Platelet-derived growth factor (PDGF) is expressed in many different tumors, but its precise roles in tumorigenesis remain to be fully defined. Here, we report on a mouse model that demonstrates dose-dependent effects of PDGF-B on glial tumorigenesis. By removing inhibitory regulatory elements in the PDGFB mRNA, we are able to substantially elevate its expression in tumor cells using a retroviral delivery system. This elevation in PDGF-B production results in tumors with shortened latency, increased cellularity, regions of necrosis, and general high-grade character. In addition, elevated PDGF-B in these tumors also mediates vascular smooth muscle cell recruitment that supports tumor angiogenesis. PDGF receptor (PDGFR) signaling appears to be required for the maintenance of these high-grade characteristics, because treatment of high-grade tumors with a small molecule inhibitor of PDGFR results in reversion to a lower grade tumor histology. Our data show that PDGFR signaling quantitatively regulates tumor grade and is required to sustain high-grade oligodendrogliomas.

Platelet-derived growth factors (PDGFs) consist of a family of four different isoforms (A, B, C, and D), and by inducing dimerization, these ligands signal through two receptor tyrosine kinase subtypes [PDGF receptor (PDGFR)-α and PDGFR-β; Ref. 1]. PDGF ligands play numerous roles throughout development and in adult tissues (2). Studies of mice with targeted deletions of PDGFA reveal a requirement for PDGF signaling in the proliferation of oligodendrocyte precursors (3, 4). The phenotypes of mice with targeted deletions of PDGFB and PDGFR-β genes are remarkably similar with embryonic lethality in both due to cardiovascular defects (5, 6). PDGFR-β signaling is particularly important in the development of vascular smooth muscle cells (vSMC), specifically in recruitment and promotion of their proliferation in support of new blood vessel formation (7, 8).

PDGF-B expression is regulated at many levels. Its mRNA contains long 5′- and 3′-untranslated regions (UTRs), and multiple elements in the 5′UTR can act as translational inhibitors (9). This complex 5′UTR can also act as an internal ribosomal entry site element that may be linked to varying levels of translation during differentiation (10). In human tumors, PDGFB mRNA splice variants are also observed, and in certain cases, shorter UTRs were associated with higher protein expression (11). However, the importance of these splicing differences in contributing to PDGF function remains unknown. Of interest, in certain forms of dermatofibrosarcomas, a translocation of PDGFB to the COL1 gene removes the 5′UTR region of PDGFB and is thought to be responsible for elevation of PDGF-B protein production, leading to unregulated autocrine stimulation (12, 13).

In gliomas, coexpression of PDGF and its receptor is often observed, establishing an autocrine loop that contributes to proliferation and perhaps also to tumor progression (14, 15). In cell culture and xenograft models, inhibition of PDGFR kinase activity by treatment with small molecule inhibitors reduces colony forming ability and tumor growth (16). However, xenograft models may not fully take into account the complex interactions between tumor and stroma arising in the brain. Previously, we described a genetically engineered mouse model for oligodendroglioma formation through retroviral delivery of oncogenes into targeted glial progenitor cells and differentiated astrocytes (17, 18). Enforced expression of PDGF-B in these cells results in oligodendrogliomas and mixed astro-oligo glial tumors, respectively. However, this vector for PDGFB contains a portion of the normal PDGFB 5′UTR. We wanted to test the effect of removal of this 5′UTR on PDGF-B expression. We also wanted to correlate the local amount of PDGF with tumor histology and to determine the requirement for continued PDGF signaling in tumor maintenance. By elevating levels of PDGF-B chain expression, we were able to generate glial tumors of higher grade and increased vascularization correlated with significant vSMC recruitment. Furthermore, treating PDGF-induced gliomas in vivo with a small molecule inhibitor that blocks both PDGFR and vascular endothelial cell growth factor receptor (VEGFR) demonstrated that PDGF signaling appears to be required for maintenance of malignant characteristics but not acutely required for tumor blood vessel maintenance.

Plasmids.

RCAS constructs were cloned in replication competent ASLV long term repeat with splice acceptor (RCAS) Bryan strain RSV pol subgroup A [BP(A)]. RCAS UTR green fluorescent protein (GFP) contains 101 nucleotides (nt) of human PDGFB 5′UTR (from −117 to −16, respective to the ATG start site) in front of GFP. RCAS GFP contains GFP alone. RCAS UTR PBIG contains PDGFB with the proximal 117-nt 5′UTR and 94-nt 3′UTR and linked to an internal ribosomal entry site GFP element. RCAS PDGFB-HA contains a 3-nt “ACC” kozak consensus sequence in front of PDGFB and a COOH-terminal hemagglutin (HA) epitope tag. For in vitro transcription and translation, the “ACC” + PDGFB-HA sequence was excised from RCAS PDGFB-HA and cloned after the T7 promoter. The UTR PDGFB sequence with the same 5′ 117-nt and 3′ 94-nt sequences was cloned after the T7 promoter. Both plasmids were linearized with ClaI before in vitro transcription and translation.

