Targeting the c-Met Pathway Potentiates Glioblastoma Responses to γ-Radiation

Materials and Methods

U1/ribozymes and expression vectors. Anti-SF/HGF and anti-c-Met U1snRNA/ribozyme/antisense chimeric transgenes (U1/ribozymes) and their expression vectors were synthesized and extensively characterized as previously described (22, 23). Endotoxin-free anti-SF/HGF, anti-Met, and control U1/ribozyme expression plasmids (designated pU1/SF, pU1/Met, or pU1/control, respectively) were complexed with liposomes containing the cationic lipid DOTIM (1-[2-[9(Z)-octadecenoy]-ethyl][-2-]([8](Z)-heptadecenyl]-3-[hydroxyethyl]imidazolinium chloride) and cholesterol (kindly provided by Dr. Gary Koe, Valentis, Inc., Burlingame, CA) for i.v. delivery to animals bearing s.c. tumors as previously described (23). Replication-defective adenoviruses expressing anti-SF/HGF and anti-Met U1/ribozymes or control U1 sequences (designated Ad-U1/SF and Ad-U1/Met and Ad-U1/control, respectively) were constructed and produced as previously described (23). Viral preparations purified by ultracentrifugation and dialysis and titered by plaque forming assays at ∼10¹² plaque-forming unit/mL were kindly provided by Dr. Andrea Gambotto (NHL Vector Core Facility, Human Gene Therapy Center, University of Pittsburgh).

Northern hybridization and immunoblottting. U87 MG human malignant glioma cells were cultured in Eagle's MEM (Cellgro Mediatech, Washington, DC) containing 10% fetal bovine serum (Cellgro Mediatech), 0.15% sodium bicarbonate (Life Technologies, Rockville, MD), 1 mmol/L sodium pyruvate (Life Technologies), 0.1 mol/L nonessential amino acids (Life Technologies), and 500 μg/mL penicillin-streptomycin (Life Technologies) at 37°C, 5% CO₂/95% air. Subconfluent cell monolayers received Ad-U1 control, Ad-U1/SF, or Ad-U1/Met (∼100 multiplicity of infection). Approximately 48 hours later, cells were harvested and total cellular RNA was purified as previously described (22). Northern analysis using ³²P-labeled cDNA probes specific for either human SF/HGF, human c-Met, or glyceraldehyde-3-phosphate dehydrogenase was done as previously described (22). For analysis of SF/HGF and c-Met protein, cells received adenovirus twice 24 hours apart and total protein was isolated in the presence of protease inhibitors 48 hours later. Western immunoblot analysis of total cellular protein (30 μg per lane) using anti-SF/HGF and anti-c-Met antibodies (Santa Cruz, Inc., Santa Cruz, CA) was done as previously described (22, 23).

In vitro cell viability. U87 MG glioblastoma cells were seeded at a density of 2,000 cells per well in 12-well tissue culture plates. After 24 hours, adherent cells were treated twice at 24 hours intervals with either Ad-U1/control or Ad-U1/ribozyme (∼100 multiplicity of infection). At 72 hours following the first adenoviral treatment, cells were subjected to 20 Gy γ-radiation (radiation source = ¹³²Cesium GammaCell Irradiator; Atomic Energy of Canada, Ltd., Mississauga, Ontario, Canada) or mock irradiated. In a subset of experiments, cultures received the caspase inhibitor Z-VAD-FMK (Sigma, St. Louis, MO) to a final concentration of 50 μmol/L (or buffer only as control) 30 minutes before the first adenoviral vector treatment and again 30 minutes before radiation treatment. At the times indicated, adherent cells were suspended by trypsinization and counted using a Beckman Coulter Counter (Fullerton, CA). Terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) immunocytochemistry was done on the pooled populations of adherent and floating cells as previously described (15).

