Glypican-1 modulates the angiogenic and metastatic potential of human and mouse cancer cells (original) (raw)

Materials. The following materials were purchased from the following companies: DMEM, RPMI 1640, and trypsin-EDTA from Mediatech Inc.; FBS from Omega Scientific Inc.; G418 from GIBCO Laboratories; Noble agar from Difco Laboratories; recombinant human EGF from Chemicon; Heparitinase and Chondroitinase ABC Protease Free from Seikagaku Corporation; One Solution reagent and anti–phospho-MAPK antibody from Promega; Immobilon-P PVDF membranes from Millipore Corp.; RNeasy Mini Kit from Qiagen; phosphatidylinositol-specific phospholipase C (PI-PLC) from Molecular Probes; magnetic dynabeads from Dynal Biotech from Invitrogen; SulfoLink Coupling Gel from Pierce Biotechnology; anti–ERK-2 antibody, anti–angiopoietin-1 and -2 antibodies, and anti-Tie2 antibody from Santa Cruz Biotechnology Inc.; anti-CD31 rat anti-mouse monoclonal antibody (catalog no. 553370) and anti-VE cadherin antibody from Pharmingen; anti-PCNA monoclonal antibody from Novocastra; Antigen Unmasking Solution from Vector Laboratories; ApopTag in situ apoptosis detection kit from Chemicon; oligonucleotide primers from Applied Biosystems; PANC-1 human pancreatic cancer cells and COS-7 cells from American Type Culture Collection. Murine B16-F10 melanoma cells were a gift from Mary Jo Turk (Dartmouth Medical School, Hanover, New Hampshire, USA) and T3M4 human pancreatic carcinoma cells were a gift from R.S. Metzger at Duke University (Durham, North Carolina, USA). Athymic nude mice were obtained from Harlan. The following recombinant human growth factors were gifts: HB-EGF and FGF2 from Scios Nova Inc. and IGF-1 and VEGF-A from Genentech.

GPC1-knockout mice were produced by targeted mutagenesis in embryonic stem cells and maintained by backcrossing for more than 9 generations onto a CD-1 background prior to establishment of a homozygous null colony. The targeting construct directed the deletion of a large portion of the first exon of the GPC1 gene, removing both the translational start site and the signal peptide, and yielded homozygous animals in which GPC1 mRNA is greatly reduced and GPC1 protein is undetectable. Homozygotes are viable and fertile.

Stable transfection. The GPC1 antisense construct was prepared by RT-PCR amplification of human placenta cDNA, as described previously (11). Stable transfection of GPC1-AS-1751 into PANC-1 cells was performed by using the lipofectamine method (12), and single clones were isolated after 3-4 weeks. After expansion, cells from each individual clone were screened for expression of GPC1 sense and antisense mRNA by northern blot analysis. Parental PANC-1 cells also were transfected with an empty expression vector carrying the neomycin-resistance gene as a control. Positive clones were routinely grown in selection medium.

Cell culture and growth assay. PANC-1 human pancreatic cancer cells were grown in DMEM supplemented with 6% FBS (complete medium). T3M4 cells were grown in RPMI 1640 supplemented with 7% FBS. COS-7 cells were grown in DMEM supplemented with 10% FBS. Murine B16-F10 melanoma cells were grown in RPMI 1640 supplemented with 5% FBS and 5 μM β-mercaptonethanol. All media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Medium for the melanoma cells also contained amphotericin B (2.5 μg/ml). All cells were cultured at 37°C in humidified air with 5% CO2.

Anchorage-dependent growth was assessed by cell counting with a hemacytometer, after plating 2.0 × 104 cells/well in 6-well plates and incubating in complete medium. Cell doubling times were then calculated. Anchorage-independent growth was assessed by double-layer soft agar assay, as previously reported (48). Briefly, 3.0 × 103 viable cells were suspended in complete medium containing 0.3% agar and seeded in triplicate in 6-well plates onto a base layer of complete medium containing 0.5% agar. Complete medium (1 ml) containing 0.3% agar was added every 5 days.