In Vitro Transcription Translation.

The reaction was performed with [35S]methionine (Amersham Biosciences) and T7-coupled reticulocyte lysate system (Promega) using manufacturer’s instructions with 100 ng of DNA template. Samples were run out on SDS-PAGE, vacuum dried onto Whatmann paper, and exposed to film overnight. Transcription-alone reactions were carried out using T7 polymerase reactions according to manufacturer’s instructions and separated on 1% agarose gels.

PDGF-BB ELISA.

DF1 cells expressing the respective PDGFB-encoding viruses were seeded at 2 × 106 cells/plate. After 24 h, cells were switched to 3 ml of serum-free medium. Conditioned medium was collected after 24 h and spun at 3000 rpm, and 100 μl were used for ELISA. To infect astrocytes, an equal number of DF1 cells bearing the respective PDGFB-encoding viruses were seeded onto plates, and virus was collected after 24 h. These viral stocks were then used to infect 3 × 105 astrocytes derived from Nestin-driven tva (N-tva) transgenic mice. Astrocytes were harvested 2 days postinfection, and protein was extracted using cold lysis. Twenty μg of protein were used for ELISA. PDGF-BB ELISA was performed using a Quantikine kit from R&D Systems according to manufacturer’s instructions. Experiments were performed in triplicate and repeated with similar results.

PDGF-BB Stimulation.

RCAS PDGFB-HA plasmid or empty RCAS plasmid was transfected into 293T cells in SFM using Fugene (Roche). The supernatant was collected after 2 days, passed through a 2-μm filter, and then applied to serum-starved NIH3T3 cells. Cells were then collected 30 min after application of conditioned medium.

Western Blot.

Whole-cell protein extracts were prepared by cold lysis of cell pellets. M-Per (Pierce) lysis buffer was supplemented with 30 mm sodium fluoride, 1 mm sodium vanadate, and protease inhibitor (Roche). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (Osmonics). Membranes were blocked with 5% nonfat milk in PBS-Tween 0.1%. Primary and secondary antibodies were diluted in the same solution. Signal was visualized using ECL chemiluminescence (Amersham Biosciences; Pierce). Glyceraldehyde-3-phosphate dehydrogenase was from ImmunoChemical (1:1000; clone 6C5), GFP from Chemicon (1:300; AB3080), p-Erk for Cell Signaling (1:500; #9101), p-Akt from Cell Signaling (1:500; #9271), antirabbit horseradish peroxidase from Amersham Biosciences (1:1000), and antimouse IgG+IgM horseradish peroxidase from Roche (1:1000).

Generation of Tumor-Bearing Mice.

DF1 cells were transfected with RCAS retroviral vectors, generating a culture of virus-producing cells. N-tva transgenic mice have been described previously (18). Using a Hamilton syringe, N-tva mice at birth received i.c. injections of 1 μl of approximately 104 DF1 cells. Mice were then followed and sacrificed early if they developed symptoms of hydrocephalus, lethargy, or cachexia. Otherwise, mice were sacrificed at the end of 12 weeks. Tumor cell density was determined by taking random samples of ×400 field images and counting nuclei. Percentage of proliferating cell nuclear antigen (PCNA)-positive cells was determined in a similar fashion. For treated tumors, PCNA analysis was done on random fields within areas of histology associated with drug treatment (i.e., round regular nuclei, vacuolated cells, and myxoid generation).

Immunohistochemical Analysis.

Mouse brains were fixed, paraffin embedded, and processed as described previously (17). Slides were stained with primary antibody overnight at 4°C. Appropriate secondary biotin-conjugated antibody was applied for 1 h at room temperature. Peroxidase signal was developed using the ABC kits (Vector Lab). For confocal microscopy, FITC antimouse was combined with biotin-antirabbit and then followed by rhodamine avidin. Smooth muscle actin (SMA) was from Dako (1:1000; clone 1A4); PCNA was from Chemicon (1:500; MAB424R); HA was from Santa Cruz Biotechnology (1:200; polyclonal sc-305); phospho-S6 ribosomal protein (pS6RP) was from Cell Signaling (1:50; #2211); and biotin antimouse (1:200), biotin antirabbit (1:200), FITC antimouse (1:100), Rhodamine avidin (1:100) were from Vector Lab. Antibodies were diluted in 5% horse serum PBS-Tween 0.1%.

