The selective Cox-2 inhibitor Celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice

Abstract

Background: The inhibition of angiogenesis is a promising strategy for the treatment of malignant primary and secondary tumors in addition to established therapies such as surgery, chemotherapy, and radiation. There is strong experimental evidence in primary tumors that Cyclooxygenase-2 (Cox-2) inhibition is a potent mechanism to reduce angiogenesis. For bone metastases which occur in up to 85% of the most frequent malignant primary tumors, the effects of Cox-2 inhibition on angiogenesis and tumor growth remain still unclear. Therefore, the aim of this study was to investigate the effects of Celecoxib, a selective Cox-2 inhibitor, on angiogenesis, microcirculation and growth of secondary bone tumors.

Methods: In 10 male severe combined immunodeficient (SCID) mice, pieces of A549 lung carcinomas were implanted into a newly developed cranial window preparation where the calvaria serves as the site for orthotopic implantation of the tumors. From day 8 after tumor implantation, five animals (Celecoxib) were treated daily with Celecoxib (30 mg/kg body weight, s.c.), and five animals (Control) with the equivalent amount of the CMC-based vehicle. Angiogenesis, microcirculation, and growth of A549 tumors were analyzed by means of intravital microscopy. Apoptosis was quantified using the TUNEL assay.

Results: Treatment with Celecoxib reduced both microvessel density and tumor growth. TUNEL reaction showed an increase in apoptotic cell death of tumor cells after treatment with Celecoxib as compared to Controls.

Conclusion: Celecoxib is a potent inhibitor of tumor growth of secondary bone tumors in vivo which can be explained by its anti-angiogenic and pro-apoptotic effects. The results indicate that a combination of established therapy regimes with Cox-2 inhibition represents a possible application for the treatment of bone metastases.

Figure 1

Graph: Tumor surface, time course. Y-axis: tumor surface (A_TUM in mm²), x-axis: time in days after implantation. ANOVA on ranks (p = 0,016); *p < 0.05 versus day 7, ^#p < 0.05 versus Control, Mann-Whitney Rank Sum Test. Significant increase in two-dimensional tumor surface in both groups between days 7 and 28 after tumor implantation. Two-dimensional tumor surface is significantly reduced in animals treated with Celecoxib compared to Controls on day 28. A, B, C, D: Photographs of the cranial window with A 549 lung carcinoma, top-view, scale bar 1000 μm. Tumor borders marked by arrows. Day 7: Celecoxib (A), Control (C). Day 28: Celecoxib (B), Control (D).

Figure 2

Graph: Functional vessel density, time course. Y-axis: functional vessel density (FVD in mm/mm²), x-axis: time in days after implantation. ANOVA on ranks (p = 0.008); *p < 0.05 versus day 7, ^#p < 0.05 versus Control, Mann-Whitney Rank Sum Test. Significant increase in functional vessel density in both groups between days 7 and 28 after tumor implantation. Functional vessel density is significantly reduced in animals treated with Celecoxib compared to Controls on day 28. A, B: Photographs from intravital fluorescence microscopy, top-view, scale bar 200 μm. Tumor borders marked by arrows. Day 28: Celecoxib (A), Control (B).

Figure 3

A, B: A 549 lung carcinoma at day 28, hematoxylin-eosin stained cross section, scale bar 500 μm. Tumor borders marked by arrows. Control (A), Celecoxib (B). C, D: A 549 lung carcinoma at day 28, TUNEL reaction, scale bar 50 μm, apoptotic cells stained red. Control (C), Celecoxib (D).