Tumor implantation and animal treatments. For s.c. tumors, U87 MG cells (4 × 10⁶ per animal) were implanted in the flanks of athymic nude mice (n = 10). When the tumors reached a size of ∼25 mm³, the animals began treatment with liposomes complexed with pU1/SF + pU1/Met ribozymes (1:1 mixture) or pU1/control (30 μg total DNA in 100 μL) with or without radiation (300 cGy/dose) delivered by a ¹³²Cesium irradiator. Liposome/DNA complexes were delivered by tail vein injection on days 5, 15, and 25 following tumor implantation as previously described (23). Tumors were irradiated on days 7, 12, 17, 22, and 27 following tumor implantation. Tumor sizes were measured every 5 days with calipers and volumes were calculated using the formula: volume = (length × width²) / 2 (9). Animals were sacrificed if tumor volume reached ∼200 mm³. For intracranial tumors, SCID/beige mice were implanted with transcranial cannulae (Plastics One, Inc., Roanoke, VA) with the proximal end within right caudate/putamen. Cannulae were used for tumor implantation and adenovirus delivery to insure precise and replicable delivery of adenoviruses directly into the tumors (24). U87 MG cells (1 × 10⁵ per animal) were implanted (n = 20) 7 days after cannulae placement. The animals subsequently received intratumoral injections of Ad-U1/SF + Ad-U1/Met (1:1 mixture) or Ad-U1/control (5 × 10⁷ plaque-forming unit in 5 μL) with or without radiation (300 cGy/dose). Adenoviruses were delivered on days 5 and 15 and radiation on days 8, 12, and 18 following tumor cell implantation. Ten mice from each treatment group were sacrificed on day 21 and brains were removed for histologic studies. The remaining 10 mice per group were monitored for survival.

Histology and immunohistochemistry. Tumor volumes were quantified by measuring tumor cross-sectional areas on 20-μm-thick H&E-stained cryostat sections from perfusion-fixed brains using computer assisted image analysis as previously described (9). Tumor cell proliferation was assessed by Ki-67 immunohistochemistry. TUNEL immunohistochemistry was done with the colorimetric TUNEL assay system (Promega, Madison, WI) according to the manufacturer's instructions. Activated caspase-3 and Ki-67 immunohistochemistry was conducted using anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) and anti MIB-1 antibodies (Dako Corp., Carpinteria, CA), respectively, as previously described (22, 23). Apoptotic and cell proliferation indices were determined by computer assisted quantification of the number of positively stained cells per microscopic field as previously described (22, 23).

Statistical analysis. Comparisons were analyzed using ANOVA followed by a post hoc Fisher's PLSD test for histopathologic end points and Bonferoni-Dunn for in vitro cell culture end points. Survival studies were analyzed with Kaplan-Meier.

Results

Scatter factor/hepatocyte growth factor and c-Met expression inhibition potentiates glioma cell radiation injury in vitro. U87 MG malignant glioma cells were treated with replication-defective adenoviral vectors coding for anti-SF/HGF or anti-Met U1/ribozymes, or with U1 lacking the antisense/ribozyme sequences as control. As shown previously (23), adenoviral vector-based delivery of anti-SF/HGF and anti-Met U1/ribozymes substantially and specifically reduced SF/HGF and c-Met mRNA levels by ∼80% to 95% (Fig. 1A). Cell-associated SF/HGF protein levels were also inhibited by ∼80% in cells treated with anti-SF/HGF U1/ribozymes (Fig. 1B). Treatment with anti-Met U1/ribozyme reduced cell-associated c-Met protein levels by 50% to 60%, consistent with the relatively longer half-life of this transmembraneous receptor in comparison with its secreted ligand (Fig. 1B).

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Fig. 1.

Chimeric U1/ribozymes inhibit glioma cell expression of SF/HGF and c-Met. U87 MG human glioblastoma cells were treated with replication-defective adenovirus coding for anti-SF/HGF U1/ribozyme (Ad-U1/SF), anti-Met U1/ribozyme (Ad-U1/Met), or control (Ad-U1). Total cellular RNA and protein were isolated and subjected to Northern and Western analyses, respectively, using specific cDNA probes and antibodies for human SF/HGF and c-Met as described in Materials and Methods. Densitometric analyses (data not shown) show that U1/ribozymes reduced cell levels of the targeted mRNA by ∼80% to 95% relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SF/HGF and c-Met proteins were reduced by ∼80% and ∼50% to 60%, respectively.

