VEGF-D expression in brain cancer

VEGF-D serves as a target for diagnosing and treating glioblastoma multiforme and related brain cancers. Cancer in a brain tissue sample is detected by analyzing expression of VEGF-D in the sample. Brain cancer is treated by modulating VEGF-D gene expression in cells of the cancer, and by inhibiting angiogenesis associated with the cancer by interfering with VEGF-D binding to a VEGF-D receptor.

EXAMPLES The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and are not to be construed as limiting the scope or content of the invention in any way. 
 Example 1 
 Materials and Methods Cell lines and tissues: Glioblastoma multiforme cells lines A-172 MG, U-251 MG, DBTRG-50 MG, U-87 MG, U-373 MG, human GBM explant cells, G48a, CSML0 and CSML110 mouse breast carcinoma cells were grown in appropriate media. The CSML0 and CSML100 mouse breast cancer cells and GBM A172 MG glioblastoma cells were grown in Dulbecco's Modified Eagle's Medium (D-MEM) with 10% Fetal Calf Serum (FCS) (Life Technologies, Rockville, Md.). U-251 MG cells were grown in D-MEM, 10% FCS, 0.1 mM MEM Non-Essential Amino Acids (NEAA) (Life Technologies), and 50 &mgr;g/ml Gentamicin Sulfate. GBM cell lines U-87 MG and U-373 MG were grown in Earle's Minimum Essential Medium (MEM), 10% FCS, 0.1 mM NEAA, 2 mM Glutamine (Life Technologies), and 100 &mgr;g/ml Sodium Pyruvate. GMB cell line DBTRG-50 MG and human explant cells were grown in RPMI-1640 (Life Technologies) 10% FCS, 100 &mgr;g/ml Sodium Pyruvate, 100 &mgr;g/ml L-Cystine (Life Technologies), 20 &mgr;g/ml L-Proline (Sigma), 1× HT Supplement, consisting of 0.1 &mgr;M Sodium Hypoxanthine and 0.016 &mgr;M Thymidine, 5 units/ml Pennicilin G and 5 units/ml Streptomycin sulfate (Penn/Strep) (Life Technologies). Normal Human Astrocytes (NHA) were grown in Astrocyte Growth Medium BulletKit R (BioWhittaker). Normal HUV-EC-C were grown in F-12 Kaighn's medium (Life Technologies) with 10% FCS, 100 &mgr;g/ml Heparin (Sigma) and 30 &mgr;g/ml Endothelial Cell Growth Supplement (ECGS) (Sigma). A retroviral vector was used to generate plasmid pMVfra-1. To produce replication-defective retroviruses, the GP&plus;E packaging cell line was employed, which was maintained in appropriate media. Successfully transfected GP&plus;E cells were selected in the presence of 800 &mgr;g/ml G418. Supernatants of virus-producing cell lines were used to infect CSML0 cells. Infected cells were selected in the presence of 400 &mgr;g/ml G418. GBM tumors and non-malignant brain tissue, the latter obtained usually from the therapeutic resections for the treatment of epilepsy, were obtained from the operating room and snap frozen immediately, as described previously. Debinski et al. (1999) Clin. Cancer Res. 5: 985-990. Ten-micron sections of GBM were thaw-mounted onto chrom-alum slides. Slides were stored at −80° C. until assayed. Sections were allowed to thaw and subsequently fixed for 10 min in acetone at −20° C. Immunostaining: GBM cells lines, human explant cells (G48a), Human Umbilical Vein Endothelial Cells (HUV-EC-C) from ATCC (Rockville, Md.), and normal human astrocytes (NHA) from BioWhittaker (Walkersville, Md.) were grown overnight on sterile glass slides in the appropriate media. Slides were washed twice in PBS and fixed for 2 min in acetone at −80° C. Slides were washed twice in PBS and either used immediately or air-dried and stored at −80° C. until assayed. In stimulation experiments, 10 4 SNB-19 GBM cells were plated on glass chamber slides and allowed to attach overnight. The cells were washed with PBS and serum-free media was applied. After 24 hr epidermal growth factor (EGF) or leukemia inhibitory factor (LIF) were added to cells at 5 and 20 ng/ml, respectively. The cells were processed for immunocytochemistry after 24 hr of stimulation period. Mouse monoclonal anti-VEGF-D (VD1) antibody was used. See, Achen et al. (2000) Eur. J. Biochem. 