PROTEASE ACTIVITY IN THE STROMAL CELLS OF GIANT CELL TUMOUR OF BONE

Abstract

Giant cell tumor (GCT) of bone is an osteolytic tumor that is locally aggressive and potentially metastatic. The pathogenesis of GCT is poorly understood. The purpose of this study was to harvest and culture primary cell lines from clinical specimens of GCT of bone and identify specific bone degradation proteases (matrix metalloproteinases: MMP-2, MMP-9) produced by the neoplastic stromal cells in vitro.

With approval by the McMaster University Biohazards and Ethics Review Boards, we acquired consent from five patients with GCT of bone, and harvested specimens intraoperatively. The specimens were chopped in DMEM containing 10% Fetal Bovine Serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. The cell suspensions were incubated at thirty-seven degrees (5% CO2 and 95% air) and cultivated. The cells were grown to confluence and taken through several passages until only proliferative cells were present. Immunocytochemistry with TRAP (Tartrate Resistant Acid Phosphatase) was used to confirm the stem cell origin of the propagative cells. Protein electrophoresis with embedded gelatin was used for detecting protease activity (MMP-2, MMP-9) on cell lysates and medium. P-aminophenyl mercuric acetate (APMA) was used to activate and ethylenediaminetetraacetic acid (EDTA) was used to block MMP-2 and MMP-9 activity. Our controls included serum free media, Human Osteosarcoma and Fibroblast cell lines.

Immunocytochemistry with TRAP confirmed that our propagative cells were not hematopoietic in origin but rather mesenchymal. Protein electrophoresis on cell lysates and medium identified the protease activity of MMP-2 and MMP-9 with lytic bands at appropriate molecular weights. APMA activated MMP-2 more than MMP-9, as indicated by increased relative density of bands. EDTA blocked the activity of both MMPs.

Our study confirmed the ability to cultivate the neoplastic stromal cells of GCT of bone from clinical specimens. Protein electrophoresis showed that activated MMP-2 and MMP-9 are secreted from the neoplastic stromal cells in vitro, suggesting a role for the tumor cells in bone destruction. These results are intriguing, as novel therapies in specific MMP inhibitors are currently underway for numerous disease processes.