treatment decreased expression of uPA and uPAR in vivo (Figure S6A). EGR1 down-regulates uPA activity uPA is produced and secreted as an inactive single-chain polypeptide, termed pro-uPA, which lacks plasminogen-activating activity. The binding of pro-uPA to uPAR induces its activation which in turn converts plasminogen to the active serine protease plasmin [19]. In this regard, we examined whether EGR1 exerts effects on uPA activity by performing uPA activity ELISA. ELISA showed that forced expression of EGR1 in osteosarcoma cell lines down-regulated the activity of uPA (Figure 6). EGR1 suppresses osteosarcoma migration into blood vessels in vivo To investigate the effects of EGR1 on osteosarcoma tumor migration and invasion in vivo, we used a novel osteosarcoma murine xenograft model with 143B cells. Intrajoint inoculation of GFP-positive 143B cells in nude mice induced primary osteosar- coma tumor formation by 2 weeks after inoculation. These primary tumors gave rise to microscopically detectible micro metastases in the lungs within 5 weeks after inoculation. Although we attempted to determine the volume of the primary tumors, we were unable to do so because tumor had extended into muscle and Chemotherapy Prevents OS Invasion by EGR1 bone. RNA was prepared from tumor formed by control vector or EGR1 expression vector transfected cells. Real-time PCR revealed that forced expression of EGR1 decreased uPA and uPAR expression in vivo (Figure S6B). After 5 weeks, we counted GFP-positive- 143B cells within 50 ml blood aspirates from hearts. The vector control group had an average of 51.2 cells, whereas the EGR1 group averaged only 18.7 cells (Figure 7A, B). Lung metastases were found in 6 of 6 control cell-inoculated mice. In contrast, there were lung metastases in 4 of 6 EGR1-expressing 143B-inoculated mice. The percent of lung metastasis area was calculated. The vector control group had an average of 0.6% metastasis area, whereas the EGR1 group averaged 0.31% metastasis area (Figure 7C). These findings show that EGR1 prevented osteosarcoma migration into blood vessel in vivo. Discussion Current standard regimens for osteosarcoma treatment include preoperative and postoperative chemotherapy. The benefits of chemotherapy have been demonstrated in many studies. Preop- erative chemotherapy induces tumor necrosis in the primary tumor facilitating surgical resection and enabling early treatment of micrometastatic disease [20,21]. Among those patients who received neoadjuvant treatment, chemotherapy-related tumor necrosis was good in 62% and poor in 38% of patients [22]. Figure 6. EGR1 decreased uPA activity. uPA activity was examined using cell lysates. Cell lysate were prepared from control vector or EGR1 expression vector transfected cells. ELISA assay showed that EGR1 decreased uPA activity 0.40-fold in 143B (A). uPA activity was decreased 0.49-fold by EGR1 in Saos-2 (B). Luciferase assay showed that EGR1 decreased uPA activity 0.57-fold in HOS (C). These experiments were in triplicate with similar results [error bars represent mean (SD)] (P,0.05). doi:10.1371/journal.pone.0016234.g006 PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16234