mL 2× TY medium and transferred to an Erlenmeyer flask containing 300 mL 2× TY medium with 100 µg/mL ampicillin and 70 µg/mL kanamycin. These samples were then incubated overnight at 37°C, with shaking at 225 rpm.

The phage particles were separated from the TG1 E. coli cells by centrifugation at 9820×g, for 30 min. The supernatant was transferred to 50 mL Falcon tubes, on ice, containing 10 mL ice-cold polyethylene glycol-6000/NaCl, and precipitated for 30 min on ice. The tubes were then centrifuged for 30 min, 2087×g, the supernatants were discarded, and the pellets (which contained the phages) were resuspended in a total volume of 1 mL phosphate-buffered saline (PBS).

Enrichment of antigen-specific nanobodies

Library screening and enrichment of nanobodies were performed according to Hassanzadeh Ghassabeh et al. [19]. Both, library panning (i.e., immunoaffinity selection) and screening were performed with the same samples prepared for different protein concentrations (200 µg/mL for panning, and 2 µg/mL for screening). No more than three rounds of pannings were carried out for each biological sample.

For the bio-panning, 100 µL of GBM protein lysate (positive) and 100 µL protein lysate from normal human brains (negative) were used. After overnight coating with protein mix (200 µg/mL) and blocking of the residual protein binding sites on the plastic with 2% casein, 10¹¹ of the phages produced were added to both coated wells, and incubated for 1 h at room temperature. The wells were washed several times with 0.1% PBS-Tween to remove unbound virions. The phages were eluted upon 10 min incubation with 100 mM triethylamine [24]. The 100 µL eluted phage solution was neutralized with 100 µL 1 M Tris-HCl (pH 7.4) and used in the next panning cycle.

The TG1 E. coli cells were grown in Luria-Bertani Miller (LB) medium for 3-4 hours at 37°C with shaking at 225 rpm. When these TG1 E. coli cells reach their exponential growth phase, they express F-pilus, which allows them to be infected with the eluted phage particles. Serial dilutions from the phage particles eluted from the positive (with antigen) and negative (without antigen) wells were made and used to infect the exponential TG1 E. coli cells. These were then incubated at 37°C for 30 min, without shaking. The TG1 E. coli cells were plated on both 120 mm square and 90 mm round Petri dishes, and incubated overnight at 37°C. The relative enrichment was determined by comparing the number of colonies (from the positive and negative wells) grown on the square Petri dishes, while the round Petri dishes were used for picking single colonies for subsequent ELISA screening.

The remaining phage particles that were eluted from the antigen-coated wells (positive) were mixed with exponential TG1 E. coli cells. After 30 min incubation without shaking, 2× TY medium containing 100 µg/mL ampicillin and 2% glucose, was added. After 20 min, 10¹¹ M13K07 helper phages were also added, to infect the TG1 E. coli cells, and these were incubated for 20 min at 37°C, without shaking, to infect cells and to start the production of fresh phage particles. The mixture was centrifuged, the pellet was resuspended in 2× TY medium containing 100 µg/mL ampicillin and 70 µg/mL kanamycin, and the phages were produced overnight at 37°C, with shaking at 225 rpm, to be used for next round of panning. The next morning, the phages were precipitated with polyethylene glycol-6000/NaCl, as described above.

Library screening with periplasmic-extract ELISA

Every second row of wells of the NUNC MaxiSorp plates (Thermo Scientific) was coated with GBM protein lysate (positive), with the rest of the wells coated with protein lysate from normal brain samples (negative), and these plates were incubated overnight at 4°C.

Single colonies from the round Petri dishes (see above) were picked after different rounds of panning, and these were grown in 24-well plates with 1 mL Terrific Broth (TB) and 100 µg/mL ampicillin per well, for 3–4 h. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to 1 mM final concentration, followed by an overnight incubation at 37°C, with shaking at 225 rpm.