To assess the mitogenic effects of growth factors, cells were plated in 1 ml of serum-free DMEM containing 0.1% BSA, 5 μg/ml transferrin, 5 ng/ml sodium selenite, antibiotics, and 0.3% agar in the absence or presence of growth factors. EGF, FGF-2, HB-EGF, or IGF-I, each a concentration of 1 nM, were added every 4 days. After 14 days, colonies consisting of more than 10 cells were counted by microscopy using an Inverted Diaphot 300 microscope (Nikon Inc.).

Generation of GPC1-knockout mice on an athymic background. The nude and GPC1 loci are on separate chromosomes, making it straightforward to generate mice that are homozygous for both. Briefly, GPC1–/– and GPC1+/+ animals were crossed with nu/nu and nu/+ animals to yield GPC1+/–nu/+ F1 hybrids. These were bred inter se, to yield an F2 generation of nu/nu and nu/+ animals from which only those that were GPC1+/+ and GPC1–/– were kept. Twenty one of each of these groups were bred inter se (avoiding brother-sister matings) to yield offspring of which only those that were GPC1+/+ nu/nu and GPC1–/– nu/nu were kept, and these F3 mice were used for experiments.

In vivo tumorigenicity assays and tissue preparation. To assess the effects of GPC1 suppression on tumorigenicity, 1 × 106 sham-transfected or GAS PANC-1 cells were injected subcutaneously into the flank region on one side of a 4- to 6-week-old, female athymic (nude) mouse. The animals were monitored for tumor formation every week and killed 8–15 weeks after injection when the tumor diameter became 15 mm3. Tumor volume was calculated as À/4 × width × height × length of the tumor, as previously reported (48). Tumors were rapidly excised and divided into 3 fragments. One tumor fragment was snap frozen in liquid nitrogen and stored at –80°C. Another tumor fragment was embedded in OCT, frozen in liquid nitrogen, and stored at –80°C for subsequent staining with anti-CD31 antibody (49). The third tumor fragment was fixed in formalin and embedded in paraffin for subsequent immunohistochemical staining.

For orthotopic inoculation of cancer cells, parental, sham-transfected or GPC1 antisense-transfected PANC-1 cells (2 × 106) or parental T3M4 cells (2 × 105) were injected into the pancreas of 4- to 6-week-old female athymic (nude) mice, using 28-gauge needles. Passage through this size needle did not alter cellular viability and was not associated with leaking from the injection site. At autopsy, the size of the intrapancreatic tumors was measured and the tumor volume was determined as above (48), and the number of metastatic lymph nodes was counted.

To perform studies with melanoma cells, exponentially growing murine B16-F10 melanoma cells were harvested, washed, and resuspended in PBS. Cells (100,000 in 250 μl of PBS) were injected into the lateral vein of the mice. Mice were watched daily starting at day 14 after injection, and in 4 separate experiments using 4 to 5 mice per group, they began to appear ill due to lung tumor burden 20–25 days following the injection. All the mice in each experiment were sacrificed on the same day, as soon as they appeared ill. The lungs were immediately removed and placed briefly in PBS containing 3% hydrogen peroxide and 10% buffered formalin. The number of pulmonary metastases was then counted. All studies with mice were approved by Dartmouth Medical School Institutional Animal Care and Use Committee.

Immunoblotting. Sham- and GAS cells and tissue samples were lysed in lysis buffer containing 50 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml benzamidine, and 1 mM PMSF (50). GPC1 generally appears on immunoblots as broad, faintly stained high-molecular-weight smears, in part due to poor binding of proteoglycans to blotting membranes (11). However, after digestion with heparitinase (to remove HSs) or heparitinase in conjunction with chondroitinaseABC (to also remove chondroitin sulfates), GPC1 migrates as a relatively distinct band on SDS-PAGE. Therefore, when performing immunoblotting for GPC1, cell lysates were first incubated in the absence or presence of these enzymes for 3 h at 37°C and subjected to SDS-PAGE and electrotransferred to Immobilon-P membrane. To study conditioned media, sham-transfected and GAS cells were incubated with PI-PLC and heparitinase for 1 h. Following concentration in a Microcon YM-10, media proteins were subjected to SDS-PAGE and electrotransferred to Immobilon-P PVDF membranes. After blocking, membranes were blotted with an affinity-purified rabbit anti-human GPC1 antibody and with a secondary horseradish peroxidase-conjugated antibody. Bound antibody was visualized using enhanced chemiluminescence.

