High VEGF Expression Correlates with Fast Growth and Early Metastasis in a Novel Model of Osteosarcoma Shang-You Yang, Haiying Yu, Jeffrey Krygier, Paul H. Wooley, and Michael P. Mott Wayne State University, Detroit, Michigan, USA

INTRODUCTION Osteosarcoma is an extremely aggressive malignant tumor of the skeleton characterized by fast growing and early hematogenic metastasizing. Osteosarcoma is an extremely aggressive malignant tumor of the skeleton characterized by fast growing and early hematogenic metastasizing. It is postulated that the growth, invasion, and metastatic potential of many solid tumors are dependent on angiogenesis and the potent proangiogenic factor - VEGF. It is postulated that the growth, invasion, and metastatic potential of many solid tumors are dependent on angiogenesis and the potent proangiogenic factor - VEGF. However there are relatively few studies and inclusive results to elucidate the correlation of VEGF expression and progression of osteosarcoma. However there are relatively few studies and inclusive results to elucidate the correlation of VEGF expression and progression of osteosarcoma.

OBJECTIVES Establish a murine model of in situ osteosarcoma and lung metastasis. Establish a murine model of in situ osteosarcoma and lung metastasis. Compare the expression of VEGF and the progression/prognosis of osteosarcoma. Compare the expression of VEGF and the progression/prognosis of osteosarcoma.

METHODS  Establishment of osteosarcoma mouse model: 1. Two methods to deliver the osteosarcoma cells to the proximal end of tibia of SCID mouse. 1)a 0.5cm incision along the lateral ligament of knee to expose the proximal end of tibia and a small hole across metaphysis was drilled. 100 µl of medium containing 10 5 osteosarcoma cells were injected into the hole. 2)100 µl of medium with 10 5 osteosarcoma cells were directed injected to proximal tibia with a larger syrange. 2. Four different osteosarcoma cell lines were tested to establish tumors in vivo.

Screened OS Cell Lines ATCC #cell typeVEGFtransplant CRL-1423fibroblast+++√ CRL-1543fibroblast-√ CRL-1544fibroblast++√ CRL-1547fibroblast+++ CRL-8303fibroblast+ HTB-85fibroblast++ HTB-86epithelial+ HTB-96epithelial+√ CRL-7511mixed+

Micro CT

VEGF Expression, IHC

High VEGF, drilled, HE 25x (CRL1423)

Higher Magnification

Moderate VEGF, Dental drilled (CRL1544)

Directed injected, High VEGF (CRL1423)

Low VEGF, directed injected (CRL1543)

Periosteal tumor growth

Lung Metastasis

All of the High VEGF osteosarcoma mice sacrificed at 8 weeks and 80% of the mice at 6 weeks developed dispersive lung metastasis lesions. All of the High VEGF osteosarcoma mice sacrificed at 8 weeks and 80% of the mice at 6 weeks developed dispersive lung metastasis lesions. Only 1 out of 7 HOS-tumor mice developed a similar dispersive pulmonary change within the same time. Only 1 out of 7 HOS-tumor mice developed a similar dispersive pulmonary change within the same time.

ELISA

Real Time PCR

Summary Successfully establishment of mouse orthotopic model of osteosarcoma. Successfully establishment of mouse orthotopic model of osteosarcoma. Pre-drilling in the bone or directed-injection of tumor cells resulted different tumor formations, osteo- or soft tissue sarcomas. Pre-drilling in the bone or directed-injection of tumor cells resulted different tumor formations, osteo- or soft tissue sarcomas. Initial tumor growth may not require VEGF, but expression of VEGF is correlated with fast growing and metastasis. Initial tumor growth may not require VEGF, but expression of VEGF is correlated with fast growing and metastasis. c-fos and c-myc expressions are elevated during the tumor development. c-fos and c-myc expressions are elevated during the tumor development.

Study in Progress Investigate the correlations of other cytokines/oncogenes with VEGF and the tumor growth/metastas. Investigate the correlations of other cytokines/oncogenes with VEGF and the tumor growth/metastas. Evaluate the gene modification of soluble Flt-1, the decoy receptor of VEGF, as a potential therapeutic avenue to block/halt the growth and metastasis of osteogenic sarcoma. Evaluate the gene modification of soluble Flt-1, the decoy receptor of VEGF, as a potential therapeutic avenue to block/halt the growth and metastasis of osteogenic sarcoma.

Acknowledgement Supported by a grant from Sarcoma Service, Karmanos Cancer Institute; and FMRE, Orthopaedic Surgery, WSU Supported by a grant from Sarcoma Service, Karmanos Cancer Institute; and FMRE, Orthopaedic Surgery, WSU