The cultures were then centrifuged at 4°C, the supernatant was removed gently and discarded, and 200 µL/well Tris/EDTA/sucrose (TES) was added. The plates were then shaken at 150 rpm for 30 min at room temperature. A minor modification was introduced using distilled (d)H₂O instead of using a 1∶4 dilution of TES in dH₂O (TES/4). Adding 300 µL/well dH₂O caused an osmotic shock that released the induced proteins present in the periplasm of the TG1 E. coli cells. This step liberates the nanobodies, as they contain a signalling peptide that transports the polypeptide into the periplasm [25]. The plates were centrifuged for 13 min at 2087×g. Then, 100 µL/well of the supernatant was transferred to both, positive and negative wells of the coated ELISA plates. Next, addition and incubation with the primary (mouse anti-HA; Sigma-Aldrich) and secondary (goat anti mouse IgG, whole molecule; Sigma-Aldrich) antibodies followed. After this, alkaline phosphatase substrate (Sigma-Aldrich) was added, which allowed visualisation of the specifically bound proteins. The absorbance was measured at 405 nm.

A clone was considered positive if its periplasmic extract yielded an ELISA signal at least twice of that without antigen coating. The TG1 E. coli colonies of the nanobodies that had good ELISA signals were amplified using colony PCR, and sequenced at the Genetic Service Facility (VIB, Antwerp).

Nanobody recloning

Three nanobodies were chosen for large-scale production, and these were recloned into the pHEN6 expression vector to extend the nanobody with a His tag [26], [27].

Single colonies of the clones were amplified by PCR using the A6E and 38 primers (framework1 and framework4 primers, respectively). The PCR products were purified using PCR purification kits, and digested overnight at 37°C with PstI and Eco91I (both from Thermo Scientific). The products were ligated for 2 h at room temperature into pHEN6 vector. Electro-competent E. coli WK6 cells were transformed with the ligation reaction. The selection was made on LB agar plates containing 100 µg/mL ampicillin and 1% glucose. Five randomly chosen clones from each nanobody were amplified by colony PCR, and sequenced at the Genetic Service Facility (VIB, Antwerp). Individual bacterial clones that had exactly the same sequence as the original nanobody clones were chosen for the large-scale expression.

Nanobody expression and purification

The expression and purification procedures were carried out as described by Vincke et al. [28] and Chames [29]. The procedure described is applicable for the expression of a single nanobody.

After recloning, five single colonies for each nanobody clone were picked and inoculated into 15 mL LB medium with 100 µg/mL ampicillin. These were grown overnight at 37°C, with shaking at 225 rpm. These five overnight cultures were pooled in one tube, and 5 mL of this culture was equally distributed into five shaker flasks, each containing 330 mL TB with 100 µg/mL ampicillin, 0.1% glucose and 2 mM MgCl₂. The flasks were shaken at 200 rpm for 3–4 h at 37°C. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM, and the flasks were shaken overnight at 28°C at 200 rpm.

Next day, the culture was centrifuged for 8 min, at 14°C, 9820×g. The supernatant was discarded, and the pellets of all five cultures pooled. They were resuspended in 20 mL TES until the suspension became homogeneous, which was then shaken for 1 h in a cold-room at 4°C. Osmotic shock was then caused by adding 40 mL TES/4 to this suspension, which was shaken for another 2 h at 200 rpm in the cold-room at 4°C. Five hundred microliters of 2 M MgCl₂ was added to the centrifuge tube, and the suspension was mixed and centrifuged for 30 min at 9820×g. The supernatant was transferred to two 50 mL tubes (Falcon) that already contained approximately 5 mL Ni⁺-NTA agarose (Ni-beads, Qiagen)/tube. The Ni-beads were previously washed three times with 20 mL PBS. The periplasmic extract was incubated with these Ni-beads overnight in a cold-room at 4°C, with shaking at 200 rpm.

A second overnight periplasmic extraction is optional, to obtain a higher protein yield. Here the samples from both extractions are pooled and equally distributed into four 50 mL tubes (Falcon) that already contained Ni-beads, for the incubation for 1 h in a cold-room at 4°C, shaking at 200 rpm. These were then centrifuged for 7 min at 394×g. The supernatant was discarded, and 45 mL/Falcon PBS was added to the beads, which were then shaken for 1 h in the cold-room at 4°C. The tubes were centrifuged and the supernatant was removed, leaving approximately 5 mL liquid on the resin.

The expressed nanobodies were purified using immobilised metal-affinity chromatography (IMAC). A filter was put in an empty PD-10 column, and the periplasmic extract with the Ni-beads was poured into the column. The column was washed with 20 mL PBS and drained. 10 mL freshly prepared PBS with 0.5 M imidazole was used to elute the expressed nanobodies, with this elution repeated three times. The protein concentration of the combined eluates was measured using a NanoDrop spectrophotometer. The proteins were then concentrated using Vivaspin concentrators to obtain a final nanobody concentration of 1 mg/mL. The expressed proteins were separated by SDS-PAGE and visualized after staining with Coomassie blue R-250.

