ROR2 regulates the survival of murine osteosarcoma cells in lung capillaries

Aim:Lung metastasis is a leading cause of death in patients with osteosarcoma(OS).No effective therapy exists that improves the five-year overall survival rate of OS patients with metastasis.Therefore,finding novel therapeutic targets will help develop new treatment strategies for OS patients with lung metastasis.Methods:Based on analysis of gene expression profiles between sublines of the Dunn OS LM8 cell line with high(LM8-H)and low(LM8-L)metastatic ability,we have identified Wnt signal-related genes that play an important role in lung metastasis of OS.Function of the genes was investigated by establishing sublines of gene knockout and assessing their metastatic ability using a mouse lung metastasis model.The molecular mechanism underlying the function of the genes was further investigated by in vitro experiments.Results:We have identified that receptor tyrosine kinase-like orphan receptor 2(ROR2),a receptor of the non-canonical Wnt signaling pathway,was involved in OS cell survival in lung capillaries during metastasis.LM8-H knocked out of Ror2(H/Ror2-KO)significantly reduced lung metastasis by decreasing the viability in lung capillaries 48 h after intravenous injection.In vitro study revealed that ROR2 increased anoikis resistance through AKT activation.Reconstitution of ROR2 expression in H/Ror2-KO cells restored their metastatic ability and viability in lung capillaries.Conclusion:The results demonstrate a novel ROR2 function in OS lung metastasis and may inform new treatment strategies for OS patients.

Multiplexed bioluminescence imaging of cancer cell response to hypoxia and inflammation in the caudal-artery injection model of bone metastasis during zoledronic acid treatment

Aim:Therapeutic agents suppressing bone remodeling have been clinically approved to delay metastatic progression and skeletal-related events in patients with bone metastasis.However,therapeutic agents including zoledronic acid(ZA)are insufficient to regress established bone metastasis.Therefore,new treatment strategies are desired,and unraveling the status of cancer cells during bone metastatic progression will help develop therapeutic strategies.Methods:We developed a unique multiplexed reporter system for bioluminescent imaging(MRS-BLI)using three luciferase reporter genes.This system allows for the noninvasive and quantitative monitoring of tumor growth and activities of nuclear factor-kappa B(NF-κB)and hypoxia-inducible factor(HIF),which are the key transcriptional factors in response to inflammation and hypoxia,respectively.PC-3/MRS-BLI,a human prostate cancer cell line that stably retains the MRS-BLI reporter genes,was applied to the caudal-artery injection model of bone metastasis to observe the status of cancer cells during bone metastasis development and ZA treatment(<1 month).Results:MRS-BLI reveals key events during the bone metastasis development:NF-κB and HIF are activated in cancer cells after migration to the bone marrow and are transiently reduced,followed by rapid activation before proliferation begins.ZA treatment suppresses the growth of metastasized cancer cells by suppressing NF-κB and HIF activities that may be indirectly induced by osteoclast activation.Conclusion:By visualizing the NF-κB and HIF activities of PC-3/MRS-BLI in bone,MRS-BLI has enabled new discoveries regarding the regulation of bone metastases.Further analysis of the progression of bone metastases using MRS-BLI may provide important information for developing new therapeutic strategies.