Fibulin-3 Is Uniquely Upregulated in Malignant Gliomas and Promotes Tumor Cell Motility and Invasion

Fibulin-3 is highly expressed in glioma tissue and cells. A. Total homogenates from nine high-grade gliomas (two grade 3 astrocytomas and seven grade 3 glioblastomas) and seven age-matched controls were probed for fibulin-3 (arrow). *, nonspecific cross-reactivity of the antibodies used. B. Quantification of results from A; notice that fibulin-3 expression is virtually absent in normal brain compared with gliomas. Integrated optical density (IOD). The accompanying Western blot shows a representative subcellular fractionation of glioma tissue revealing the distribution of fibulin-3. Proliferating cell nuclear antigen, microsomal ribophorin, and soluble α-tubulin were used as specific markers for the nuclei-enriched (N), membrane-enriched (M), and soluble (S) subcellular fractions, respectively. C. Expression of fibulin-3 in the conditioned medium and total lysates of glioma cell lines (U87, U251, and U373), primary cultures of glioma xenografts (X12 and X14), and cultured normal human astrocytes (NHA). Equal protein loading for culture media was determined by protein measurement and by comparable amido black staining of the blot membranes prior to incubation with antibody. D. Analysis of relative migration of fibulin-3 from soluble fraction of glioma tissue (○, tissue) or medium from U251-MG glioma cells (▵, cells) indicated a single form of ∼54 kDa molecular weight, as expected. No other fibulin-3 bands were observed. *, antibody cross-reactivity as indicated above. E. Detection of fibulin-3 mRNA in human glioma cell lines, rodent glioma cell lines (rat CNS-1, mouse GL-261, and KR-158), and NHA. RT-PCR in the absence of template (right). F. Results from qRT-PCR show the comparative expression of fibulin-3 mRNA (columns, mean; bars, SEM) in NHA (control = 1) versus glioma cell lines and glioma neurospheres prepared from patient specimens (OG2, OG6, and OG12) as indicated in Materials and Methods. Gliomaspheres cultured in serum-free conditions express fibulin-3 at levels comparable to those of typical glioma cell lines, suggesting that expression of fibulin-3 in culture is not caused by serum-induced mesenchymal drift.

Fibulin-3 has a predominant isoform in glioma cells. A. Two possible isoforms of human fibulin-3 have been proposed: Fib3L (long, 53-55 kDa) and Fib3S (short, 43-45 kDa). B. Both isoforms were created by PCR, tagged with a COOH-terminal V5 epitope, and expressed in rat CNS-1 glioma cells. Blots were probed with anti-V5 antibody and with antibody against human fibulin-3 that does not cross-react with endogenous rat fibulin-3. Results obtained with both antibodies strongly suggest that recombinant Fib3S is produced much less efficiently than Fib3L and secreted at very low or undetectable amounts to the culture medium. Note that the V5 antibody detected a number of faint bands above the position of Fib3L in the culture medium, which may correspond to glycoforms not detected by the anti–fibulin-3 mAb3-5 antibody.

Expression of fibulin-3 increases substrate-dependent glioma cell adhesion. A. Human (U87MG and U251MG) and rat (CNS-1) glioma cells stably overexpressing fibulin-3 were plated and quantified on multiwell plates coated with fibronectin (FN), type I laminin (LN), high–molecular weight hyaluronic acid (HA), or left uncoated and blocked with bovine albumin (BSA). B. Nontransfected cells were plated as above on multiwell plates coated with purified fibulin-3 (white columns) or left uncoated (BSA, black columns). Fibulin-3 did not act as a proadhesive substrate for any of the cell lines tested. All experiments were repeated at least thrice with three to six replicates per condition. Data (columns, mean; bars, SEM) were analyzed by two-way ANOVA (***, P < 0.001). Results on poly-l-lysine–coated wells were undistinguishable from those on uncoated surfaces (data not shown). C. Relative expression levels of fibulin-3 in conditioned medium from control and fibulin-3–overexpressing U87MG and U251MG glioma cells were determined by Western blotting and confirmed to be within the physiologic range observed in clinical specimens, as described in Materials and Methods. Overexpression of fibulin-3 in transfected rat CNS-1 cells was quantified by qRT-PCR (data not shown) and found comparable with mRNA overexpression levels in transfected human glioma cells.

