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PDGF-C induces maturation of blood vessels in a model of glioblastoma and attenuates the response to anti-VEGF treatment.

di Tomaso E, London N, Fuja D, Logie J, Tyrrell JA, Kamoun W, Munn LL, Jain RK - PLoS ONE (2009)

Bottom Line: PDGF-C overexpressing tumors had smaller vessel diameters and lower vascular permeability compared to the parental or siRNA-transfected tumors.Treatment with DC101, an anti-VEGFR-2 antibody, induced decreases in vessel density in the parental tumors, but had no effect on the PDGF-C overexpressing tumors.These results suggest that PDGF-C plays an important role in glioma vessel maturation and stabilization, and that it can attenuate the response to anti-VEGF therapy, potentially contributing to escape from vascular normalization.

View Article: PubMed Central - PubMed

Affiliation: E.L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Recent clinical trials of VEGF inhibitors have shown promise in the treatment of recurrent glioblastomas (GBM). However, the survival benefit is usually short-lived as tumors escape anti-VEGF therapies. Here we tested the hypothesis that Platelet Derived Growth Factor-C (PDGF-C), an isoform of the PDGF family, affects GBM progression independent of VEGF pathway and hinders anti-VEGF therapy.

Principal findings: We first showed that PDGF-C is present in human GBMs. Then, we overexpressed or downregulated PDGF-C in a human GBM cell line, U87MG, and grew them in cranial windows in nude mice to assess vessel structure and function using intravital microscopy. PDGF-C overexpressing tumors had smaller vessel diameters and lower vascular permeability compared to the parental or siRNA-transfected tumors. Furthermore, vessels in PDGF-C overexpressing tumors had more extensive coverage with NG2 positive perivascular cells and a thicker collagen IV basement membrane than the controls. Treatment with DC101, an anti-VEGFR-2 antibody, induced decreases in vessel density in the parental tumors, but had no effect on the PDGF-C overexpressing tumors.

Conclusion: These results suggest that PDGF-C plays an important role in glioma vessel maturation and stabilization, and that it can attenuate the response to anti-VEGF therapy, potentially contributing to escape from vascular normalization.

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Related in: MedlinePlus

PDGF-C expression in human gliomas – autocrine & paracrine signaling.A: Expression of PDGF-C in human samples of gliomas. 27 samples of Grade IV glioma (Tissue Microarrays, kind gift from Dr. David Louis, MGH) were screened for expression of PDGF-C using immunohistochemistry (PDGF-C monoclonal antibody from R&D, used at 1/100). 23 out of 27 specimens expressed PDGF-C in tumor cells and/or in the peri-vascular area. Panels I, II and III are 3 different patients; panel IV is normal brain tissue present on the same array. In I and III positive tumor cells can clearly be identified; positive endothelium is evident in I and II, and perivascular cells also stain in panel II. In all panels, brown color (DAB) shows positive labeling for PDGF-C while blue (hematoxilin) stains nuclei. B: PDGF-C RNA expression in several common glioma cell lines. We chose U87 since it has a relatively low level expression compared to other cell lines and is thus amenable to downreglation. C: Lack of autocrine effect of PDGF-C on U87MG cells. Qt-RTPCR of PDGFRα in U87 cells shows that RNA is present, but there is no protein detectable by Western blot (not shown). PDGFRβ protein expression in parental (U87MG), PDGF-C overexpressor (U87-C), Mock-transfected (M) or ShRNAi cells (si). PDGFRβ phosphorylation. Cells were treated overnight with one of the following: recombinant PDGF-B (rBB, positive control); conditioned medium from cells overexpressing PDGF-C (CC); fresh DMEM culture medium (DMEM). PDGF-C did not induce phosphorylation above the baseline level. D: Effect of PDGF-C on 10T1/2 myofibroblasts. Panel I, Top: Phosphorylation of PDGFRβ. Cells were stimulated overnight with fresh DMEM culture medium (DMEM), recombinant PDGFBB (rBB; positive control), U87-C conditioned medium (CC), or conditioned medium from mock-transfected cells (M). PDGF-C did not stimulate this receptor. Bottom: phosphorylation of PDGFRα on 10T1/2 cells in culture. Cells were stimulated overnight with conditioned medium from U87si (si), U87-CC (CC), mock transfected cells (M), or fresh DMEM culture medium (DMEM). Recombinant PDGFAA was used as a positive control (rAA). The phosphorylation induced by the mock and U87si media is likely due to PDGF-A produced by these cells. Treatment of 10T1/2 cells with PDGF-C conditioned medium results in faster migration (Boyden chamber assay; panel II) and proliferation (WST-1 assay; panel III).
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pone-0005123-g001: PDGF-C expression in human gliomas – autocrine & paracrine signaling.A: Expression of PDGF-C in human samples of gliomas. 27 samples of Grade IV glioma (Tissue Microarrays, kind gift from Dr. David Louis, MGH) were screened for expression of PDGF-C using immunohistochemistry (PDGF-C monoclonal antibody from R&D, used at 1/100). 23 out of 27 specimens expressed PDGF-C in tumor cells and/or in the peri-vascular area. Panels I, II and III are 3 different patients; panel IV is normal brain tissue present on the same array. In I and III positive tumor cells can clearly be identified; positive endothelium is evident in I and II, and perivascular cells also stain in panel II. In all panels, brown color (DAB) shows positive labeling for PDGF-C while blue (hematoxilin) stains nuclei. B: PDGF-C RNA expression in several common glioma cell lines. We chose U87 since it has a relatively low level expression compared to other cell lines and is thus amenable to downreglation. C: Lack of autocrine effect of PDGF-C on U87MG cells. Qt-RTPCR of PDGFRα in U87 cells shows that RNA is present, but there is no protein detectable by Western blot (not shown). PDGFRβ protein expression in parental (U87MG), PDGF-C overexpressor (U87-C), Mock-transfected (M) or ShRNAi cells (si). PDGFRβ phosphorylation. Cells were treated overnight with one of the following: recombinant PDGF-B (rBB, positive control); conditioned medium from cells overexpressing PDGF-C (CC); fresh DMEM culture medium (DMEM). PDGF-C did not induce phosphorylation above the baseline level. D: Effect of PDGF-C on 10T1/2 myofibroblasts. Panel I, Top: Phosphorylation of PDGFRβ. Cells were stimulated overnight with fresh DMEM culture medium (DMEM), recombinant PDGFBB (rBB; positive control), U87-C conditioned medium (CC), or conditioned medium from mock-transfected cells (M). PDGF-C did not stimulate this receptor. Bottom: phosphorylation of PDGFRα on 10T1/2 cells in culture. Cells were stimulated overnight with conditioned medium from U87si (si), U87-CC (CC), mock transfected cells (M), or fresh DMEM culture medium (DMEM). Recombinant PDGFAA was used as a positive control (rAA). The phosphorylation induced by the mock and U87si media is likely due to PDGF-A produced by these cells. Treatment of 10T1/2 cells with PDGF-C conditioned medium results in faster migration (Boyden chamber assay; panel II) and proliferation (WST-1 assay; panel III).