These IMAC-purified nanobodies were additionally cleaned using size-exclusion chromatography, with an ÄKTA purifier (GE Healthcare) and the Unicorn software. The samples were run on a Sephadex S75 column using filtered PBS (pH 8.0) as the mobile phase, at a flow-rate of 2 mL/min. The eluate was collected in 1.5 mL microcentrifuge tubes (Eppendorf), and those corresponding to the nanobodies were collected in separate 50 mL tubes (Falcon) for each nanobody. The protein concentration was measured on a Synergy H4 Hybrid Reader. Using dilution in PBS, the nanobodies were brought to a final concentration of 1 mg/mL (when possible), and were stored at 4°C.

Obtaining nanobody:antigen pairs

Sample preparation and MS analysis was performed according to the Generic IP protocol established by the Proteomic Facility at the International Centre for Genetic Engineering and Biotechnology (ICGEB) in Trieste, Italy (https://sites.google.com/site/icgebproteomics/documents).

Two of the nanobodies were used at 1 mg/mL, and one at 0.2 mg/mL. The concentration of the extracted membrane proteins from the GBM stem-like cell lines was 3 mg/mL.

Ni-beads (400 µL) were washed three times with 1 mL PBS. After the last wash, the Ni-beads were resuspended in 2 mL PBS and equally distributed into six microcentrifuge tubes (Eppendorf). The nanobodies were incubated with the Ni-beads for 1 h at 4°C on a rotating platform (positive samples). Three of the microcentrifuge tubes were left without nanobodies, considered as ‘negative’ samples, and these were treated identically to the Ni-beads with the nanobodies. The Ni-beads with the nanobodies were briefly centrifuged, the supernatant was discarded, and the extracted membrane proteins were added. Here, 500 µL/tube protein lysate was added to one ‘positive’ and one ‘negative’ microcentrifuge tube of the same nanobody. These were incubated again at 4°C for 1 h, while rotating. After incubation, both suspensions were centrifuged, the supernatants (protein lysate) were placed into new microcentrifuge tubes (for the second nanobody) and incubated for 1 h in the cold-room at 4°C, while rotating. The pellets (the nanobody:antigen pair) from the positive and negative microcentrifuge tubes of the first nanobody were washed three times with 1 mL 0.1% PBS-Tween, transferred to new tubes, and washed three times again with 1 mL PBS. The microcentrifuge tubes were briefly centrifuged, the supernatants were removed, and the pellets were kept on ice. The same procedure was repeated with the second and third nanobody.

Sample preparation for mass spectrometry analysis

All of the pellet samples from the nanobody:antigen pairs were digested with trypsin solution (2 µL porcine trypsin (Promega) in 100 µL 20 mM triethylammonium hydrogen carbonate buffer), overnight at room temperature.

After the overnight digestion, the samples were centrifuged and the supernatants were transferred to a clean microcentrifuge tube. Then 25 µL 20 mM triethylammonium hydrogen carbonate buffer was added to the Ni-beads, which were centrifuged, and these supernatants were combined with the first supernatants. Then, 1 µL Tris(2-carboxyethyl)phosphine (as a reducing agent; Thermo Scientific) was added to each of the microcentrifuge tubes, which were incubated for 5 min at room temperature. Then, 1 µL chloroacetamide was added to each sample, and these were incubated for 1 h at room temperature. In order to elute any tryptic fragments that remained bound to the nanobodies, the pelleted Ni-beads were washed once with 1 mL PBS/tube, centrifuged, and the supernatants were removed. Then 25 µL 6 M urea was added to each microcentrifuge tube. The samples were centrifuged briefly, and the supernatants containing the eluted antigens were transferred to clean microcentrifuge tubes. The same volume of urea was added two more times to each microcentrifuge tube, and after brief centrifugation, the supernatants that were treated with the same nanobody were pooled. All of the supernatants were purified using Empore C18 filters. These filters were inserted into pipette tips, wet with acetonitrile, and equilibrated with 0.1% trifluoroacetic acid in water. The samples were loaded onto the filters, washed with 0.1% trifluoroacetic acid and eluted into new microcentrifuge tubes using 65% acetonitrile with 0.1% trifluoroacetic acid. The samples were finally dried for 5 min at 95°C. Prior to the analysis with MS, each sample was resuspended in 25 µL 0.2% trifluoroacetic acid.