Treatment of Tumor-Bearing Mice.

Symptomatic mice were scanned with magnetic resonance imaging (MRI) images and contrast injection. For imaging, mice were anesthetized with isoflurane. Images were obtained on a 1.5-T General Electric LX Echo Speed Signa scanner using a home-built solenoid coil and procured in a fashion similar to that described previously (19). Low-resolution sagital scout images were obtained initially followed by T2-weighted axial images [repetition interval (TR), 3000–3500 ms; effective echo time (TE), 102 ms; spatial resolution, 1.5-mm slice thickness × 156 μm × 156 μm in plane resolution). These were followed by pre- and post-contrast axial T1-weighted images (TR, 300 ms; TE, 14 ms; spatial resolution, 1.5-mm slice thickness × 156 μm × 208 μm in plane resolution, and three excitations/phase-encoding step). For contrast enhancement, T1-weighted images were obtained after injection of contrast (0.2 mm/kg). Mice with enhancement were treated with 100 mg/kg PTK787/ZK222584 daily with i.p. injections for 7 days. PTK787/ZK222584 was resuspended in 5% DMSO and 1% Tween 80. For non-MRI-imaged mice, treatment was initiated when mice became symptomatic.

The PDGFB 5′UTR Can Act in an Inhibitory Fashion on Expression Levels.

The PDGFB cDNA used in our original retroviral gene delivery system RCAS encodes portions of the 5′UTR and 3′UTR (117 and 94 nt, respectively) as well as being linked to an internal ribosomal entry site GFP transcript (RCAS UTR PBIG). Previous studies by Horvath et al.(20) mapped this portion of the PDGFB 5′UTR within a region of translational inhibition. We assessed whether this UTR region can inhibit protein expression in our viral system by adding 100 nt of the PDGFB 5′UTR in front of GFP in the RCAS vector (RCAS UTR GFP; Fig. 1,A). In cell line DF1, in which ALV viruses are replication competent, GFP protein is produced at a much higher level in cells infected by RCAS GFP compared with RCAS UTR GFP, implying that the PDGFB 5′UTR can inhibit protein production expressed through retroviral delivery (Fig. 1,B). To test the effects of elevating PDGF-B expression in our model, we constructed a new vector for PDGFB lacking the inhibitory UTR regions and instead containing a 3-nt “ACC” kozak consensus sequence (RCAS PDGFB-HA; Fig. 1,A). An HA tag added to the COOH terminus allows for immunohistochemical analysis in subsequent experiments. In reticulocyte-based in vitro transcription and translation labeling reactions in which both constructs are expressed by the T7 promoter, both yielded similar amounts of mRNA, but the new “ACC” PDGFB-HA construct expressed substantially more protein (Fig. 1,C). When infected with the new RCAS PDGFB-HA virus, DF1 cells secrete more PDGF-BB in the supernatant than cells infected with RCAS UTR PBIG (325 versus 175 pg/ml; P < 0.01; Fig. 1,D). There was also a 4–15-fold higher expression of PDGF-BB in lysates from astrocytes infected by the new vector compared with the original vector (Fig. 1 E; one representative experiment is shown).

Although the COOH terminus of PDGF-B peptide is thought to be cleaved in one secreted form, the addition of an HA tag to PDGF-B may affect expression of a functional ligand (21). To exclude this possibility, we stimulated serum-starved NIH 3T3 cells with supernatant collected from 293T cells transfected with RCAS PDGFB-HA or control vector. The RCAS PDGFB-HA-conditioned supernatant transiently induced Akt and Erk phosphorylation above control levels, indicating that the HA tag did not block production of active ligand (Fig. 1 F). In addition, the ELISA used to quantitate PDGF-BB levels captures ligand with a recombinant PDGFRβ-Fc fusion, indicating that the PDGF-BB produced can bind PDGFRβ receptors. Although we cannot formally rule out an effect of the UTR or HA sequences on viral production or PDGF-B activity, it appears that substantially higher levels of PDGF-B protein production results from deleting the UTR sequences in our retroviral delivery system.

Elevated Levels of PDGF-B Expression in CNS Progenitors in Vivo Leads to Increases in Both Incidence and Grade of Tumor.

