Signaling pathways in which the candidate genes are involved.

<p>An extensive literature search revealed that the candidate genes are cross-linked to 3 signaling pathways: NF-κB, Akt and MAPK, which all play a role in cancer. NF-κB signaling pathway has a crucial role in regulating immune responses, whereas Akt signaling has been shown to inhibit the growth of GBM cells and GBM stem-like cells that may also be impaired by MAPK signaling disruption. Because of the RT-qPCR results, <i>CTSK</i>'s role has been examined and it was found via cross linking to other candidate genes obtained via osteopontin (<i>OPN</i>) gene functions.</p

RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain <i>(in vivo)</i>.

<p>(A) Upregulated expression of seven genes (<i>TFRC</i>, <i>CTSK</i>, <i>GFPT2</i>, <i>ERAP2</i>, <i>GPR56</i>, <i>CD74</i>, <i>PI3</i>) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the <i>CTSK</i> gene using GBM tissues and cell lines with reference genes <i>TBP</i> and <i>HPRT1</i> in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.</p

Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.

<p>ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. <b>Please note</b> that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.</p

Scheme of cellular processes and activities involving overexpressed protease and protease inhibitor genes in GBM.

<p>The overexpressed protease and inhibitor genes in GBM tissues and cells were queried by the Biomine search engine which identified processes and activities ascribed with KEGG and GO identifiers (in circles) in which selected genes (in bold caption) are involved.</p

Analysis of Glioblastoma Patients' Plasma Revealed the Presence of MicroRNAs with a Prognostic Impact on Survival and Those of Viral Origin

<div><p>Background</p><p>Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.</p><p>Experimental Procedures</p><p>MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits.</p><p>Results</p><p>We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.</p><p>Conclusion</p><p>The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.</p></div

Relative band intensities of antigens validated with Western blot.

<p>The relative band intentisy of the antigens, TRIM28 and β-actin, was calculated as the ratio between arbitrary units of the band of the antigen and the arbitrary units of the band of the internal control, GAPDH [AU(antigen)/AU(GAPDH)]. Samples: GSCc – cytosolic/nuclear protein fraction isolated from GBM stem-like cell lines; GBMc – cytosolic/nuclear protein fraction isolated from GBM tissues; NBTc – cytosolic/nuclear protein fraction isolated from human brain samples.</p

Schematic presentation of the whole workflow.

<p>1. Material preparation – expanding a GBM cell line, laminin grown and enriched in stem-like cells, 2. Llama immunization with whole GBM cells, 3. Construction of a nanobody library, 4. Phage display cycle – enrichment of antigen specific nanobodies on various biological samples (whole protein extract from GBM tissues, membrane protein-enriched fractions from GBM tissues and GBM stem-like cell lines), 5. ELISA - selection of GBM specific nanobodies, 6. Nanobody production – large scale production and purification of specific nanobodies, 7. Nanobody:antigen pairs – immobilizing the nanobodies and binding to the corresponding antigens, 8. Antigen identification by mass spectrometry, 9. Antigen validation by Western blot. See Materials and methods for additional experimental details.</p

Hierarchical clusters of rules for the validated targets of miRNAs detected in the plasma samples of GPs.

<p>Hierarchical clustering of the top 100 statistically significant rules (p≤0.05) is presented. The SegMine rules were derived from genes, representing validated targets of the GBM-related plasma miRNAs. Euclidian distance and Ward’s linkage criteria were used to compute the hierarchy.</p

MiRNAs with altered plasma levels in the GPs group vs. the HIo group.

<p>MiRNAs with altered plasma levels in the GPs group vs. the HIo group.</p

Sequences of the nanobodies that were chosen for the large-scale production.

<p>The amino acid sequence of the CDR3 regions of the nanobodies is given in alphabetic order.</p