Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate - PubMed (original) (raw)

. 1998 Jul 21;95(15):8795-800.

doi: 10.1073/pnas.95.15.8795.

R L Kendall, G Cabrera, L Soroceanu, Y Heike, G Y Gillespie, G P Siegal, X Mao, A J Bett, W R Huckle, K A Thomas, D T Curiel

Affiliations

Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate

C K Goldman et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.

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Figures

Figure 1

Figure 1

Transient transfection of HT-1080 cells with sflt-1 cDNA inhibits lung implantation and growth. Human HT-1080 fibrosarcoma cells were transiently transfected with plasmid expressing sFLT-1 or the corresponding control plasmid and 8 × 105 cells in 0.3 ml were injected via the tail vein (n = 9/group). After 30 days lungs were removed and examined histologically to detect tumor nodules, defined by clusters containing ≥6 tumor cells. The statistical difference between groups was computed using χ2 statistics (∗, P < 0.02).

Figure 2

Figure 2

Transient transfection of HT-1080 cells with sflt-1 cDNA inhibits the growth of s.c. nodules. Human HT-1080 fibrosarcoma cells were transiently transfected with plasmid expressing sFLT-1 (filled circles) or the corresponding control plasmid (open circles). Cells (3 × 106) transfected with either psflt-1 (n = 5) or pcDNA3 (n = 7) were injected into the flanks of athymic nude mice. Nodule dimensions were used to compute tumor volume. The statistical difference between groups was computed using Student’s t test (∗, P < 0.03).

Figure 3

Figure 3

Expression of sflt-1 mRNA and sFLT-1 protein by stably transfected HT-1080 tumor cells. In each panel samples derived from control cells stably transfected with pcDNA3 and clones C4 and B3, each stably transfected with pCIsflt-1, are in lanes 1, 2, and 3, respectively. (A) Total cellular RNA was analyzed for the presence of sflt-1 mRNA on a Northern blot hybridized with a specific sflt-1 probe. (B) A Western blot of heparin-binding proteins from media conditioned by these cells was probed with an antisera directed against the N-terminal polypeptide sequence of sFLT-1. (C) Heparin-binding proteins isolated from media conditioned by these cells were covalently crosslinked to 125I-labeled VEGF and analyzed by SDS/PAGE and autoradiography. Arrows indicate the migration positions of free VEGF (V) and VEGF crosslinked to either one (M, monomer) or two (D, dimer) molecules of sFLT-1.

Figure 4

Figure 4

In vitro and in vivo growth of HT-1080 clones stably transfected with sflt-1 cDNA. HT-1080 tumor cells were stably transfected with control pcDNA3 plasmid (open square) or the corresponding pCIsflt-1 plasmid incorporating HCMV intron A (pCIsflt-1) from which clones C4 (open circles) and B3 (filled circles) were selected. (A) In vitro the control (pcDNA3), moderate (C4) sFLT-1-expressing, and high (B3) sFLT-1-expressing clones grew with equivalent rates in culture. (B) In nude mice (n = 6/group) tumors developing from two s.c. injections totaling 3 × 106 cells/animal of either the control (pcDNA3), moderate sFLT-1-expressing (C4), or high (B3) sFLT-1 expressing clones grew at high, intermediate, and low rates, respectively. (C) Tumors are shown in situ 19 days after injections of HT-1080 cells either stably transfected with control plasmid DNA (pcDNA3, top) or clones stably expressing intermediate (C4, middle) and high (B3, bottom) levels of sFLT-1.

Figure 5

Figure 5

Survival kinetics of SCID mice injected intracranially with D54-MG human glioma cells stably transfected with sflt-1 cDNA. D54-MG human glioma cells (5 × 105) either not transfected (open squares) or stably transfected with either pcDNA3 (stippled squares) or psflt-1 (filled circles) were stereotactically injected into brains of SCID mice (n = 5/group) to a depth of 2.5 mm after which they were monitored twice daily. Survival of animals injected with tumor cells expressing the sflt-1 gene was significantly (P < 0.005; Kaplan–Meier survival analysis, Mantel–Cox log rank test) longer than those injected with tumor cells transfected with either control pcDNA3 or no plasmid which were not different from each other.

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