Chemotherapy Prevents OS Invasion by EGR1 Figure 7. EGR1 prevents osteosarcoma cell migration into blood vessel in vivo. GFP expression virus-transfected 143B cells were inoculated into the knee joint. To examine tumour cell invasion of blood vessels, we counted GFP- positive- 143B cells within 50 ml blood aspirates from hearts at 5 weeks after inoculation (A). The number of GFP- positive cells in blood was decreased in EGR1 -expressing 143B- inoculated mice (B) [error bars represent mean (SD)] (P,0.05). Metastatic nodules in lungs were evaluated under fluorescence microscopy. Six of 6 control cell-inoculated mice exhibited lung metastases. Four of six (66.7%) EGR1-expressing cell-inoculated mice exhibited lung metastases. The percent of lung metastasis area was calculated by Lumina Vision (C). doi:10.1371/journal.pone.0016234.g007 These findings showed that one-third of osteosarcoma patients are even poor responders to chemotherapy. Even though chemother- apy is quite benefit for osteosarcoma patients, administration of preoperative chemotherapy results in delay of surgical resection of primary tumor. It is possible that new lung metastases will develop during preoperative chemotherapy in poor or non-responders. We showed that low-dose anti-tumor agent treatment up-regulated EGR1 expression and that EGR1 prevented osteosarcoma invasion via uPA/uPAR down-regulation. These findings suggest that preoperative chemotherapy prevents the development of new lung metastases in poor or non-responders. In addition, osteosar- coma incidence rates in the United States peak in adolescence and in the elderly [23]. Many elderly patients cannot tolerate aggressive chemotherapy. Our findings suggest that low-dose chemotherapy might be useful for elderly osteosarcoma patients by preventing new metastasis when used in combination with radiation therapy or as maintenance therapy. EGR1 has received much attention recently because of its wide range of activities as a transcription factor. Remarkably, EGR1 can exert effects as either a growth promoter or a tumor suppressor. EGR1 may induce or suppress cell proliferation or induce apoptosis of cancer cells [10,16,24–27]. Our MTT assay PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16234