267: 2505-2515. It was employed at a final dilution of 1:500 (7.5 &mgr;g/ml). Other primary antibodies including rabbit polyclonal Fra-1 (1:100), c-Fos (1:100), c-Jun (1:150), and mouse monoclonal JunB (1:75) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.); and mouse monoclonal Factor VIII (1:150) and rabbit GFAP (1:500) were purchased from DAKO Chemical (Carpinteria, Calif.). Slides were washed in two changes of PBS and blocked for 30 min with 10% (v/v) normal goat serum (NGS) in PBS at room temperature. Primary antibody was diluted in 1.5% NGS/PBS and incubated at room temperature for either 1 hr (VEGF-D, Factor VIII, and GFAP) or 2 hr (Fra-1, JunB, c-Fos, and c-Jun). Slides were washed in three changes of PBS for 10 min each. Secondary antibody, goat anti-rabbit Rhodamine (1:150), Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa.) or sheep anti-mouse Cy3 (1:250), Sigma (St. Louis, Mo.) was diluted in 1.5% NGS/PBS and incubated in the dark at room temperature for 45 min. For double-labeling experiments, the secondary antibodies were goat anti-mouse Oregon Green R (1:200) (Molecular Probes, Oregon) and goat anti-rabbit Rhodamine (1:150). Slides were washed in 3 changes of PBS for 10 min each and mounted with Gel-Mount, Biomeda Corp. (Foster City, Calif.). Some slides were counterstained with Hoechst No. 33258 Nuclear Counterstain (DAPI). Photomicrographs were taken at 40× magnification in all cases with a Hamamatsu C2400 digital camera. Background was normalized to the samples without primary antibody. Each set of images was taken exactly at the same exposure settings. Images were processed with Paint Shop Pro V 6.0 (Jasc software Inc., Eden Prairie, Minn.). Western Blots: Cell lysates were prepared from sub-confluent cultures. Cells were washed twice in PBS and lysed in RIPA buffer (PBS, 1% Igepal CA-630; ICN Biomedicals, Inc. Costa Mesa, Calif.), 0.5% sodium deoxycholate (Fisher Scientific, Fair lawn, N.J.), 0.1% SDS containing Mammalian Protease Inhibitor Cocktail (Sigma). GBM and non-malignant brain tumor samples were minced into small pieces while frozen and thawed in RIPA buffer with Mammalian Protease Inhibitor Cocktail. Lysates were passed through a 21-gauge needle to shear the DNA. 1 mM PMSF (Sigma) was added and the lysates were incubated on ice for 30-60 min. Non-solubilized debris was pelleted at 10,000×g for 10 min. The supernatant was collected, aliquoted, and stored at −80° C. until use. Normal human brain lysates were also purchased from Chemicon International, Inc. (Temecula, Calif.) and Clontech. Lysates were run on either 12% or 15% SDS-PAGE. Proteins were transferred to PVDF membrane (Pierce, Rockford, Ill.) and blocked for 1 hr with 5% blotto (5% dry milk, PBS, 0.05% Tween-20). Membranes were incubated with primary antibody diluted in blotto for 40 min at room temperature while shaking. Antibodies included: anti-mouse VEGF-D antibody (40% cross-reactivity with human VEGF-D; 1:500) from R&D Systems, and Fra-1 (1:100) from Santa Cruz Research Antibodies. Following three five-minute washes in PBS/0.05% Tween-20, membranes were incubated in secondary antibody conjugated with horseradish peroxidase (goat anti-mouse IgG or goat anti-rabbit IgG) at a dilution of 1:10,000 or 1:15,000 in 5% blotto for 40 min at room temperature while shaking. Membranes were washed in several changes of PBS and detection was performed using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). Membranes were exposed to autoradiographic film X-OMAT AR for up to 5 min. Films were scanned in a transparency scanner at a pixel size of 88×88 micron (Molecular Dynamics, Sunnyvale, Calif.). The images were compiled in Paint Shop Pro V 6.0. cDNA arrays: Atlas Oncogene/Tumor Suppressor Arrays were purchased from Clontech and 1 &mgr;g of poly(A)&plus;RNA was labeled with &lsqb;&agr;- 33 P&rsqb;dATP according to the manufacturer. Membranes were pre-hybridized overnight at 68° C. in ExpressHyb (Clontech) containing 0.1 mg/ml sheared salmon sperm DNA. Labeled CDNA probe was denatured and added to the pre-hybridization solution and the membranes were hybridized overnight at 68° C. Membranes were then washed twice in 2×SSC/1% SDS for 20 min followed by two washes in 0.1% SSC/0.5% SDS at 68° C. The membranes were exposed to autoradiographic film for up to 10 days at −70° C. The arrays contain cDNA specific fragments for oncogenes, such as c-fos, junB, and c-myc. Housekeeping genes included ubiquitin, liver glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and phospholipase. RNA used for the cDNA micro-array assays was isolated from sub-confluent cultures of GBM cells using the acid-guanidium isothiocyanate-phenol-chloroform method. Chomczynski P, and Sacchi N (1987) Analyt. Biochem. 162: 156-159. Poly(A)&plus;RNA was further isolated using the Oligotex mRNA Kit (Qiagen Inc, Valencia, Calif.). Normal Human Brain Poly(A)&plus;RNA was purchased from Clontech (Clontech Laboratories, Inc., Palo Alto, Calif.). Karyotyping: The karyotypes of HGA cells analyzed in this study were performed in a blinded fashion by clinical cytogeneticists at the Cancer Genetics Laboratory, Genetics & IVF Institute, Fairfax, Va. 