Mass spectrometry analysis and antigen identification

The MS analysis and antigen identification were performed at the Proteomic Facility at the ICGEB in Trieste (Italy). The purified samples were analysed by liquid chromatography–tandem MS (LC-MS/MS), using an Easy-nLC system connected to an Amazon electron-transfer dissociation ion trap (both instruments from Bruker). The LC was developed using a 75 min gradient from 0% to 80% methanol in 0.1% formic acid. The resulting MS/MS spectra were searched against a human database using the X!tandem and MASCOT search engines, allowing for a 5% false-discovery rate. This led to the identification of two antigens.

Antigen validation

The antigens captured by the nanobodies for the MS identification were validated by Western blotting (WB). Approximately 10 µg protein extracts from the biological samples (from the GBM tissues, the GBM stem-like cell lines, and the post-mortem human brain samples) were separated by 12% SDS PAGE. An Immobilion-P Transfer Membrane (Millipore) was activated by incubating it for 15 s in methanol, followed by 2 min incubation in dH₂O, and then placing it in WB transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) until use. The WB transfer was approximately 1 h at 100 V.

The proteins on the WB membrane were stained using Ponceau S and blocked in 5% PBS-milk for 1 h at room temperature, with shaking. To detect TRIM28 in the selected biological samples, the membrane was incubated with the SAB1404789 monoclonal anti-tripartite motif containing protein (TRIM28) antibody (1∶1500; Sigma Aldrich) at 4°C, overnight with shaking. After overnight incubation, the Western blot membrane was incubated with the A5441 monoclonal anti-β-actin antibody (1∶1500; Sigma Aldrich) for 1 h at room temperature, with shaking. In order to have an internal control, the Western blot membrane was incubated with the monoclonal anti-GAPDH antibody produced in mouse (1∶3000; Sigma Aldrich) for 1 h at room temperature, with shaking. The incubation with the secondary antibody, a goat anti-mouse IgG (H+L) horse radish peroxidase-conjugated antibody (1∶5000; Jackson ImmunoResearch), was also for 1 h at room temperature. SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific) was spread over the membrane after the last incubation, and the bands were visualised using chemiluminescence and a LAS-4000 CCD camera (Fujifilm; Tokyo, Japan). The bands were analysed with the Multi Gauge version 3.2 software. The band intensities of the antigens, β-actin and TRIM28, were normalized to the band intensities of the internal control, GAPDH. The numerical value of the relative band intensity of the antigens was calculated as the ratio between the arbitrary units (AU) of the antigens and the arbitrary units of the internal control.

TRIM28 as a tumour marker

TRIM28 – also known as TIF1β (transcriptional intermediary factor 1-β) and KAP1 (KRAB-interacting protein 1), is a protein that belongs to the tripartite motif (TRIM) family (also known as the RBCC family) that at the N-terminal region is composed of three conserved zinc-binding domains, a RING finger (R), B-boxes (B-box Type 1 (B1) and B-box Type 2 (B2)), and a coiled-coil region (CC). To date, about 70 members of the TRIM family are known. Their structures were explained in detail by Micale et al. [42]. TRIM proteins vary in the number of B-boxes and the C-terminal domain. Indeed, the TRIM family can be subdivided into 11 different groups based on the composition of the highly variable C-terminal region. The 6^th subfamily of the TRIM proteins, due to the presence of C-terminal BROMO and PHD domains (Figure 5), consists of three TIF1 members, TIF1α, TIF1β, TIF1γ, which are also known as TRIM24, TRIM28 and TRIM33, respectively, and found to display high sequence homology. Their well-conserved PHD and BROMO domains were found widely distributed among nuclear proteins acting at the chromatin level and involved in the control of gene expression through regulation of the transcriptional activity of numerous sequence-specific transcription factors [43], [44]. Alteration of these proteins can affect transcriptional regulation, cell proliferation and apoptosis, and members of the 6^th TRIM subfamily, otherwise involved in an assortment of cellular processes such as cell growth, development and differentiation [45] are also thought to be important regulators of carcinogenesis. Namely, TRIM24 and TRIM33 have been described to acquire oncogenic activity upon chromosomal translocations when the RBCC motif is fused to another protein [46], [47], whereas TRIM28 is believed to be mediator of p53 suppression by promoting p53 ubiquitylation and degradation [48].