Previously, we described a transgenic mouse model in which an avian retrovirus receptor, TVA, is expressed in specific glial lineages using targeted promoters, N-tva and GFAP-tva. We demonstrated that by i.c. infection of RCAS avian retroviruses carrying the human PDGFB gene, oligo and mixed astro-oligo glial tumors develop. To determine whether the elevated level of PDGF-B expression by our new vector has any effect on tumorigenesis, we injected cells producing either virus into two cohorts of N-tva mice. Mice infected with virus coding for higher PDGF-B expression developed tumors with both higher incidence and shorter latency. Injection of RCAS PDGFB-HA generated tumors at 97% incidence (28 of 29), compared with a 55% incidence (26 of 47) obtained by injection of RCAS UTR PBIG. The resulting Kaplan-Meier curve is significant for decreased survival (P < 0.001; Fig. 2,A). In addition, the tumors generated by higher expression of PDGF-B are also of higher grade. Twenty-nine percent of the tumors (8 of 28) have regions of necrosis compared with only 4% (1 of 26) from the original vector. Furthermore, within a given tumor, regions of pseudopalisading necrosis, a feature of high-grade glial tumor histology, correlate with relatively higher levels of PDGF-B chain expression as determined by immunostaining for the HA epitope tag (Fig. 2,B; Fig. 3, A, D, and E).

The RCAS PDGFB-HA-induced tumors have increased cellularity and tumor burden. These tumors had on average 292 ± 11 nuclei/×400 field compared with 168 ± 8 nuclei/×400 field for RCAS UTR PBIG-induced tumors, a 70% increase (P < 0.001; Fig. 2 B). The increase in PDGF-B levels also generates larger tumors. At 4 weeks, a subset of mice from each group succumbs to hydrocephalus and tumor. At this time, RCAS UTR PBIG-induced oligodendroglioma tumor cells are confined to the ventricles or have limited infiltration. In contrast, RCAS PDGFB-HA-induced tumors have infiltrated extensively throughout the brain parenchyma (data not shown).

Tumors induced by elevation of PDGF-B expression also have more pronounced angiogenesis. Whereas RCAS UTR PBIG-induced oligodendrogliomas have a typical chicken-wire fine capillary network seen in low grade human oligos, 61% (17 of 28) of RCAS PDGFB-HA-induced oligodendrogliomas develop arteriole-like vessels or larger diameter capillaries compared with only 15% (4 of 26) of RCAS UTR PBIG-induced tumors (Fig. 2,B; Fig. 3, B and C). Because PDGF-BB is known to promote proliferation of vSMC and vSMC support arteriole formation, we looked for the presence of these cells within the tumor. RCAS PDGFB-HA-induced tumors have significant vSMC infiltration, whereas RCAS UTR PBIG tumors have limited or no vSMC infiltration (Fig. 4, C and D). Furthermore, within a given tumor, regions of vSMC proliferation (as determined by SMA marker staining) correlate with levels of PDGF-B chain expression (Fig. 3, F and G). These regions of vSMC are associated with vessels (Fig. 4 C). However, the infiltration of vSMCs seems more extensive than endothelial cells, suggesting that high PDGF concentrations within the tumor may separate a portion of the vSMC recruitment and actual blood vessel formation. In sum, these results demonstrate that by increasing levels of PDGF-B production, a tumor may achieve higher grade characteristics.

PDGF May Act in a Paracrine Fashion in High PDGF-B-Expressing Tumors.

We wanted to test the hypothesis that by further elevating levels of PDGF-BB, tumor cells can sustain an autocrine loop but perhaps also secrete PDGF acting on other cells. We performed immunohistochemical analysis of high-grade tumors looking at the pattern of PDGF-B expression. Within a field of oligodendroglioma cells, there was varying expression of PDGF-B chain ranging from robust detection of HA to low or undetectable levels (Fig. 4 A). Therefore, high-grade tumors did not develop with uniformly high-expressing cells but instead with tumor cells of unequal PDGF-B expression. We also noted the presence of diffuse HA staining that could indicate incomplete processing and secretion of PDGF ligands. Together, the varying levels of expression and the diffuse HA staining suggest that high-expressing cells may produce and secrete enough ligand to increase the local concentration of PDGF.