 Example 2 
 Normal Brain Versus GBM Immunoreactivity for VEGF-D To establish the expression of VEGF-D in normal brain, immunofluorescence studies were performed using sections of non-malignant brain tissue. Using ten randomly selected specimens of human brain, some immunoreactivity could be detected in normal brain using an anti-VEGF-D antibody VD1. Some tissue sections of non-malignant brain showed a weak staining for VEGF-D, which was localized primarily to the cell cytoplasm and cell processes. The remaining samples showed sporadic and even poorer staining, if at all. On the contiguous sections of the same piece of non-malignant brain, the staining of endothelial cell-associated von Willebrand Factor (Factor VIII) was evident, scattered and corresponded to the expected picture of vessel density and size seen in non-malignant brain. Small caliber vessels imprinted by the staining with the antibody against Factor VIII were seen in all sections. The background fluorescence in these experiments, i.e. without primary antibody added, was low. Using exactly the same conditions as for immunofluorescence studies in non-malignant brain, a distinctively different picture of VEGF-D staining in malignant brain tissues has emerged. GBM sections demonstrated high levels of immunofluoresence for VEGF-D when compared with non-malignant brain and controls. VEGF-D showed a pattern of staining most compatible with cytoplasmic localization, i.e. diffused within the malignant astrocytic cells and also concentrated in their processes. All of the ten randomly selected tumors showed this pattern suggesting a remarkably high prevalence rate of VEGF-D over-production/accumulation in GBMs. GBMs are known to be highly vascular, which can be demonstrated by staining an antigen inherent to endothelial cells. Therefore, an antibody against Factor VIII was used in parallel immunofluorescence studies with tissue specimens; it was isotype-matched to the anti-VEGF-D antibody VD1. Anti-Factor VIII antibody immunofluorescence staining showed a characteristic pattern of hyperplastic vessels present on contiguous tissue sections of the same GBM as used for VEGF-D staining. Factor VIII staining demonstrated larger and more complex blood vessels in GBM tissue than seen in non-malignant brain. The pattern of VEGF-D staining in GBM was strongly suggestive of astrocytoma cells expressing this angiogenic factor. In order to document this directly, a double-staining experiment was performed using several GBM specimens. GBM sections were stained for glial-fibrillary acidic protein (GFAP), which is the most specific cytoplasmic marker for astrocytoma cells available. See, McKeever PE (1998) J. Histochem. Cytochem. 46: 585-594. GFAP staining of GBM revealed a typical cytoplasmic localization that outlines both astrocytoma cell bodies and their at times tube-like processes. VEGF-D staining co-localized with the staining for GFAP. GBM specimen &num;8 was stained with DAPI, a nuclear counter-stain, which further underlined the cytoplasmic localization of VEGF-D. Thus, a prominent presence of VEGF-D in the cytosol of astrocytoma cells of GBM was established in situ. 
 Example 3 
 Astrocytoma Cells Synthesize VEGF-D The pattern of VEGF-D staining in GBM tissue specimens indicated that the tumor astrocytoma cells were the principal source of in situ-detected VEGF-D. In order to document that astrocytoma cells in fact express VEGF-D, several GBM cell lines were analyzed for both VEGF-D and Factor VIII immuno-reactivity. Six tumor cell lines studied showed a prominent immunofluorescent staining for VEGF-D. GBM cells, such as A-172 MG, G48a, U-87 MG, DBTRG-50 MG, U-251 MG, and U-373 MG demonstrated a diffuse cytoplasmic staining pattern. This diffuse cytoplasmic pattern provided a characteristic nuclear outline generated by VEGF-D. In contrast, the analysis of the non-malignant cell lines, human endothelial umbilical vein cells (HUV-EC-C) and normal human astrocytes (NHA), demonstrated low levels of immunoreactivity for VEGF-D, although this was more pronounced in NHA than in HUV-EC-C. Furthermore, HUV-EC-C stained prominently for Factor VIII in the cytoplasm and was the only cell line positive for this endothelial cell marker used in the experiment. Background fluorescent staining was low in all specimens and cell lines examined when using an irrelevant mouse IgG 1 or no primary antibody. VEGF-D is an X-linked factor and therefore the karyotypes of astrocytoma cells used in experiments described herein were analyzed. Five out of six established cell lines exhibited abnormal ploidy for chromosome X, the most frequent alteration being an additional chromosome X. The high content of VEGF-D immunoreactivity in GBM tumor specimens and GBM cell lines indicated VEGF-D protein therein. Cell lysates of four of the cell lines were analyzed by SDS-PAGE and Western blotting performed with the anti-VEGF-D antibody. A single protein band was detected with an approximate molecular weight of 48 kDa. This protein band corresponds to the full-length VEGF-D. Stacker et al. (1999) J. Biol. Chem. 274: 32127-32136. The GBM cell lines U-251 MG and DBTRG-50 MG possessed this protein, but it was not found in the lysates from either the astrocyte (NHA) or endothelial (HUV-EC-C) cell lines. These data, taken together with the specific immunostaining of tissues and cells, indicate that GBM tumor cells produce abundant amounts of VEGF-D, an X-linked angiogenic factor. 
 Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.