To assess the consequences of altered GPC1 levels on MAPK activation, membranes were stripped for 30 min at 50°C in buffer containing 2% SDS, 62.5 mM Tris (pH 6.7), and 100 mM 2-mercaptoethanol and blotted with an anti–phospho-ERK (anti–active MAPK) antibody. To confirm equal loading of lanes, membranes were stripped again and blotted with an anti-ERK2 antibody.

Immunohistochemistry of paraffin-embedded and frozen tissues. Paraffin-embedded tumor tissues were sectioned (5 μm thick), mounted on poly-l-lysine-coated glass slides, and allowed to dry overnight at 23°C. These sections were used for detection of PCNA and apoptosis by the ApopTag TUNEL assay, as previously reported (50, 51).

PCNA, angiopoietin-1, angiopoietin-2, and Tie2 immunostaining was performed using the streptavidin-peroxidase technique (VECTASTAIN ABC Kit; Vector Labs). After deparaffinization, antigen retrieval was performed by heating in a microwave oven (95°C) for 5 min in 10 mM citrate buffer at pH 6.0 (PCNA) or by using a 2100-Retriever (PickCell Laboratories) and Antigen Unmasking Solution (angiopoietin-1, angiopoietin-2, and Tie2). Sections were then incubated for 10 min with 3% hydrogen peroxide to block endogenous peroxidase activity, for 30 min at 23°C with 5% normal goat serum, and overnight at 4°C with a monoclonal anti-PCNA antibody (2 μg/ml). Bound antibody was detected with biotinylated anti-mouse IgG and VECTASTAIN ABC Reagent (Vector Labs) complex, using diaminobenzidine tetrahydrochloride (DAB) as the substrate (50). Some sections were incubated without primary antibodies and did not yield positive immunoreactivity.

Two types of immunostaining procedures were performed to assess microvessel density. First, for tumor tissues frozen in OCT, sections (5 μm thick) were mounted on poly-l-lysine–coated glass slides and air-dried overnight at 23°C (49, 50). Frozen tissue sections were fixed in acetone for 5 min, and endogenous peroxidase activity was blocked by incubation for 10 min with 3% hydrogen peroxide. Samples were then incubated for 30 min at 23°C with 5% normal rabbit serum in PBS and overnight at 4°C with monoclonal anti-CD31 antibody (1 μg/ml). Bound antibody was detected with biotinylated rabbit anti-rat IgG secondary antibody and streptavidin-peroxidase complex, using DAB as the substrate. Second, for paraffin embedded tissues, endothelial cells were detected with the anti–VE cadherin antibody. Sections in both of the above procedures were counterstained with Mayer's hematoxylin.

To identify individual apoptotic cells in tissue sections, the TUNEL method consisting of the ApopTag in situ apoptosis detection kit (BD Pharmingen) that specifically recognizes 3′-OH DNA ends generated by DNA fragmentation was used according to the manufacturer’s directions, as previously described (51). Briefly, deparaffinized and proteinase K–digested tissue sections were incubated for 60 min at 37°C with terminal deoxynucleotidyl transferase (TdT) enzyme and digoxigenin-dUTP after blocking of endogenous peroxidase activity. Digoxigenin residues catalytically bound to the DNA 3′-OH ends by TdT were detected by antidigoxigenin antibodies conjugated with peroxidase, using DAB as the substrate, followed by counterstaining with methyl green. Omission of TdT enzyme did not yield any immunoreactivity.

To perform quantitative morphometry, the stained slides were analyzed with Image-Pro Plus (Media Cybernetics), as previously reported (50). Three fields at ×100 magnification were chosen randomly from each tumor for CD31 staining, and 5 fields at ×200 magnification were analyzed following TUNEL and PCNA staining.

Anti-peptide antibodies. Anti-GPC1 antibodies were generated by Genemed Synthesis Inc., using a peptide corresponding to amino acid residues 343 to 360 of human GPC1 (CGNPKVNPQGPGPEEKRR) that was synthesized and conjugated to keyhole limpet hemocyanin as previously reported (52). Following purification over Sephadex G-25 column, the peptide conjugate was injected intradermally into rabbits, using complete Freund’s adjuvant.

