Expression of proteases and Pearson correlation.

(A) Comparison of the relative amounts of ADAM (a disintegrin and metalloprotease domain) proteases as well as BACE (beta-secretase) and MMP2 (matrix metallopeptidase 2) in the SLGC lines indicated (data from proteome array). Based on the assumption that the antibodies spotted on the filters of the proteome array possessed similar KDs, the relative expression of the proteases was calculated. The sum of the signals of all proteases was arbitrary set at 100%. The bars indicate to what percentage the individual proteases contribute to this expression. (B) Similar assay as in (A). The relative Cathepsin D levels were compared to the relative expression of all other proteases, depicted in panel (A). (C) Similar analysis as in (A) comparing the relative expression of the protease inhibitors TIMP (tissue inhibitor of metalloproteinases) 1, 2, 3 and 4.–Parts D and E graphically summarize some of the correlation data shown in Figs 8 and 9. In (B) the coefficients are indicated on the y-axis, highlighting expression levels with a positive correlation to the Sox2 (blue) or CD133 (violet) dots. In (C) the Pearson correlation coefficients were calculated relative to the levels of the neural proteins Tau and GFAP, as well as the hyaluronan receptor CD44, the integrin αv, and the N- and E-cadherin, respectively. In all cases, the expression levels that entered the calculations were determined with the same whole cell extracts. The calculations were confirmed with biological replicates. (TIF)</p

Growth factor requirements and growth curves.

(A) Expression (RT-PCR analysis) of mRNAs coding for EGF (epidermal growth factor), TGFα (transforming growth factor α), HB-EGF (Heparin-binding EGF-like growth factor), and bFGF (basic fibroblast growth factor). Bars depict the mean of a minimum of three replicates, whiskers the standard deviation. Significant differences relative to the expression in the T1338 cell line are indicated (**, pB) Effects of growth factor depletion on proliferation (BrdU ELISA). The combination of growth factors added into the medium is indicated by distinct levels of gray(white: EGF/bFGF; light grey: EGF; dark grey: bFGF; black: no growth factor added). The bars depict mean values and standard deviations. Significant reduction of BrdU incorporation is indicated by p-values (**, pC) Growth curves of the SLGC lines indicated were performed in the presence of EGF (red line), bFGF (black line) or both (blue line), or in the absence of growth factors (violet line). After six days (d6) cells were re-plated for studies with extended incubation times. Values for d9 were determined from both the original and the replated cultures. Significant differences between the growth curves at specific time points are indicated by p-values (*, pD) Growth factor ELISA. The amounts of EGF and bFGF present in T1371 and T1447 cultures were determined at days d2 and d9 after plating. The assays were performed in DMEM/Ham’s F12 containing fetal calf serum (10% FCS) or the serum supplements BIT (bovine albumin, insulin, and transferrin) and B27, respectively. The presence of exogenous growth factors is indicated by EGF/bFGF. Each assay encompassed a minimum of four replicates. Significant differences are indicated (**, p (TIF)</p

Expression of integrins.

(A) Comparison of the relative amounts of α-integrins expressed in the SLGC lines indicated (data from proteome array). Based on the assumption that the antibodies spotted on the filters of the proteome array possessed similar KDs, the relative expression of the integrins was calculated. The sum of the signals of all integrins was arbitrary set at 100%. The bars indicate to what percentage the individual α-integrins contribute to this expression. (B) Similar analysis as in (A) comparing the relative expression of the integrins β1, β2, β3, β4, β5, and β6. (C) Western blot analysis using antibodies directed against the integrins αv, β1, β3, and β5. The loading controls Actin and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) are shown below the corresponding blots. (D) Flow cytometry analysis experiment using early passages of SLGCs. The established non-SLGC cells lines U87 (glioblastoma) and CaCo2 (colon carcinoma) lack stemness. (E) Expression of mRNAs coding for the β-Integrins indicated. Total RNA was isolated from SLGC lines, subjected to reverse transcription and qRT- PCR analysis. Signal intensities were normalized against gapdh. (F) A similar experiment as in (E) investigating the effect of serum (10% fetal calf serum) on the expression of α- and β-integrin mRNAs, respectively. In order to highlight serum-mediated changes, the relative expression obtained with RNAs isolated from the corresponding serum-free cultures was arbitrary set at 1.- SC, derived from an orthotopic tumor. (TIF)</p

Assignment of proteins to metaprofiles and crossvalidation.