Mentions: To assess whether PDGF-C is expressed in human glioblastomas, we first examined a series of 27 surgical specimens of human glioblastomas from MGH patients. In 23 of these samples, we detected PDGF-C in cancer cells but also in endothelial and perivascular cells (Figure 1A). In contrast, there was no detectable PDGF-C in normal brain; this suggested that PDGF-C might play a role in human gliomas.


PDGF-C induces maturation of blood vessels in a model of glioblastoma and attenuates the response to anti-VEGF treatment.

di Tomaso E, London N, Fuja D, Logie J, Tyrrell JA, Kamoun W, Munn LL, Jain RK - PLoS ONE (2009)

PDGF-C expression in human gliomas – autocrine & paracrine signaling.A: Expression of PDGF-C in human samples of gliomas. 27 samples of Grade IV glioma (Tissue Microarrays, kind gift from Dr. David Louis, MGH) were screened for expression of PDGF-C using immunohistochemistry (PDGF-C monoclonal antibody from R&D, used at 1/100). 23 out of 27 specimens expressed PDGF-C in tumor cells and/or in the peri-vascular area. Panels I, II and III are 3 different patients; panel IV is normal brain tissue present on the same array. In I and III positive tumor cells can clearly be identified; positive endothelium is evident in I and II, and perivascular cells also stain in panel II. In all panels, brown color (DAB) shows positive labeling for PDGF-C while blue (hematoxilin) stains nuclei. B: PDGF-C RNA expression in several common glioma cell lines. We chose U87 since it has a relatively low level expression compared to other cell lines and is thus amenable to downreglation. C: Lack of autocrine effect of PDGF-C on U87MG cells. Qt-RTPCR of PDGFRα in U87 cells shows that RNA is present, but there is no protein detectable by Western blot (not shown). PDGFRβ protein expression in parental (U87MG), PDGF-C overexpressor (U87-C), Mock-transfected (M) or ShRNAi cells (si). PDGFRβ phosphorylation. Cells were treated overnight with one of the following: recombinant PDGF-B (rBB, positive control); conditioned medium from cells overexpressing PDGF-C (CC); fresh DMEM culture medium (DMEM). PDGF-C did not induce phosphorylation above the baseline level. D: Effect of PDGF-C on 10T1/2 myofibroblasts. Panel I, Top: Phosphorylation of PDGFRβ. Cells were stimulated overnight with fresh DMEM culture medium (DMEM), recombinant PDGFBB (rBB; positive control), U87-C conditioned medium (CC), or conditioned medium from mock-transfected cells (M). PDGF-C did not stimulate this receptor. Bottom: phosphorylation of PDGFRα on 10T1/2 cells in culture. Cells were stimulated overnight with conditioned medium from U87si (si), U87-CC (CC), mock transfected cells (M), or fresh DMEM culture medium (DMEM). Recombinant PDGFAA was used as a positive control (rAA). The phosphorylation induced by the mock and U87si media is likely due to PDGF-A produced by these cells. Treatment of 10T1/2 cells with PDGF-C conditioned medium results in faster migration (Boyden chamber assay; panel II) and proliferation (WST-1 assay; panel III).
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getmorefigures.php?uid=PMC2662415&req=5