Previous work shows that PDGF-BB promotes vSMC recruitment and proliferation in support of angiogenesis (7, 22). Therefore, we looked for the interaction between PDGF-B and vSMC in our tumors. As noted before, vSMC infiltration is seen in areas of highest PDGF-B concentration (Fig. 4,B). In confocal images, one can also observe tumor cells with high levels of PDGF-B surrounding the vasculature (Fig. 4, EG). Proliferative and migratory effects on smooth muscle cells by PDGF-BB and other factors are thought to be mediated by the phosphatidylinositol 3′-kinase-Akt pathway (23, 24, 25). Because PDGF is known to activate Akt leading to downstream p70S6 kinase activation, we looked to see whether ribosomal component S6 is phosphorylated in the vasculature. Indeed, we found extensive pS6RP activation in vSMC (Fig. 4, HJ). These data suggest that PDGF-BB acts to induce vSMC tumor infiltration and in the process activates the AKT pathway.

Tumor Cell Response to PDGFR Blockade.

To test whether signaling through the PDGFR is required for maintenance of PDGF-induced gliomas, we sought to treat mice harboring high-grade gliomas with a small molecule PDGFR tyrosine kinase inhibitor, PTK787/ZK222584. This compound was designed as a VEGFR family inhibitor but has been shown to be also effective against PDGFR. Furthermore, we previously showed that PTK787/ZK222584 is able to inhibit and reverse the transforming effects of PDGFB on glial cells in vitro(17). Mice infected with RCAS PDGFB-HA virus were screened for the development of high-grade, contrast-enhancing lesions using MRI (Fig. 5, A and B; Ref. 26). We identified eight mice with contrast-enhancing lesions. Three mice were sacrificed after imaging to determine the histology for these high-grade lesions. The five remaining mice were then treated with PTK787/ZK222584 at 100 mg/kg/day for 7 days. Two of these mice expired during treatment presumably due to the aggressiveness and size of these lesions. For the three mice that survived throughout the treatment procedure, there was a marked decrease or loss of contrast enhancement on re-imaging with contrast MRI (a finding consistent with lower grade gliomas; Fig. 5,C). These three mice were sacrificed, and their brains were analyzed histologically along with seven additional tumor-bearing mice treated in a similar fashion but without MRI imaging. Eight mice were also treated with vehicle alone, and results from these mice were similar to untreated controls. High-grade RCAS PDGFB-HA-induced tumors typically have nests of oligodendroglioma cells with irregularly shaped nuclei and a high proportion of PCNA staining (Fig. 6, A and D). These characteristics are in contrast to the 10 tumors after treatment with PTK787/ZK222584. All of the treated RCAS PDGFB-HA-induced tumors contained large regions of lower grade histological appearance with vacuolated cells, round regular nuclei, increased mucomyxoid generation, and decreased PCNA staining (Fig. 6, B and E). This histology is found only in small regions of either vehicle-treated or untreated tumors. We note, however, that in treated mice, the entire tumor does not respond, and approximately 30–60% of the tumor area on histological section achieves a lower grade histological appearance (Fig. 6,C). This lower grade histological appearance corresponded to a decrease in cell proliferation. In untreated high-grade oligodendrogliomas, 59 ± 4% of the cells are PCNA positive, whereas in regions associated with treatment histology, only 15 ± 3% of tumor cells remain PCNA positive (P < 0.001; Fig. 6,F). In certain treated tumors, there is also a significant decrease in tumor cell density and loss of tumor mass. Despite this loss of cellular density, tumor cells were not appreciably increased in tunnel staining for apoptosis (Fig. 6,G). Staining for HA demonstrates that tumor cells in treatment-associated regions of the tumor continue to express PDGF-B, confirming that PTK787/ZK222584 blocks signaling at the receptor level (Fig. 6 H).

Tumor Vasculature Maintenance Does Not Require Sustained PDGFR Signaling.

Because extensive angiogenesis is seen in high-grade oligodendrogliomas, we were interested in the roles of VEGFR and PDGFR on the tumor vasculature. Treatment with PTK787/ZK222584 did indeed reduce contrast enhancement, indicating a change in vessel integrity (Fig. 5). This is in agreement with the reported vascular permeability changes in clinical trials with PTK787/ZK222584 (27). However, despite in vitro activity against both PDGFR and VEGFR, PTK787/ZK222584 did not appear to disrupt the maintenance of existing vasculature. Arterioles and larger vessels persisted in the tumor mass in regions where oligodendroglioma cells were less proliferative and had treatment-associated histology (Fig. 6 I). First, this suggests that in this model, the observed effect of PTK787/ZK222584 is largely through blockade of PDGFR in tumor cells and not through disruption of the vasculature. Second, within the time frame of treatment (7 days), PDGFR and VEGFR signaling do not appear to be required to sustain the tumor vasculature. This is in agreement with observations by Cao et al.(28) in which PDGF-BB can act along with other angiogenic factors to stimulate vessel formation, but vessels persist even after growth factor withdrawal. However, we cannot rule out a lack of in vivo efficacy against blood vessels due to insufficient PTK787/ZK222584 levels to achieve the required VEGFR inhibition.

