Determining the role of thrombospondin-1 in a mouse model of breast cancer
Authors: Karen O. Yee, Caitlin M. Connolly, Jack W. Lawler
Journal: Cancer Research (2006)

1039  Thrombospondin-1 (TSP-1) has been shown to be an effective anti-angiogenic and anti-tumorigenic protein in vitro and in many xenograft mouse models. These models are limited in the information they provide because they don’t recapitulate the natural progression of the tumor at the correct body site. We were interested to know how TSP-1 affects breast cancer progression in vivo. The approach we decided to take was to develop a transgenic mouse model for breast cancer in order to enable us to study tumor progression at different stages (ie. initiation to metastasis). We crossed TSP-1 +/+ and TSP-1 -/- female mice with male polyomavirus middle T Antigen (PyT)/mouse mammary tumor virus (MMTV) transgenic mice to generate progeny that are PyT +/-, TSP-1 +/+ and PyT +/-, TSP-1-/-. At 60 days of age when tumors are becoming palpable, we found that tumors in the PyT +/-, TSP-1 -/- mice are 35% larger than tumors found in the PyT +/-, TSP-1+/+. In our preliminary analysis on blood vessel density, TSP-1+/+ mice at 60 days of age have fewer tumor vessels per mm2. This suggests that TSP-1 may play role in inhibiting early tumor progression through an anti-angiogenic mechanism. By contrast, at 90 days of age when tumors are well established, tumors from the PyT +/-, TSP-1 -/- mice are only slightly larger than tumors found in the PyT +/-, TSP-1 +/+ mice, and while vessel density isn’t significantly different, vessels are larger in the PyT+/-, TSP-1-/- mice. Surprisingly, we found that there is a higher incidence of overt lung metastasis in the PyT +/-, TSP-1+/+ mice suggesting that TSP-1 plays an important role during lung metastasis in this model. This data has been confirmed using real time PCR on RNA isolated from the lungs and we are able to detect higher PyT expression in PyT +/-, TSP-1+/+ mice at 60 and 90 days of age. Furthermore, we were only able to detect PyT expression in cells isolated from the blood of 45 day old PyT +/-, TSP-1+/+ mice indicating that TSP-1 may be enabling intravasation during early tumor growth. Since TSP-1 is known to activate TGF-beta, we determined the level of both active TGF-beta and latent TGF-beta using an ELISA. We find that while active TGF-beta levels did not differ between the two genotypes at 90 days of age, the level of latent TGF-beta is significantly higher in the PyT+/-, TSP-1+/+ tumors. Finally, we have stained tumor sections with the F4/80 antibody to detect the presence of macrophages in the tumors. At 90 days of age, PyT +/-, TSP-1 +/+ tumors have a large amount of necrosis and the macrophages are found predominantly within the tumor mass either in the matrix or in the cystic, necrotic lesion. By contrast, the PyT+/-, TSP-1 -/- mice contain fewer cystic and necrotic tumors and most of the macrophages are found in the periphery of the tumor. In conclusion, we have demonstrated that TSP-1’s role in tumor progression is very complex and differs at key points during tumor progression and endogenous TSP-1 appears to support breast cancer metastasis.