Proc Amer Assoc Cancer Res, Volume 45, 20041756 Clinical relevance: With advances in detection and treatment of primary tumors, secondary tumor (metastases) are becoming larger contributors to cancer patient mortality. Animal models can be useful for the understanding of possible mechanisms of seeding and growth of secondary tumors in various organs as well as for testing efficacy of possible treatments. Purpose: The goal of the studies described herein was to understand the role of transforming growth factor-beta (TGF-b) in cancer cell growth and metastasis. Background: TGF-b is a multifunctional cytokine which can affect tumors in multiple ways. Its direct effects include modulation of cell growth and differentiation, while its indirect activities include regulation of immune function, extracellular matrix production, and angiogenesis. Experimental Procedures: Anti-TGF-b antibodies were tested in syngeneic immunocompetent mice using cell lines derived from spontaneous tumors or oncogene-driven tumors. We have also established quantitative endpoints to measure tumor number and size as well as destruction of soft and hard tissues. Results: Inhibition of TGF- b has distinct effects on primary tumors and secondary lung and bone tumors in a manner related to the nature of the animal model. Thus, our models have allowed us to distinguish between the various tumor-associated biological activities of TGF-b. Conclusions: Similar to the heterogeneity observed in human tumors, animal models also show varied responses to treatment. Our results have important implications for the use of animal models for testing various inhibitors. Use of different in vivo systems may help elucidate specific pathways for tumor growth and metastasis and help discover how to stratify patients in order to help personalize cancer therapies.

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. in these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteohlastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.

With advances in detection and treatment of primary tumors, secondary tumors (metastases) are largely responsible for cancer patient mortality. While transplants of human tumor cells have been useful for studying factors which inhibit primary tumor growth, newer animal models allow for studies of seeding and growth of secondary tumors in various organs as well as measures of the effects of possible treatments on tumors in multiple sites. Of the many molecules which have been implicated in metastasis, transforming growth factor beta, TGF-‚, is of particular interest in studies of cancer-related bone destruction because of its known involvement in bone development and turnover 1-4 as well as in bone repair 5 . Consistent with its pleiotropic activities, TGF-‚ can affect tumors in multiple ways 6-12 . Its direct effects include modulation of tumor cell growth, differentiation, and migration, while its indirect activities include regulation of immune function, extracellular matrix production, and angiogenesis 6-12 . In order to better understand the role of TGF-‚ in cancer, we set up and characterized several animal models and established quantitative endpoints for the analysis of primary and secondary tumors. To this end, anti-TGF-‚ antibodies were tested in syngeneic, immunocompetent mice using cell lines derived from spontaneous tumors or oncogene-driven tumors. Quantitative endpoints were used to measure tumor number and size as well as destruction of soft and hard tissues. Using these animal models, we found that inhibition of TGF-‚ has distinct effects on primary tumors and secondary lung and bone tumors in a manner related to the nature of the animal model. Thus, our models have allowed us to distinguish between the various tumor-associated biological activities of TGF-‚. In conclusion, similar to the heterogeneity observed in human tumors, animal models also show varied responses to treatment. Our results have important implications for the use of animal models for testing various possible cancer treatments. Use of different in vivo systems may assist in the elucidation of specific pathways for tumor growth and metastasis as well as discovery of methods to stratify patients as part of personalized cancer therapies.

Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.

Fish stanniocalcin (STC) inhibits uptake of calcium and stimulates phosphate reabsorption. To determine the role of the highly homologous mammalian protein, STC-1, we created and characterized transgenic mice that express STC-1 under control of a muscle-specific promoter. STC-1 transgenic mice were smaller than wild-type littermates and had normal growth plate cartilage morphology but increased cartilage matrix synthesis. In STC-1 mice, the rate of bone formation, but not bone mineralization, was decreased. Increased cortical bone thickness and changes in trabeculae number, density, and thickness in STC-1 mice indicated a concomitant suppression of osteoclast activity, which was supported by microcomputed tomography analyses and histochemistry. Skeletal muscles were disproportionately small and showed altered function and response to injury in STC-1 mice. Electron microscopy indicated that muscle mitochondria were dramatically enlarged in STC-1 mice. These changes in STC-1 mice could not be explained by deficits in blood vessel formation, as vascularity in organs and skeletal tissues was increased as was induction of vascularity in response to femoral artery ligation. Our results indicate that STC-1 can affect calcium homeostasis, bone and muscle mass and structure, and angiogenesis through effects on osteoblasts, osteoclasts, myoblasts/myocytes, and endothelial cells.