Our work shows that PDGF-B may play dose-dependent roles in glial tumorigenesis. In our model at lower doses, PDGF establishes an autocrine loop to promote gliomagenesis. As the level of PDGF-B expression is increased, concentration-dependent effects become observable in vivo. Increased cellularity results from high-expressing PDGF-B tumors. If PDGF acts only in an autocrine fashion in high-grade tumors, one would expect PDGF-B expression to be elevated in the vast majority of tumor cells because these cells would achieve a competitive growth advantage. However, on closer examination, we find that individual cells within the tumor express varying levels of PDGF-B. This effect may have been difficult to observe in previous xenograft models that begin with a clonal population of tumor cells. The varying levels of expression in our model could better mirror the situation seen in glioblastomas that are known to have complex patterns of gene expression. Because PDGF is limiting for oligodendrocyte precursor cell growth, our observations suggest that high expressing cells may stimulate growth of neighboring cells, inducing proliferation and hypercellularity in regions of the highest PDGF concentration. Moreover, within a tumor area, high PDGF-B expression regions correlate with areas of necrosis. Thus, elevated concentrations of PDGF may lead to increased cell density and to higher grade histological features in glial tumors.

The interaction between a tumor and its in situ environment is critical for tumor development. PDGF-BB may play an important role in neovascularization by supporting vSMC recruitment. By elevating expression of PDGF-B in tumor cells, PDGF appears to act in a fashion similar to that found in the early development of normal blood vessels. We observe in high-grade tumors an increase in vSMC infiltration correlating with more extensive angiogenesis. We also observed an activated Akt pathway in the recruited vSMC consistent with other studies showing that PDGF-BB stimulation of vSMC is dependent on prolonged Akt signaling (29). Therefore, by elevating PDGF-B expression, a tumor is able to recruit vSMC in support of arteriole and large vessel formation.

In our model, we find that inhibiting PDGFR activity can convert tumors from high to lower grade. The overall tumor mass responded to treatment as detected by loss of contrast enhancement, whereas the tumor cells themselves responded by exhibiting more round, regular nuclei and having a lower percentage of PCNA-positive cells. Although there was a decrease in cell density, no significant apoptosis was observed. This is in agreement with other studies in which inhibition of PDGFR signaling led to cell cycle arrest rather than apoptosis (16). Alternatively, the kinetics of apoptosis may prevent its detection at the time the assay was performed, or tumor cells may die through necrosis. Nevertheless, this treatment-induced shift from high- to low-grade character supports the idea that PDGFR signaling is required for maintenance of high-grade characteristics of oligodendrogliomas. Because PDGF-BB also has a role in angiogenesis, we were interested in its requirement for tumor vascular maintenance. The integrity of the vessels changed with treatment as MRI contrast enhancement significantly decreased, potentially a result of either PDGFR or VEGFR inhibition. However, the vessels themselves persisted along with the supporting vSMC. The data imply that in our model PDGF-BB may act only for vSMC recruitment and is not acutely required for blood vessel maintenance over a treatment period of 7 days. Thus, there is an acute, persistent requirement for PDGF-B in oligodendroglioma cell proliferation but perhaps not in maintaining the tumor vasculature.

Our results demonstrate that elevation of PDGF-B expression has relevant effects on the mode of PDGF action. Thus, it may be important in clinical evaluations to detect not only the presence of PDGF but also its relative level of expression. The mechanism for this overexpression by tumor cells may be achieved not only on a strictly transcriptional level but perhaps also by splicing and translational effects. Therapeutically, treatment outcome from blocking PDGFR signaling may be in the form of converting high-grade regions to lower grade characteristic and preventing new recruitment of vSMC for tumor angiogenesis.

Grant support: NIH Medical Scientific Training Program Grant GM07739 (A. Shih), the Robert Wood Johnson Endowment Fund (A. Shih), the Serrossi Foundation, Bressler Foundation, Kirby Foundation, and NIH Grants UO1CA894314-1, RO1 CA099489 (E. Holland), and R24 CA83084 (J. Koutcher).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Eric C. Holland, 1275 York Avenue, New York, NY 10021. Fax: (646) 422-2062; E-mail: hollande@mskcc.org

Fig. 1.

