Tyrosine kinase inhibitor SU6668 represses chondrosarcoma growth via antiangiogenesis in vivo

Abstract

Background: As chondrosarcomas are resistant to chemotherapy and ionizing radiation, therapeutic options are limited. Radical surgery often cannot be performed. Therefore, additional therapies such as antiangiogenesis represent a promising strategy for overcoming limitations in chondrosarcoma therapy. There is strong experimental evidence that SU6668, an inhibitor of the angiogenic tyrosine kinases Flk-1/KDR, PDGFRbeta and FGFR1 can induce growth inhibition of various primary tumors. However, the effectiveness of SU6668 on malignant primary bone tumors such as chondrosarcomas has been rarely investigated. Therefore, the aim of this study was to investigate the effects of SU6668 on chondrosarcoma growth, angiogenesis and microcirculation in vivo.

Methods: In 10 male severe combined immunodeficient (SCID) mice, pieces of SW1353 chondrosarcomas were implanted into a cranial window preparation where the calvaria serves as the site for the orthotopic implantation of bone tumors. From day 7 after tumor implantation, five animals were treated with SU6668 (250 mg/kg body weight, s.c.) at intervals of 48 hours (SU6668), and five animals with the equivalent amount of the CMC-based vehicle (Control). Angiogenesis, microcirculation, and growth of SW 1353 tumors were analyzed by means of intravital microscopy.

Results: SU6668 induced a growth arrest of chondrosarcomas within 7 days after the initiation of the treatment. Compared to Controls, SU6668 decreased functional vessel density and tumor size, respectively, by 37% and 53% on day 28 after tumor implantation. The time course of the experiments demonstrated that the impact on angiogenesis preceded the anti-tumor effect. Histological and immunohistochemical results confirmed the intravital microscopy findings.

Conclusion: SU6668 is a potent inhibitor of chondrosarcoma tumor growth in vivo. This effect appears to be induced by the antiangiogenic effects of SU6668, which are mediated by the inhibition of the key angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRbeta and FGFR1. The experimental data obtained provide rationale to further develop the strategy of the use of the angiogenesis inhibitor SU6668 in the treatment of chondrosarcomas in addition to established therapies such as surgery.

Figure 1

A: Functional vessel density, time course. Y-axis: functional vessel density (FVD in mm/mm²), x-axis: time in days after tumor implantation. SU6668 induced a regression of functional vessel density (FVD), while FVD in Controls increased between day 7 and 14 after tumor implantation, remaining constant thereafter. B: Tumor surface, time course. Y-axis: tumor surface (A_TUM in mm²), x-axis: time in days after tumor implantation. In Controls, tumor surface continuously increased over the period of investigation. In contrast to this, SU6668 induced a growth arrest of chondrosarcomas beginning on day 14 after tumor implantation. A and B: Treatment started on day 7 after tumor implantation. Results are given as median with 25% quartiles and 75 % quartiles. Mann-Whitney rank sum test, ^#p < 0.05 versus Control, *p < 0.05 versus day 7 after tumor implantation. C and D: Immunohistochemistry for CD31 (PECAM-1) on day 28 after tumor implantation confirmed the results of the in vivo quantification of the functional vessel density with intravital microscopy. Untreated Controls showed uniformly distributed CD31 positive cells (brown) within the implanted tumors (C). In SU6668 treated tumors, CD31 positive cells were rare and only detectable in peripheral areas of the tumor residues (D). Scale bars represent 50 μm.

Figure 2

SW 1353 chondrosarcoma at day 28 after tumor implantation, hematoxylin-eosin stained cross section from representative tissue samples of the cranial window preparation. A, D: 4-fold magnification, scale bar 500 μm. B, C, E, F: 20-fold magnification, scale bar 100 μm. In Controls, the overview magnification shows a large tumor formation with high cellular density (A). Enlarged cutaway views (B, C) from the framed regions show high cellular density and destructive tumor growth with resorption of the surrounding bone (arrows, tips pointing to resorption lacunae). In SU6668, tumor size was markedly reduced (D). Enlarged cutaway views (E, F) from the framed regions show fibrous "scar" tissue with residual tumor cells and low cellular density and regenerative bone formation at sites of previous bone resorption (double arrows, distal tip pointing to former resorption front, proximal tip pointing to new mineralization front).