We examined the effects of SF/HGF and c-Met expression knockdown on the response of U87 MG cells to γ-radiation (Fig. 2). Cultures were pretreated with either Ad-U1/control or active Ad-U1/ribozymes (1:1 mixture of anti-SF/HGF and anti-Met U1/ribozymes) on culture days 1 and 2 followed by either γ-radiation (20 Gy) or mock radiation as shown in Fig. 2A. As shown in Fig. 2B, cells were statistically significantly more sensitive to γ-radiation following SF/HGF and c-Met expression knock down when compared with controls as evidenced by the number of viable adherent cells remaining 96 hours after radiation relative to the number of cells immediately before radiation (43% versus 84%, respectively; P < 0.005). These quantitative data was supported by the appearance of substantially more floating cells following radiation in the U1/ribozyme-treated cultures (data not shown). TUNEL analysis showed statistically significantly more apoptotic cell death in cultures treated with both U1/ribozymes and radiation than in cultures treated with either modality alone (P < 0.001; Fig. 2C). Pretreating cells with the pan-caspase inhibitor Z-VAD-FMK resulted in 3- to 4-fold more cytoprotection (P < 0.005) in cells treated with U1/ribozymes + radiation injury in comparison with cells treated with either U1/ribozymes alone or radiation alone (Fig. 2D). These results show that U1/ribozyme-based targeting of the endogenous autocrine SF/HGF/c-Met pathway sensitizes U87 MG glioma cells to radiation cytotoxicity and apoptosis and synergistically activates radiation-induced caspase-dependent cell death.

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Fig. 2.

A, growth curve of U87 MG cells treated with Ad-U1/control (-▪-), Ad-U1/ribozyme (1:1 mix of anti-SF/HGF and anti-Met Ad-U1/ribozymes; - ▴-), Ad-U1/control plus radiation (-□-), and Ad-U1/ribozyme plus radiation (-▵-) as described in Materials and Methods. Cells were treated with viral vectors on days 1 and 2 and irradiated on day 4 (20 Gy). The adherent cells were counted daily. B, cells were treated as in (A); the number of adherent cells 24 to 96 hours after radiation was compared with that immediately before radiation. The percentage cell number relative to cell numbers before radiation (set at 100%) is shown. Radiation alone and Ad-U1/ribozyme alone induced a slight cell number loss by 96 hours (14% and 16%, respectively). Combining Ad-U1/ribozyme and radiation caused a substantially greater cell loss (57%) compared with either treatment alone, *, P < 0.005). C, cultures were treated as in (A) and the adherent and floating cells at day 8 were collected and subjected to TUNEL staining for detection of apoptosis. Percentage of TUNEL-positive cells in each treatment group above that measured in the Ad-U1/control group is shown. Combining Ad-U1/ribozymes with radiation generated significantly more TUNEL-positive cells than either treatment alone (*, P < 0.001). D, cells were treated with the caspase inhibitor Z-VAD-FMK (50 μmol/L) or buffer prior to exposure to U1/ribozymes and radiation as described in Materials and Methods. The change in adherent cells between culture days 4 and 8 (i.e., following radiation) was determined for each experimental group. Z-VAD-FMK had a 4- to 5-fold greater cytoprotective effect on cells exposed to Ad-U1/ribozyme + radiation in comparison to cells exposed to either Ad-U1/ribozyme or radiation alone (*, P < 0.005). Mean increase in adherent cells measured 4 days after radiation in the presence of Z-VAD-FMK relative to control (i.e., no Z-VAD-FMK treatment) is shown for each experimental group. Experiments were repeated three times with representative results shown; bars, ± SE.