The PDGFB UTR regions can act to regulate expression levels. A, 1.RCAS GFP, 2.RCAS UTR GFP, 3.RCAS PDGFB-HA, 4.RCAS UTR PBIG constructs. Bold segments denote UTR regions. L, Ltr; GPE, gag, pol, env; ACC, 3-nt kozak consensus; IGFP, internal ribosomal entry site GFP. B, Western blot for GFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in DF1 cells infected with RCAS GFP or RCAS UTR GFP. C, top, in vitro transcription and translation reaction with [35S]methionine (35S-met) comparing “ACC” PDGFB-HA and UTR PDGFB constructs. Middle, linearized DNA T7 promoter templates. Bottom, RNA products from in vitro transcription reaction. D, levels of PDGF-BB in supernatant of DF1 cells infected with RCAS PDGFB-HA or RCAS UTR PBIG (∗, P < 0.01, n = 5, SE). E, relative levels of PDGF-BB in extracts of mouse astrocytes infected with RCAS PDGFB-HA or RCAS UTR PBIG in one representative experiment (∗, P < 0.001, n = 3, SE). F, phospho-Akt (pAkt) and phospho-Erk (pErk) Western blot of 3T3 cells stimulated with supernatant from RCAS PDGFB-HA or control-transfected 293T cells (∗, nonspecific cross-reacting band).

Fig. 1.

The PDGFB UTR regions can act to regulate expression levels. A, 1.RCAS GFP, 2.RCAS UTR GFP, 3.RCAS PDGFB-HA, 4.RCAS UTR PBIG constructs. Bold segments denote UTR regions. L, Ltr; GPE, gag, pol, env; ACC, 3-nt kozak consensus; IGFP, internal ribosomal entry site GFP. B, Western blot for GFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in DF1 cells infected with RCAS GFP or RCAS UTR GFP. C, top, in vitro transcription and translation reaction with [35S]methionine (35S-met) comparing “ACC” PDGFB-HA and UTR PDGFB constructs. Middle, linearized DNA T7 promoter templates. Bottom, RNA products from in vitro transcription reaction. D, levels of PDGF-BB in supernatant of DF1 cells infected with RCAS PDGFB-HA or RCAS UTR PBIG (∗, P < 0.01, n = 5, SE). E, relative levels of PDGF-BB in extracts of mouse astrocytes infected with RCAS PDGFB-HA or RCAS UTR PBIG in one representative experiment (∗, P < 0.001, n = 3, SE). F, phospho-Akt (pAkt) and phospho-Erk (pErk) Western blot of 3T3 cells stimulated with supernatant from RCAS PDGFB-HA or control-transfected 293T cells (∗, nonspecific cross-reacting band).

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Fig. 2.

Higher levels of PDGF-B production leads to decreased survival and increased tumor incidence. A, Kaplan-Meier tumor-free survival curve of N-tva mice that received injections of cells producing RCAS PDGFB-HA virus or RCAS UTR PBIG virus (∗, P < 0.001). Mice were sacrificed at the end of 12 weeks (wks). B, table comparing tumor characteristics (∗∗, P < 0.001; ∗, P < 0.05, SE). t test performed for cells/×400 field (n = 22, 23); χ2 analysis for necrosis and large vessel characteristics.

Fig. 2.

Higher levels of PDGF-B production leads to decreased survival and increased tumor incidence. A, Kaplan-Meier tumor-free survival curve of N-tva mice that received injections of cells producing RCAS PDGFB-HA virus or RCAS UTR PBIG virus (∗, P < 0.001). Mice were sacrificed at the end of 12 weeks (wks). B, table comparing tumor characteristics (∗∗, P < 0.001; ∗, P < 0.05, SE). t test performed for cells/×400 field (n = 22, 23); χ2 analysis for necrosis and large vessel characteristics.

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Fig. 3.

Higher levels of PDGF-B increases the grade of the tumor. Regions of necrosis (A; ×50) and angiogenesis (B; ×400) in RCAS PDGFB-HA-induced tumor. C, Typical fine capillary found in RCAS UTR PBIG-induced tumor (×400). D and F, HA immunostaining of RCAS PDGFB-HA-induced tumor. E, H&E of boxed region in D (×25). Inset, higher magnification of region of pseudopalisading necrosis in region indicated by arrow. G, SMA immunostaining of boxed regions in F (×50).

Fig. 3.