(A) Protein-specific characterization of the metaprofiles (MPs). Each of the five plots decomposes each metaprofile along its signature proteins. Proteins that showed no significance (B) Comparison of the mean squared reconstruction error of the original data (SLGC lines) with two non-SLGC lines U87 (established glioblastoma cell line without stemness features) and CaCo2 (established colon cancer cell lines with >95% CD133-positive cells). A two-fold cross-validation leads to slightly higher training and test errors than the average training error on all expression data. The test on the non-SLGCs shows that the learned features are rather SLGC-specific.</p

Expression of SLGC and GBM subtype markers.

(A-C) Western blot analyses. The loading controls Actin (42 kDa) or GAPDH (glyceraldehyde-3 phosphate dehydrogenase; 36 kDa) are shown below the corresponding blots. Brackets indicate that the same nitrocellulose filter was used for the respective detections. (D) Immunofluorescence analysis of the expression of the platelet-derived growth factor receptors-1 and -2 (PDGFR-α, -β). The primary antibodies were revealed with goat anti-mouse DyLight®488 (green) and goat anti-rabbit Cy3 (red), respectively. K58 served as a marker for the Golgi apparatus.—Bars, 50 μm; DAPI, 4′,6-diamidino-2-phenylindole.–Akt, protein kinase B; CD133, Prominin-1; CD44, cell surface receptor that engages extracellular matrix components such as hyaluronan; CDK6, cyclin-dependent kinase 6; EGFR, epidermal growth factor receptor; FABP7, fatty acid binding protein 7; MERTK, tyrosine-protein kinase Mer; Musashi, RNA binding protein; Nanog, DNA binding homeobox transcription factor; PDGFR-α, -β, platelet-derived growth factor receptor α and β; PTEN, phosphatase and Tensin homolog; p53, tumor suppressor p53; Sox2, SRY-box transcription factor 2; SC, derived from orthotopic tumor.</p

Pearson correlation coefficients (k): SLGC markers.

In (A) cell lines were ranked according to their Sox2 expression at the time of analysis. The Actin-normalized relative expression of the respective proteins is indicated in the plots. Group 1 displayed significantly higher Sox2 levels than Group 2. Group E comprises the established cell lines U87 and CaCo2, both of which lack Sox2 expression. The ranking was as follows: [T1452, T1371, T1495-SC, T1586, T1440, T1587, T1447-SC, T1495, T1447, T1338], [T1522, T1389, T1467, T1442, T1464, T1454, T1600, T1439], [CaCo2, U87]. In (B) cell lines were ranked according to their CD133 expression at the time of analysis. The Actin-normalized relative expression of the respective proteins is indicated in the plots. The CD133 levels were highest in the CaCo2 cell line, followed by the SLGCs assigned to groups with decreasing CD133-levels. The non-SLGC line CaCo2, which encompasses >90% of CD133 positive cells, is indicated on the very left. The ranking was as follows: [CaCo2], [T1452, T1333, T1495-Sc], [T1447-SC], [T1586, T1442, T1587, U87, T1600, T1464, T1447, T1495, T1440], [T1522, T1371, T1389, T1467, T1439, T1454].–For abbreviations, see the legends to Figs 5 and 6.</p

Expression of cell surface proteins.

(A, B, C) Flow cytometry analyses of SLGCs expanded in serum-free (N) and serum-containing (F) medium. Fluorochrome-coupled antibodies directed against CD133/Prominin-1, CD44, integrin α6/CD49f, and integrin αv/CD51, as well as the endothelial CAM (cell adhesion molecule) PECAM/CD31 or the endothelial cadherin VE-cadherin/CD144 were used. A minimum of 10,000 cells was analysed. Assays with corresponding N- and F cultures were done in parallel. Bars represent mean values, whiskers represent the variation between technical replicates. (D) Box plots summarizing the real-time PCR (qRT-PCR) analyses using primers directed against integrins (Int) or cadherins (Cdh). The plots indicate the mean values calculated from the qRT-PCR data of 12 distinct SLGC lines, in which each SLGC line was analyzed in three individual reactions. The standard deviations are indicated by the upper and lower borders of the boxes, and the median is symbolized by the central line. The range of minimal and maximal values is represented by the whiskers. The qRT-PCR values were normalized against the reference genes gapdh, ubiquitin ligase, and 18 s r-rna prior to calculation of the Box plots.</p

Characteristics of SLGC lines and correlations.