Systemic scatter factor/hepatocyte growth factor and c-Met U1/ribozymes synergize with γ-radiation against s.c. glioma xenografts. We have previously shown that i.v. liposome-based U1/ribozyme delivery reduces the levels of both SF/HGF and c-Met mRNA in U87 glioma xenografts (22, 23). We used this same regimen to determine if targeting the SF/HGF/c-Met pathway with U1/ribozymes sensitizes glioma xenografts to γ-radiation in vivo, similar to that observed in vitro. Animals bearing preestablished s.c. U87 MG glioma xenografts underwent a treatment regimen consisting of i.v. injections of liposomes complexed with either control plasmid DNA (pU1/control) or plasmid DNA coding for anti-SF/HGF + anti-Met U1/ribozymes (1:1 mixture) interspersed with fractionated γ-irradiation (300 cGy/dose) or mock irradiation as described in the Materials and Methods and Fig. 3A. All therapy ended on post-implantation day 27. Tumor growth rates were inhibited in animals treated with either radiation alone or U1/ribozymes alone. Neither treatment alone stopped tumor growth or caused tumor regression. Tumor growth returned to control rates following a brief delay after the last dose of each of these single treatment modalities. In contrast, tumors in animals treated with both U1/ribozymes and radiation regressed with no evidence of tumor growth through post-glioma cell implantation day 46, 20 days following the last day of therapy. Furthermore, 40% of animals treated with both U1/ribozymes and radiation experienced complete tumor regression. These animals were euthanized on post-implantation day 60, autopsied, and examined carefully for tumor. None was found to have detectable tumor. Thus, combining radiation with U1/ribozyme therapy markedly enhanced the antitumor response in a highly cooperative and potentially synergistic fashion.

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Fig. 3.

Anti-SF/HGF and anti-Met U1/ribozymes markedly enhance the antitumor response to γ-radiation in preestablished s.c. and intracranial glioblastoma xenografts. S.c. (A) and intracranial (B) human U87 MG glioblastoma xenografts (n = 10) were initiated on day 0 as described in Materials and Methods. Beginning on post-implantation day 5, animals received control expression vectors (U1/control) or active anti-SF/HGF + anti-c-Met U1/ribozymes (1:1 ratio) followed by either γ-radiation (300 cGy/dose) or mock irradiation (arrows) as described in Materials and Methods. A, s.c. tumor growth was quantified by serial caliper measurements. Animals without measurable subcutaneous tumor on post-implantation day 60 were sacrificed and subjected to autopsy. Those without histopathological evidence of tumor (40% of animals treated with the combination of U1/ribozyme + radiation) were designated cured. B, Kaplan Meier survival curves of animals bearing orthotopic intracranial tumor xenografts treated by direct intratumoral injection of Ad-U1/ribozymes ± radiation (arrows) as described in Materials and Methods. Statistically significant survival prolongation resulted from active U1/ribozymes alone (P < 0.03) and from the combination of U1/ribozymes + radiation (P < 0.0005).

Intratumoral scatter factor/hepatocyte growth factor and c-Met U1/ribozymes synergize with γ-radiation to inhibit intracranial glioma growth and enhance long-term animal survival. We next asked if anti-SF/HGF and anti-Met U1/ribozymes would enhance the antitumor effects of γ-radiation in an orthotopic intracranial glioma. Intracranial glioma xenografts were established as described in Materials and Methods. Animals began treatment 5 days later with radiation, intratumoral adenoviruses expressing U1/ribozymes (anti-SF/HGF + anti-Met, 1:1 mixture), or a combination of both radiation and U1/ribozymes as described in Materials and Methods. Animal survival was monitored and Kaplan-Meier survival curves are shown in Fig. 3B. Treatment with either radiation or U1/ribozymes alone resulted in modest prolongations in median survival from 30 days in controls to 45 and 53 days in each monotherapy treatment group, respectively (P < 0.1 for radiation and P < 0.03 for U1/ribozymes compared with control). There were no long-term survivors in control animals or in animals treated only with radiation. U1/ribozyme therapy resulted in 20% long-term survival. Animals treated with both radiation and U1/ribozymes experienced a marked improvement in survival (P < 0.0005). Eighty percent of animals treated with this combination survived through post-implantation day 92 at which time all surviving animals were sacrificed for histopathologic examination. In addition, a subset of animals were sacrificed on post-implantation day 21 (3 days following completion of therapy) and their brains were subjected to histologic examination. Tumor sizes on post-implantation day 21 were consistent with the animal survival responses (Fig. 4A and B). There was a trend toward smaller tumors in response to radiation alone (P < 0.28). Tumors were modestly but statistically significantly smaller in response to U1/ribozyme monotherapy (P < 0.04) and markedly smaller in response to combination therapy (P < 0.01). None of the long-term survivors sacrificed at post-implantation day 92 was found by histologic examination to harbor active tumor (Fig. 4C).

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Fig. 4.