For affinity purification of the antibody, the purified peptide immunogen (10 mg) was coupled to SulfoLink Coupling Gel (Pierce Biotechnology) according to the manufacturer’s protocol. The anti-GPC1 antibody was purified using a 0.5-ml column equilibrated with PBS. The antibody was eluted with 50 mM glycine/0.15 M NaCl (pH 2.5) and neutralized with 1 M Tris buffer. The fractions with the highest reading at 280 OD were pooled (final pH 8.5), and the antibody was dialyzed overnight in PBS.

COS-7 cells were transiently transfected using lipofectamine with a construct encoding GPC1 fused to alkaline phosphatase, termed “APT6-GPC1” (13). The medium was collected 3–5 days after transfection, filtered, and stored at –80°C until use. An antigen-capture ELISA was performed to determine the affinity of the purified antibody, using 96-well Nunc Immunoplates (Nunc) that were coated with Protein A (50 μg/ml) for 1 h at 37°C. After washing with PBS, medium from the APT6-GPC1–transfected COS-7 cells that had been heat inactivated for 10 min (65°C) was added to the plates, and incubation was continued for 2 h at 37°C. After washing, p-nitrophenol phosphate (in 2 M diethanolamine, pH 8.8, 1 mg/ml BSA, and 1 mM MgCl2) was added to each well. Incubations were continued at 37°C for 20 min and reactions terminated by adding 0.5 M NaPO4 (pH 8.0). OD readings were performed on a microplate reader at 405 nm.

Endothelial cell isolation and proliferation assay. Primary endothelial cells were isolated from adult mouse livers following mechanical mincing and collagenase (2 mg/ml) digestion for 30 min at 37°C. Cells were isolated by incubating for 30 min at 23°C with magnetic Dynabeads (Invitrogen) labeled with anti-CD31 antibodies and using a magnetic separator (53). Cells were then incubated in DMEM supplemented with 20% FBS, penicillin/streptomycin (100 U/ml), heparin (100 μg/ml), endothelial cell growth factor (100 μg/ml), 1× nonessential amino acids, 2 mM l-glutamine, 1× sodium pyruvate, gentamicin (58 μg/ml), and amphotericin B (25 ng/ml), termed “complete endothelium medium.” To assess the effects of VEGF-A on endothelial cell proliferation, cells from passage 1 or 2 were plated for 4 h in 96-well cell culture plates coated with fibronectin (5 μg/ml) at a density of 4,000 cells per well. The medium was then replaced with fresh complete endothelium medium containing 2% FBS and 0.5% BSA. Cells were incubated at 37°C for 48 h in the absence or presence of 50 ng/ml VEGF-A, prior to the addition of One Solution reagent (Promega) for 2 h. Optical density was then determined at 490 nm on a Molecular Devices microplate reader.

Preparation of mouse embryonic fibroblasts. Primary MEFs were isolated from 13.5-day-old embryos by trypsin digestion. The cells were resuspended in DMEM containing 8% FBS, 100 U/ml penicillin/streptomycin, and 25 ng/ml amphotericin B. Following plating on tissue culture plates and incubation at 37°C with 5% CO2, cells were collected for RNA isolation.

Quantitative real-time PCR. To assess expression of proangiogenic factors in the tumors, RNA extraction, RT-PCR, and first-strand cDNA synthesis for quantitative real-time PCR analysis (Q-PCR) were carried out as described previously (54, 55). Target gene sequences were from the National Center for Biotechnology Information GenBank databases. Q-PCR was performed using an ABI PRISM 7300 sequence detection system (Applied Biosystems). RNA expression was calculated based on a relative standard curve representing 4-fold dilutions of human cDNA. Q-PCR data were expressed as a relative quantity based on the ratio of the fluorescent change observed with the target gene to the fluorescent change observed with 18S ribosomal subunit. Hepatic endothelial cell and MEF RNA samples were isolated with the Qiagen RNeasy kit following the manufacturer’s directions, and Q-PCR was performed as described above, using the Applied Biosystems TaqMan Assay with prevalidated murine probes and primer sets. A dilution series was carried out for each gene, and the 18S ribosomal subunit was used as an internal control.

Statistics. Unless otherwise indicated, Student’s t test was used for statistical analysis, with P < 0.05 defined as significant.