pone-0005123-g001: PDGF-C expression in human gliomas – autocrine & paracrine signaling.A: Expression of PDGF-C in human samples of gliomas. 27 samples of Grade IV glioma (Tissue Microarrays, kind gift from Dr. David Louis, MGH) were screened for expression of PDGF-C using immunohistochemistry (PDGF-C monoclonal antibody from R&D, used at 1/100). 23 out of 27 specimens expressed PDGF-C in tumor cells and/or in the peri-vascular area. Panels I, II and III are 3 different patients; panel IV is normal brain tissue present on the same array. In I and III positive tumor cells can clearly be identified; positive endothelium is evident in I and II, and perivascular cells also stain in panel II. In all panels, brown color (DAB) shows positive labeling for PDGF-C while blue (hematoxilin) stains nuclei. B: PDGF-C RNA expression in several common glioma cell lines. We chose U87 since it has a relatively low level expression compared to other cell lines and is thus amenable to downreglation. C: Lack of autocrine effect of PDGF-C on U87MG cells. Qt-RTPCR of PDGFRα in U87 cells shows that RNA is present, but there is no protein detectable by Western blot (not shown). PDGFRβ protein expression in parental (U87MG), PDGF-C overexpressor (U87-C), Mock-transfected (M) or ShRNAi cells (si). PDGFRβ phosphorylation. Cells were treated overnight with one of the following: recombinant PDGF-B (rBB, positive control); conditioned medium from cells overexpressing PDGF-C (CC); fresh DMEM culture medium (DMEM). PDGF-C did not induce phosphorylation above the baseline level. D: Effect of PDGF-C on 10T1/2 myofibroblasts. Panel I, Top: Phosphorylation of PDGFRβ. Cells were stimulated overnight with fresh DMEM culture medium (DMEM), recombinant PDGFBB (rBB; positive control), U87-C conditioned medium (CC), or conditioned medium from mock-transfected cells (M). PDGF-C did not stimulate this receptor. Bottom: phosphorylation of PDGFRα on 10T1/2 cells in culture. Cells were stimulated overnight with conditioned medium from U87si (si), U87-CC (CC), mock transfected cells (M), or fresh DMEM culture medium (DMEM). Recombinant PDGFAA was used as a positive control (rAA). The phosphorylation induced by the mock and U87si media is likely due to PDGF-A produced by these cells. Treatment of 10T1/2 cells with PDGF-C conditioned medium results in faster migration (Boyden chamber assay; panel II) and proliferation (WST-1 assay; panel III).
Mentions: To assess whether PDGF-C is expressed in human glioblastomas, we first examined a series of 27 surgical specimens of human glioblastomas from MGH patients. In 23 of these samples, we detected PDGF-C in cancer cells but also in endothelial and perivascular cells (Figure 1A). In contrast, there was no detectable PDGF-C in normal brain; this suggested that PDGF-C might play a role in human gliomas.

Bottom Line: PDGF-C overexpressing tumors had smaller vessel diameters and lower vascular permeability compared to the parental or siRNA-transfected tumors.Treatment with DC101, an anti-VEGFR-2 antibody, induced decreases in vessel density in the parental tumors, but had no effect on the PDGF-C overexpressing tumors.These results suggest that PDGF-C plays an important role in glioma vessel maturation and stabilization, and that it can attenuate the response to anti-VEGF therapy, potentially contributing to escape from vascular normalization.

View Article: PubMed Central - PubMed

Affiliation: E.L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Recent clinical trials of VEGF inhibitors have shown promise in the treatment of recurrent glioblastomas (GBM). However, the survival benefit is usually short-lived as tumors escape anti-VEGF therapies. Here we tested the hypothesis that Platelet Derived Growth Factor-C (PDGF-C), an isoform of the PDGF family, affects GBM progression independent of VEGF pathway and hinders anti-VEGF therapy.

Principal findings: We first showed that PDGF-C is present in human GBMs. Then, we overexpressed or downregulated PDGF-C in a human GBM cell line, U87MG, and grew them in cranial windows in nude mice to assess vessel structure and function using intravital microscopy. PDGF-C overexpressing tumors had smaller vessel diameters and lower vascular permeability compared to the parental or siRNA-transfected tumors. Furthermore, vessels in PDGF-C overexpressing tumors had more extensive coverage with NG2 positive perivascular cells and a thicker collagen IV basement membrane than the controls. Treatment with DC101, an anti-VEGFR-2 antibody, induced decreases in vessel density in the parental tumors, but had no effect on the PDGF-C overexpressing tumors.

Conclusion: These results suggest that PDGF-C plays an important role in glioma vessel maturation and stabilization, and that it can attenuate the response to anti-VEGF therapy, potentially contributing to escape from vascular normalization.

Show MeSH
Related in: MedlinePlus