Treatment of experimental brain tumors with trombospondin-1 derived peptides: an in vivo imaging study

Abstract

Antiangiogenic and antiproliferative effects of synthetic D-reverse peptides derived from the type 1 repeats of thrombospondin (TSP1) were studied in rodent C6 glioma and 9L gliosarcomas. To directly measure tumor size and vascular parameters, we employed in vivo magnetic resonance (MR) imaging and corroborated results by traditional morphometric tissue analysis. Rats bearing either C6 or 9L tumors were treated with TSP1-derived peptide (D-reverse amKRFKQDGGWSHWSPWSSac, n=13) or a control peptide (D-reverse amKRAKQAGGASHASPASSac, n=12) at 10 mg/kg, administered either intravenously or through subcutaneous miniosmotic pumps starting 10 days after tumor implantation. Eleven days later, the effect of peptide treatment was evaluated. TSP1 peptide-treated 9L tumors (50.7+/-44.2 mm3, n=7) and C6 tumors (41.3+/-34.2 mm3, n=6) were significantly smaller than tumors treated with control peptide (9L: 215.7+/-67.8 mm3, n=6; C6: 184.2+/-105.2 mm3, n=6). In contrast, the in vivo vascular volume fraction, the mean vascular area (determined by microscopy), and the microvascular density of tumors were not significantly different in any of the experimental groups. In cell culture, TSP1, and the amKRFKQDGGWSHWSPWSSac peptide showed antiproliferative effects against C6 with an IC of 45 nM for TSP1. These results indicate that TSP1-derived peptides retard brain tumor growth presumably as a result of slower de novo blood vessel formation and synergistic direct antiproliferative effects on tumor cells. We also show that in vivo MR imaging can be used to assess treatment efficacy of novel antiangiogenic drugs non-invasively, which has obvious implications for clinical trials.

Figure 1

C6 glioma cells (5000/well in DMEM containing 1% FBS) were plated in 96 well tissue culture plates in the presence of the indicated concentrations of human platelet TSP1 (closed circles), Ficoll-conjugated peptides D-reverse-amKRFKQDGGWSHWSPWSS-thiopropionyl-AECMficoll (open circles) and D-reverse-amKRAKQAGGWSHWSPWSSC-AECMficoll (closed triangle), and free peptides D-reverse-amKRAKQAGWSHWAAac (open triangle), D-reverse-amKRAKQAGGWSHWSPWSSac (open squares), and D-reverse-amKRAKQAGGASHASPASSac (filled squares). Cells were grown for 72 hours at 37°C in a humidified incubator with 5% CO₂. Proliferation was quantified using the Promega Celltiter assay. Results are presented as a percentage of control proliferation determined without inhibitors, mean±SD, n = 3.

Figure 2

Distribution of fluorescent-labeled TSP1 derived peptide in gliosarcoma and normal brain as determined by fluorescent microscopy of 9L-GFP-34-1 tumor sections (thickness-8 µm). Tumors were implanted in the brain of Fischer rats stereotactically as described in Materials and Methods. Rhodamine-X-labeled P1 (rv-amKRAKQAGGWSHWSPWSSac) was injected IV at 10 mg/kg. Animals were sacrificed at 1 hour after administration of the peptide and brain tissue was processed for microscopy. (A) Dual channel microscopy of a blood vessel at the tumor/normal brain interface. Red—Rhodamine-X-peptide, green—GFP expressing 9L cells. Magnification 250x. (B) Distribution of Rhodamine-X-peptide in 9L tumor. Magnification, 60x. (C) Distribution of Rhodamine-X-peptide in contralateral normal brain. Magnification 60x.

Figure 3

Non-enhanced and contrast-enhanced representative MR axial maps of intracerebrally implanted tumors in animals treated with control and experimental (TSP1 derived peptide). (A) 9L-GFP-34-1 tumor. (B) C6 tumor.

Figure 4

Fluorescence microscopy and immunohistology of 9L-GFP-34-1 tumor sections (8 µm). Tumors were implanted in the brain of Fischer rats stereotactically as described in Materials and Methods. (A) Double-channel fluorescent microscopy of brain tissue isolated from rats that received injection of Hoechst dye immediately before euthanasia. Green—GFP; Blue—Hoechst. (B) Anti-CD31 immunohistology of a parallel section. Blood vessels are delineated using secondary antibody-peroxidase/DAB stain with subsequent counter staining with hematoxylin. Magnification 100x.

Figure 5

Cross-sectional vessel area measured using fluorescent 9L-GFP-34-1 tumor section images. Sections were prepared from the animals treated with control and experimental peptides (n=300 in each data set). Numbers represent mean±SEM.