Histopathologic analysis of antitumor responses in animals bearing intracranial glioblastoma xenografts. Mice received U87 MG human glioblastoma cells by intracranial injection on day 0. Mice were then treated with Ad-U1/control or Ad-U1/ribozymes ± γ-radiation (300 cGy/dose) from post-implantation days 5 to 18 as described in Materials and Methods and in Fig. 3B. A and B, animals (n = 10) were sacrificed on post-implantation day 21 and tumors analyzed in 20-μm-thick H&E-stained brain sections. A, representative brain sections show tumors that are demarcated by arrows. Encephalomalacia adjacent to tumor resulted from withdrawal of the transcranial cannula during tissue processing. B, computer-assisted tumor size quantification. *, P < 0.01, compared with U1/control group. C, animals surviving beyond 90 days in the experiment shown in Fig. 3B were sacrificed on post-implantation day 92. Brain sections (20-μm-thick) from all animals were stained with H & E and examined for tumor. A representative animal treated with the combination of active U1/ribozymes and radiation reveals no evidence of surviving tumor.

Scatter factor/hepatocyte growth factor/c-Met pathway targeting and γ-radiation cooperatively inhibit tumor cell proliferation and synergistically enhance tumor cell apoptosis. We show above that combining anti-SF/HGF and anti-Met U1/ribozymes with radiation causes glioma regression, durable antitumor responses, and a high percentage of long-term survivors. These in vivo antitumor responses suggested that targeting the SF/HGF/c-Met pathway in conjunction with radiation enhances tumor cell growth arrest and/or tumor cell death. To examine these possible mechanisms in more detail, animals bearing intracranial tumors were treated with either U1/ribozymes, radiation, or both as described above. Tumor cell proliferation was identified by anti-MIB1 (Ki-67) immunohistochemistry. The tumor proliferative index was not affected by radiation alone, was modestly reduced by ∼28% in response to U1/ribozyme monotherapy, and was more substantially reduced by ∼68% in response to combination therapy (Fig. 5). Thus, these two therapies cooperated to inhibit tumor cell proliferation.

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Fig. 5.

SF/HGF/c-Met targeting potentiates the effect of γ-radiation on tumor cell proliferation inhibition. Mice received U87 MG cells by intracranial injection on day 0. They were then treated with control vectors or active U1/ribozymes ± radiation and sacrificed on post-implantation day 21 as described in Fig. 3B. Tumors were then examined for cell proliferation using Ki-67 immunohistochemistry as described in Materials and Methods (A). The number of labeled cells per high-powered microscopic field was quantified by computer-assisted image analysis. *, P < 0.0001 compared with Ad-U1/control. +, P < 0.001 compared with either Ad-U1/ribozyme or Ad-U1/ribozyme + radiation.

We also examined the effect of radiation alone, U1/ribozymes alone, and their combination on in vivo tumor cell apoptosis using TUNEL and anti-cleaved caspase-3 immunohistochemistry. Each treatment alone increased ∼3-fold the percentage of cells containing detectable activated cleaved caspase-3. Combining the two treatments increased this index of apoptosis activation by ∼40-fold (Fig. 6). Similar results were seen using TUNEL staining. Monotherapy with either radiation or U1/ribozyme therapy alone increased the percentage of TUNEL-positive tumor cells 2- and 3-fold, respectively. Combination therapy increased the TUNEL index 14-fold (Fig. 6). These findings are consistent with a synergistic activation of glioma cell apoptosis.

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Fig. 6.

SF/HGF/c-Met targeting and γ-radiation synergistically enhance tumor cell apoptosis. Intracranial U87 MG xenografts were established and treated as in Fig. 5. Post-implantation day 21 tumors were then examined for cell apoptosis by imunohistochemistry for activated caspase-3 (A) and TUNEL staining (C). Cell labeling was quantified by computer-assisted image analysis (B and D). Differences in blue-green staining between panels in (A) represent variations in methyl green counterstaining. As expected anti–activated caspase-3generates a predominantly cytoplasmic stain relative to TUNEL that labels condensed/fragmented nuclei. *, P < 0.0001.