Glioblastoma multiforme (GBM) and the GBM variant gliosarcoma (GS) are among the tumors with the highest morbidity and mortality, providing only palliation. Stem-like glioma cells (SLGCs) are involved in tumor initiation, progression, therapy resistance, and relapse. The identification of general features of SLGCs could contribute to the development of more efficient therapies. Commercially available protein arrays were used to determine the cell surface signature of eight SLGC lines from GBMs, one SLGC line obtained from a xenotransplanted GBM-derived SLGC line, and three SLGC lines from GSs. By means of non-negative matrix factorization expression metaprofiles were calculated. Using the cophenetic correlation coefficient (CCC) five metaprofiles (MPs) were identified, which are characterized by specific combinations of 7–12 factors. Furthermore, the expression of several factors, that are associated with GBM prognosis, GBM subtypes, SLGC differentiation stages, or neural identity was evaluated. The investigation encompassed 24 distinct SLGC lines, four of which were derived from xenotransplanted SLGCs, and included the SLGC lines characterized by the metaprofiles. It turned out that all SLGC lines expressed the epidermal growth factor EGFR and EGFR ligands, often in the presence of additional receptor tyrosine kinases. Moreover, all SLGC lines displayed a neural signature and the IDH1 wildtype, but differed in their p53 and PTEN status. Pearson Correlation analysis identified a positive association between the pluripotency factor Sox2 and the expression of FABP7, Musashi, CD133, GFAP, but not with MGMT or Hif1α. Spherical growth, however, was positively correlated with high levels of Hif1α, CDK4, PTEN, and PDGFRβ, whereas correlations with stemness factors or MGMT (MGMT expression and promoter methylation) were low or missing. Factors highly expressed by all SLGC lines, irrespective of their degree of stemness and growth behavior, are Cathepsin-D, CD99, EMMPRIN/CD147, Intβ1, the Galectins 3 and 3b, and N-Cadherin.</div

GBM; glioblastoma multiforme; GS, gliosarcoma; GS*, recurrent gliosarcoma; suffix “SC”, indicates that the cell lines was established from an orthotopic tumor grown in a SCID mouse (SC2, was derived from xenotransplanted T1495-SC); PTEN, <i>Phosphatase and tensin homolog;</i> +/loss*, PTEN status in T1440 subpopulations is +/+, +/- or -/-; T1389 mutant*, subpopulations with mixed Tp53 status in exons 5 and 6; T1338 WT*, a subpopulation of T1338 cells is heterozygote for Tp53 mutation; RTK, receptor tyrosine kinase, IDH, isocitrate dehydrogenase; E, [EGFR] epidermal growth factor receptor; E^§, amplification of truncated EGFR; Pα, Pβ, platelet-derived growth factor [PDGF] receptors α and β; MERTK, tyrosine protein kinase Mer—n.t., not tested.

GBM; glioblastoma multiforme; GS, gliosarcoma; GS*, recurrent gliosarcoma; suffix “SC”, indicates that the cell lines was established from an orthotopic tumor grown in a SCID mouse (SC2, was derived from xenotransplanted T1495-SC); PTEN, Phosphatase and tensin homolog; +/loss*, PTEN status in T1440 subpopulations is +/+, +/- or -/-; T1389 mutant*, subpopulations with mixed Tp53 status in exons 5 and 6; T1338 WT*, a subpopulation of T1338 cells is heterozygote for Tp53 mutation; RTK, receptor tyrosine kinase, IDH, isocitrate dehydrogenase; E, [EGFR] epidermal growth factor receptor; E§, amplification of truncated EGFR; Pα, Pβ, platelet-derived growth factor [PDGF] receptors α and β; MERTK, tyrosine protein kinase Mer—n.t., not tested.</p

MGMT-status.

(A) RT-PCR analysis of mgmt mRNA expression; gapdh served as a control. T1371 was studied during passages p2, p6, and p26. (B, C) Examples of methylation-specific PCR (MSP) using gDNA from tumor tissue as well as SLGC lines (mc) or clones (cl) obtained by a limited dilution assay. M (methylated status), PCR using the primers MGMTmeth-F/MGMTmeth-R; U (unmethylated status), PCR using the primers MGMTun/MGMTun-R. Arrows point to the specific PCR products, *, labels unused primers/frequently seen byproducts of m- or u- status, including SLGCs as well as the non-SLGC GBM line U87 and the colon cancer cell line CaCo2, which served as controls in various experiments. (D) Summary of the quantification of the MSP data; mc/SC, SLGC/SCID lines; cl, clones derived from SLGC lines by means of limited dilution assays.–For mc/SC the mean values ± SEM from a minimum of three biological replicates are shown. For clones, the bars represent the mean values calculated from all clones together, and whiskers indicate the variations between the relative percentages of the PCR products determined for the clones. Altogether, six T1338, four T1371, five T1452, three T1447, six T1464, seven T1522 and five T1586 clones were included in the study. (E) Western blot analysis of the expression of the MGMT protein.–Altogether, six T1338, four T1371, three T1447, six T1464, seven T1522 and five T1586 clones were included in the study. (TIF)</p