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Figure 5. Schematic presentation of the structure of TRIM28.

The conserved RBCC motif is present at the N-terminal domain; the variable C-domain includes the PHD and BROMO domains. Image adapted from Hatakeyama et al. [45]. Author's approval was obtained for the use of this image; original image is given in Figure S2.

https://doi.org/10.1371/journal.pone.0113688.g005

In 2012, Chen et al. [49] hypothesised that in the early stages of lung adenocarcinomas, the levels of TRIM28 are up-regulated for protective reasons; i.e., to control cell growth and suppress tumour development. More recently, Liu et al. [50] reported that TRIM28 is a possible marker for the detection of circulating cancer cells, and provided evidence for a role for TRIM28 in metastasis development and prognosis in early stage non–small-cell lung cancer (NSCLC). In this study, 35 of 48 patients with NSCLC had overexpressed TRIM28 mRNA levels, while the rest of these showed underexpressed TRIM28 mRNA levels, compared to non-cancerous tissue. Liu et al. [50] thus proposed that TRIM28 might be involved in tumour-cell migration, and tumour invasion and progression. Even though the findings of Liu et al. [50] and Chen et al. [49] are contradictory, as lung cancers are the cause for the occurrence of 65% secondary GBMs [51], the correlation of TRIM28 to GBM shown in the present study is an interesting discovery that deserves further being investigated.

β-actin as a tumour marker

β-actin is a highly abundant cytoskeletal protein spread throughout the peripheral cell regions and the leading edges of motile cells. It is involved in cell migration and division, wound healing, embryonic development, and immune responses [52]. As a result of its constant expression levels, β-actin has been used as an internal control for normalisation in gene expression studies [53]. However, more recently, its use as an internal control has been questioned due to the growing evidence that the expression levels of β-actin vary under different conditions. β-actin has been shown to be up-regulated in liver, gastric, colorectal, lung cancers and in melanomas [54]. The overexpression of β-actin in cancers suggests that it may have altered function in carcinogenesis [55], including of glioblastoma as lung cancers and melanomas are primary tumours that are responsible for the occurrence of secondary GBMs.

Cell migration is important during the whole of the life span of vertebrates, and especially during embryonic development. This is a process that requires changes in the actin cytoskeleton, and β-actin polymerisation and formation of invading structures. Recent studies have shown that β-actin polymerisation can promote cell motility, invasiveness and metastasis. Le et al. [56] showed that β-actin is localised in the pseudopodia of invasive cells, but absent in non-invasive cells. These data are further supported by the studies of Nowak et al. [57], [58] and Popow et al. [59], who reported increased β-actin levels in two invasive cell lines, the LS180 human colon adenocarcinoma cell line, and Morris 5123 rat hepatoma cells, respectively. This suggested possible roles for β-actin in tumour metastasis formation.

In 1998, Fages et al. [60] examined the β-actin distribution in C6 glioma cells. They showed localised β-actin mRNA at the leading edges in 80% of cells analysed. On the other hand, Panopoulos et al. [61] demonstrated that GBM cells can show exceptional motility that can even occur in the absence of polymerised actin. They concluded that even though actin polymers are not totally necessary for glioma cell migration, they do promote the migration of these cells.

By investigating different brain regions of five GBM patients, Com et al. [62] identified 24 proteins that were up-regulated in the glioblastoma region, one of which was β-actin. To our knowledge, this was the first demonstration of a direct correlation between β-actin and GBM.

Although data the literature are still controversial, our findings support the idea that β-actin might have a role in GBM cell migration, a finding that should now be considered and carefully examined. As tumour invasion is a disease of dysregulated cell motility, β-actin might play a crucial part in this process.

In conclusion, by using technology based on camelid heavy-chain antibodies we produced a phage-displayed VHH library and after a number of immunoaffinity selections we identified two new potential biomarkers, TRIM28 and β-actin, specific to GBM stem-like cells. The advantage of the approach is the simultaneous acquisition of both antigen and corresponding nanobody that can be used for further development of diagnostic and/or targeting strategies.