Fibulin-3 enhances radial glioma cell dispersion and individual cell migration. A. Representative image of control and fibulin-3–expressing cell aggregates (CNS-1 cells) seeded on brain slices and cultured for 5 d (bars, 200 μm). B. The dispersion of the invasive cell lines CNS-1 and X12 on brain slices was analyzed by two-way ANOVA for repeated measures followed by post hoc Bonferroni's test (**, P < 0.01; ***, P < 0.001). Area ratio = area occupied by cells at each time point relative to the original area (points, mean; bars, SEM). C. Semiautomated tracking of CNS-1 cells was used to calculate mean cell velocity (average of frame-to-frame velocity for each cell) and net migrated distance (net difference in cell position between first and last frame). Representative tracks of individual cells imaged overnight. Mean cell velocities remained stable but showed a trend to increase toward the end of the cell-tracking experiment, probably due to cumulative tissue disruption by migrating cells (bar, 200 μm).

Knockdown of fibulin-3 reduces glioma cell adhesion and motility. A. Reduction of fibulin-3 in the conditioned medium of the cell line U87MG after transient transfection with two independent siRNA sequences. Housekeeping control proteins were quantified in cell lysates. Total protein content was 15 μg/lane in all cases; samples were processed 48 h after transfection. B. Knockdown of fibulin-3 in the cell line CNS-1, monitored by qRT-PCR, following the same procedure as in A. C. Transient knockdown of fibulin-3 reduces cell adhesion to all substrates tested. Transfected cells were analyzed as indicated in Fig. 4. Black columns, control siRNA; white columns, fibulin-3 siRNA (***, P < 0.001 by two-way ANOVA). Results were virtually identical with the cell line U251MG (data not shown). D. CNS-1 cells were transiently transfected with fibulin-3 siRNAs 48 h before seeding on cultured brain slices. Follow-up over 5 d, as indicated in Fig. 5, showed a significant reduction in the total area occupied by dispersed cells (*, P < 0.025, ***, P < 0.001 by two-way ANOVA for repeated measures).

Fibulin-3 increases tumor dispersion in vivo. A. Representative control and fibulin-3–overexpressing tumors 15 d after intracranial injection of CNS-1 cells revealed by Nissl staining (top images) or detection of GFP-fluorescent tumor cells (bottom images; bar, 1 mm). Fibulin-3 tumors are larger and less compact than controls. B. Higher magnification of Nissl-stained tumors. Fibulin-3–overexpressing tumors show more diffuse borders and large clusters separated from the tumor mass (bar, 200 μm). C. Tumor area (in mm²) was quantified every fourth section and plotted against the rostrocaudal extension of the tumor, centered on the injection site. Notice the increase in caudal invasion of fibulin-3–expressing tumors, probably due to invasion of thalamoreticular tracts. D. Cell clusters were counted and measured every fourth section as described (61) and their cumulative area quantified using image analysis software (***, P < 0.001 by Student's t test). E. Total tumor volumes were calculated using Cavalieri's estimator of morphometric volume and compared by Student's t test (**, P < 0.01).

Expression of fibulin-3 does not affect cell proliferation. A. Representative staining for the proliferation marker Ki67 around the core of control or fibulin-3–overexpressing tumors formed by CNS-1 cells. The proliferative index (percentage of Ki67-positive nuclei relative to total number of 4′,6-diamino-2-phenylindole–stained nuclei in each field; columns, mean; bars, SEM) was calculated by automated scoring of at least 300 nuclei per field (using ImageJ software) and compared by Student's t test (P = 0.228, no significant differences). Note the higher cell density in control tumors (bars, 50 μm). B. Proliferation rates of CNS-1 cells stably transduced with fibulin-3 (•) or a control (○) cDNA were analyzed using a metabolic assay for reduction of tetrazolium. Proliferation curves were repeated twice using triplicates for each day. The absence of significant differences was verified in a second glioma cell line (U251MG).