Higher levels of PDGF-B increases the grade of the tumor. Regions of necrosis (A; ×50) and angiogenesis (B; ×400) in RCAS PDGFB-HA-induced tumor. C, Typical fine capillary found in RCAS UTR PBIG-induced tumor (×400). D and F, HA immunostaining of RCAS PDGFB-HA-induced tumor. E, H&E of boxed region in D (×25). Inset, higher magnification of region of pseudopalisading necrosis in region indicated by arrow. G, SMA immunostaining of boxed regions in F (×50).

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Fig. 4.

Vascular recruitment by PDGF in RCAS PDGFB-HA-induced tumors. A, HA immunostaining, varying levels of HA expression among tumor cells, and also diffuse staining (×400). B, HA immunostaining, high levels of HA staining in tumor cells (×200). Arrow, blood vessel, low or negative for HA staining. C, SMA immunostaining in RCAS PDGFB-HA-induced tumor (×200). Arrow, blood vessel associated with vSMC. D, SMA immunostaining in RCAS UTR PBIG-induced tumor (×200). EJ, confocal images (×630). E, HA; F, SMA; G, composite 4′,6-diamidino-2-phenylindole, HA, and SMA; H, pS6RP; I, SMA; J, composite 4′,6-diamidino-2-phenylindole, pS6RP, and SMA.

Fig. 4.

Vascular recruitment by PDGF in RCAS PDGFB-HA-induced tumors. A, HA immunostaining, varying levels of HA expression among tumor cells, and also diffuse staining (×400). B, HA immunostaining, high levels of HA staining in tumor cells (×200). Arrow, blood vessel, low or negative for HA staining. C, SMA immunostaining in RCAS PDGFB-HA-induced tumor (×200). Arrow, blood vessel associated with vSMC. D, SMA immunostaining in RCAS UTR PBIG-induced tumor (×200). EJ, confocal images (×630). E, HA; F, SMA; G, composite 4′,6-diamidino-2-phenylindole, HA, and SMA; H, pS6RP; I, SMA; J, composite 4′,6-diamidino-2-phenylindole, pS6RP, and SMA.

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Fig. 5.

T1-MRI images of mice. A, pre-contrast images. Post-contrast enhancement images of mice before (B) and after (C) treatment with PTK787/ZK222584. Arrows indicate regions of contrast enhancement.

Fig. 5.

T1-MRI images of mice. A, pre-contrast images. Post-contrast enhancement images of mice before (B) and after (C) treatment with PTK787/ZK222584. Arrows indicate regions of contrast enhancement.

Close modal
Fig. 6.

Tumor response associated with small molecule PDGFR inhibitor treatment. PDGFB-HA tumors untreated (A and D) and treated with PTK787/ZK222584 (B, C, E, G, H, and I). A, H&E (×200); B, H&E, increased vacuolization and mucomyxoid material (blue regions) in treated tumor (×200). C, H&E cross-section of mouse brain with enhancing tumor treated with PTK787/ZK222584. Red arrow, treatment-associated region. Green arrow, nonresponsive or minimally responsive region. D, PCNA staining, pleiomorphic nuclei (×400). E, PCNA staining, more round and regular nuclei (×400). F, percentage of PCNA-positive cells in untreated and treated tumors (∗, P < 0.001, n = 14, SE). G, Tunnel staining (×400); H, HA staining of treatment-associated tumor regions (×400); I, persistence of vessels within tumor treatment-associated region (×400).

Fig. 6.

Tumor response associated with small molecule PDGFR inhibitor treatment. PDGFB-HA tumors untreated (A and D) and treated with PTK787/ZK222584 (B, C, E, G, H, and I). A, H&E (×200); B, H&E, increased vacuolization and mucomyxoid material (blue regions) in treated tumor (×200). C, H&E cross-section of mouse brain with enhancing tumor treated with PTK787/ZK222584. Red arrow, treatment-associated region. Green arrow, nonresponsive or minimally responsive region. D, PCNA staining, pleiomorphic nuclei (×400). E, PCNA staining, more round and regular nuclei (×400). F, percentage of PCNA-positive cells in untreated and treated tumors (∗, P < 0.001, n = 14, SE). G, Tunnel staining (×400); H, HA staining of treatment-associated tumor regions (×400); I, persistence of vessels within tumor treatment-associated region (×400).

Close modal

We thank Ed Nerio for his technical assistance, Jennifer Doherty for immunohistochemistry work, and Peter Traxler (Novartis) for reading of the manuscript. The PDGFR kinase inhibitor PTK787/ZK222584 was a gift of Jeanette Wood (Novartis).

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