Therapeutic Synergy of TNP-470 and Ionizing Radiation: Effects on Tumor Growth, Vessel Morphology, and Angiogenesis in Human Glioblastoma Multiforme Xenografts

Therapeutic Synergy of TNP-470 and Ionizing Radiation: Effects on Tumor Growth, Vessel Morphology, and Angiogenesis in Human Glioblastoma Multiforme Xenografts1

Institute of Molecular Pathology, University of Copenhagen, DK-2100 Copenhagen, Denmark

Abstract

We examined the effect on tumor growth, vessel morphology, and expression of angiogenic factors of combining radiotherapy and antiangiogenesis in the human glioblastoma line U87 grown in the flank or intracranially in the nude mouse. The antiangiogenic agent TNP-470 was given 6.7 mg/kg s.c. daily on day 1–7 starting 1 week after transplantation. Irradiation (IR), 10 Gy × 1, was administered on day 7. A series of tumors were excised 8 and 48 h after the end of treatment. The vascular morphology was evaluated in CD31 immunostained cryosections and by electron microscopy, and the pattern of expression of angiogenic factors (mRNA and protein) was quantitatively analyzed by phosphorimaging of Northern blots and Western blots. Significant inhibition of s.c. flank tumor growth relative to untreated controls was achieved by monotherapy with both TNP-470 (P < 0.001) and IR (P < 0.001). A significant enhancement of this effect was obtained by combining TNP-470 and IR (P < 0.05). We saw no effect of TNP-470 either alone or in addition to the effect of IR on the survival of mice with intracranial tumors. CD31 immunostaining of s.c. tumors showed acute endothelial swelling and luminal protrusion in irradiated tumor vessels but never in tumors pretreated with TNP-470, and not in the untreated controls. The vessel density (Chalkley point counts) was unchanged by TNP-470 therapy. In the TNP-470-treated tumors, we observed a distinct broadening of the endothelial basement membrane by an approximately 400–700-nm-thick electron-dense yet uncharacterized fibrillar material. TNP-470 treated tumors +/− IR also had a significantly increased mRNA expression of angiopoietin-1, whereas angiopoietin-2, vascular endothelial growth factor and basic fibroblast growth factor mRNA were unchanged by the treatments. In conclusion, TNP-470 significantly enhanced the tumor effect of ionizing IR, and our findings strongly indicate that acute microvascular damage after IR is effectively prevented by concurrent TNP-470 treatment. A significant up-regulation of angiopoietin-1 seems to play a role in this protective mechanism, which as yet is not fully elucidated.

Footnotes

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↵1 Supported by The Danish Cancer Research Foundation, The Danish Medical Research Council (Grant 9700929), and Danish Cancer Society (Grant 9810034).
↵2 To whom requests for reprints should be addressed, at Molecular Pathology, University of Copenhagen, Frederik V’s Vej 11, DK-2100 Copenhagen, Denmark. Phone: 45-3532-6006; Fax: 45-3532-6081; E-mail: paulk{at}pai.ku.dk
↵3 The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
↵4 B. Eyden. Perivascular amorphous matrices containing laminin and type IV collagen not organized as a conventional basal lamina: identification by electron microscopy and implications for the control of cell biological processes, submitted for publication.
- Accepted December 20, 1999.
- Received September 30, 1999.
- Revision received December 20, 1999.