Discussion

There are likely multiple mechanisms for the enhanced tumor cell growth arrest and death observed in vivo in this study. Glioma cell–derived SF/HGF activates c-Met receptors present on the tumor cells in an autocrine fashion and on host parenchymal cells (e.g., endothelial cells), in a paracrine fashion. Interfering with the autocrine loop is expected to directly down-regulate glioma cell cycle and cytoprotective pathways (6). The in vitro antiproliferative and cell death responses of U87 MG cells to autocrine loop inhibition using U1/ribozymes in this present study essentially confirm that such direct antitumor effects can occur. These results are also consistent with our previous findings that knocking down c-Met receptor expression in glioma cells lacking an autocrine loop blocks the ability of recombinant SF/HGF to protect against chemotherapy-induced cytotoxicity (5). Inhibiting the paracrine SF/HGF/c-Met loop in tumor xenografts including U87 MG glioma generates an antiangiogenic response that can also enhance tumor responses to cytotoxic therapeutics in vivo as originally proposed by Teicher et al. and recently confirmed in clinical trial (25–28). The relative contributions of autocrine and paracrine pathway inhibition to the potentiation of γ-radiation by SF/HGF/c-Met pathway targeting have not been specifically defined and may vary between tumor types, organ sites, and mode of pathway inhibition (i.e., systemic versus direct intratumoral).

We found that anti-SF/HGF and anti-Met U1/ribozymes enhance antitumor radiation responses in both s.c. and intracranial tumor xenografts, tissue sites with distinct environmental features. These distinctions include vascular phenotype, immunologic environment, extracellular matrix, and the nature of peritumoral mesenchymal cell types (e.g., glial cells being unique to the central nervous system). Thus, the radiosensitizing response to SF/HGF/c-Met pathway inhibition in other c-Met-dependent tumors, many of which commonly metastasize to multiple organ sites, is not likely to be organ specific. Our findings also show that the therapeutic responses to the U1/ribozymes are independent of delivery route (direct intratumoral or systemic) and specific delivery system (plasmid DNA/liposome complexes versus adenovirus). Therefore, nonspecific affects such as those potentially seen from vector-specific host interactions are not likely to account for the antitumor responses observed in our experiments.

The molecular mechanisms by which SF/HGF directly protects against DNA damaging agents have been examined in multiple malignant and nonneoplastic cell lines (15–17). Cytoprotection partially requires signaling through phosphatidylinositol-3′-kinase and the downstream serine/threonine kinase c-Akt, a pathway potently activated by c-Met, in breast carcinoma, glioblastoma, and nonneoplastic Madin-Darby canine kidney cell epithelial cells. Activation of this pathway is accentuated in cells lacking the dual phosphatase MMAC1/PTEN (mutated in multiple aggressive cancers/phosphatase and tensin homologue), a tumor suppressor frequently absent in glioblastoma multiforme and absent in the U87 MG cell line used in this study. Fan et al. (29) have recently found that nuclear factor-κB downstream of c-Akt- and p21-activated kinase (Pak-1) can be additional mediators of SF/HGF-dependent cytoprotection. This recent study further implicated nuclear factor-κB–mediated transcription of at least two genes, TRAF-2 and cIAP-2, in tumor cytoprotection by SF/HGF. A comprehensive understanding of c-Met-dependent cytoprotection, particularly the transcriptional-dependent aspects, remains to be fully determined.

In summary, we show that a molecular therapeutic strategy that targets SF/HGF and c-Met generates potent antitumor activity when combined with γ-radiation. End points supporting the cooperative, and by certain criteria, synergistic antitumor response from combining these two modalities include the regression and apparent cure of s.c. glioma xenografts, the long-term survival and apparent cure of animals bearing intracranial glioma xenografts, and histologic evidence of tumor cell growth arrest and apoptosis. The pronounced cooperative therapeutic responses presented here are significant and timely in light of the expectation that pharmacologic inhibitors of c-Met activation, such as small molecule c-Met kinase inhibitors, receptor antagonists such as truncated SF/HGF proteins (e.g., NK4), decoy soluble chimeric c-Met receptors, or humanized neutralizing anti-SF/HGF and anti-c-Met antibodies, will soon be available for clinical testing (30–33). Our results highlight the need to ultimately evaluate the clinical efficacy of such reagents in conjunction with other cytotoxic regimens such as ionizing radiation therapy and chemotherapy. These would be particularly practical treatment strategies in patients with newly diagnosed malignant glioma, essentially all of who receive radiation often with chemotherapy as first-line therapy (34).