In vitro and in vivo effects of CXCR4 inhibition in osteosarcoma

Proc Amer Assoc Cancer Res, Volume 47, 2006 285 Background The chemokine receptor CXCR4 has been implicated in many metastatic cancers and its expression in osteosarcoma correlates with poor prognosis. CXCL12 (SDF-1), the ligand for CXCR4, is expressed at high levels in lung, bone marrow and liver, all of which are sites for osteosarcoma metastasis. We have previously shown that CTCE-9908, a small peptide antagonist of CXCR4, reduces the number of lung metastases in a murine model of osteosarcoma. In an effort to elucidate the mechanism by which this occurs, we have explored the effect of CTCE-9908 in vitro and in vivo. Methods K7M2 murine osteosarcoma cells were treated with CTCE-9908 (100 ug/ml) and compared with untreated controls. In vitro experiments included the growth rate of cells as determined by MTT assays, cell morphology analysis, apoptosis assays utilizing annexin/7AAD, and determination of binding to extracellular matrix protein components. In addition, K7M2 cells were injected into the tail vein of Balb/C mice following a brief pretreatment with CTCE-9908 or control. Mice were then treated with CTCE-9908 or normal saline, once daily x5, for 4 weeks. All of the animals were sacrificed by day 28 and the number of surface lung metastases were counted and confirmed by histology. Results K7M2 cells that were treated with CTCE-9908 showed a decrease in growth rate in vitro (27 vs 20 hours, p-value <0.01). This was not accompanied by any gross morphological changes. Cells that were treated with CTCE-9908 showed increased apoptosis as determined by an increased percentage of cells that were annexin-positive and 7AAD-negative. In addition, treated cells showed a marked decrease in adhesion to plates coated with selected extracellular matrix proteins, including laminin and fibronectin. Mice that were treated with CTCE-9908 had a 50% decrease in the number of gross metastatic lung nodules (94.8 vs 189.4 nodules, p-value 0.01). In addition, histological evaluation showed that mice treated with CTCE-9908 had a marked reduction in micrometastatic disease burden. Conclusions Mice treated with the CXCR4 inhibitor, CTCE-9908, have a decrease in the number of both gross and micrometastatic lung nodules. The possible mechanisms for this anti-metastatic effect include reduced proliferative rate, increased apoptosis and decreased adhesion to extracellular matrix proteins. Additional work to determine which of these pathways is most important in metastatic osteosarcoma is currently being performed.