PTHrP Induces Autocrine/Paracrine Proliferation of Bone Tumor Cells through Inhibition of Apoptosis

Giant Cell Tumor of Bone (GCT) is an aggressive skeletal tumor characterized by local bone destruction, high recurrence rates and metastatic potential. Previous work in our lab has shown that the neoplastic cell of GCT is a proliferating pre-osteoblastic stromal cell in which the transcription factor Runx2 plays a role in regulating protein expression. One of the proteins expressed by these cells is parathryroid hormone-related protein (PTHrP). The objectives of this study were to determine the role played by PTHrP in GCT of bone with a focus on cell proliferation and apoptosis. Primary stromal cell cultures from 5 patients with GCT of bone and one lung metastsis were used for cell-based experiments. Control cell lines included a renal cell carcinoma (RCC) cell line and a human fetal osteoblast cell line. Cells were exposed to optimized concentrations of a PTHrP neutralizing antibody and were analyzed with the use of cell proliferation and apoptosis assays including mitochondrial dehydrogenase assays, crystal violet assays, APO-1 ELISAs, caspase activity assays, flow cytometry and immunofluorescent immunohistochemistry. Neutralization of PTHrP in the cell environment inhibited cell proliferation in a consistent manner and induced apoptosis in the GCT stromal cells, with the exception of those obtained from a lung metastasis. Cell cycle progression was not significantly affected by PTHrP neutralization. These findings indicate that PTHrP plays an autocrine/paracrine neoplastic role in GCT by allowing the proliferating stromal cells to evade apoptosis, possibly through non-traditional caspase-independent pathways. Thus PTHrP neutralizing immunotherapy is an intriguing potential therapeutic strategy for this tumor

A short-term in vivo model for giant cell tumor of bone

<p>Abstract</p> <p>Background</p> <p>Because of the lack of suitable <it>in vivo </it>models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus <it>in vitro </it>testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.</p> <p>Methods</p> <p>Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using <it>in vivo </it>biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence <it>in situ </it>hybridization (FISH).</p> <p>Results</p> <p>The suspension of all 10 patients formed solid tumors when grafted on the CAM. <it>In vivo </it>microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells whichwere generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.</p> <p>Conclusions</p> <p>A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first <it>in vivo </it>model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.</p