High VEGF Expression High VEGF Expression Correlates with Fast Growth Correlates with Fast Growth

and Early Metastasis in a and Early Metastasis in a Novel Model of Novel Model of OsteosarcomaOsteosarcoma

Shang-You Yang, Haiying Yu, Jeffrey Shang-You Yang, Haiying Yu, Jeffrey Krygier, Paul H. Wooley, and Michael P. Krygier, Paul H. Wooley, and Michael P.

MottMott Wayne State University, Detroit, Michigan, USAWayne State University, Detroit, Michigan, USA

INTRODUCTIONINTRODUCTION Osteosarcoma is an extremely aggressive Osteosarcoma is an extremely aggressive

malignant tumor of the skeleton malignant tumor of the skeleton characterized by fast growing and early characterized by fast growing and early hematogenic metastasizing. hematogenic metastasizing.

It is postulated that the growth, invasion, It is postulated that the growth, invasion, and metastatic potential of many solid and metastatic potential of many solid tumors are dependent on angiogenesis and tumors are dependent on angiogenesis and the potent proangiogenic factor - VEGF. the potent proangiogenic factor - VEGF.

However there are relatively few studies However there are relatively few studies and inclusive results to elucidate the and inclusive results to elucidate the correlation of VEGF expression and correlation of VEGF expression and progression of osteosarcoma. progression of osteosarcoma.

OBJECTIVESOBJECTIVES Establish a murine model of in situ Establish a murine model of in situ

osteosarcoma and lung metastasis.osteosarcoma and lung metastasis. Compare the expression of VEGF Compare the expression of VEGF

and the progression/prognosis of and the progression/prognosis of osteosarcoma. osteosarcoma.

METHODSMETHODS Establishment of osteosarcoma mouse model:Establishment of osteosarcoma mouse model:1.1. Two methods to deliver the osteosarcoma cells Two methods to deliver the osteosarcoma cells

to the proximal end of tibia of SCID mouse.to the proximal end of tibia of SCID mouse.1)1) a 0.5cm incision along the lateral ligament of knee a 0.5cm incision along the lateral ligament of knee

to expose the proximal end of tibia and a small to expose the proximal end of tibia and a small hole across metaphysis was drilled. 100 µl of hole across metaphysis was drilled. 100 µl of medium containing 10medium containing 1055 osteosarcoma cells were osteosarcoma cells were injected into the hole.injected into the hole.

2)2) 100 µl of medium with 10100 µl of medium with 1055 osteosarcoma cells osteosarcoma cells were directed injected to proximal tibia with a were directed injected to proximal tibia with a larger syrange. larger syrange.

2.2. Four different osteosarcoma cell lines were Four different osteosarcoma cell lines were tested to establish tumors in vivo.tested to establish tumors in vivo.

Screened OS Cell LinesScreened OS Cell Lines

ATCC # cell type VEGF transplantCRL-1423 fibroblast +++ √

CRL-1543 fibroblast - √

CRL-1544 fibroblast ++ √

CRL-1547 fibroblast +++

CRL-8303 fibroblast +

HTB-85 fibroblast ++

HTB-86 epithelial +

HTB-96 epithelial + √

CRL-7511 mixed +

Micro CTMicro CT

VEGF Expression, IHCVEGF Expression, IHC

High VEGF, High VEGF, drilled, HE 25xdrilled, HE 25x(CRL1423)(CRL1423)

Higher MagnificationHigher Magnification

Moderate VEGF, Moderate VEGF, Dental Dental drilleddrilled(CRL1544)(CRL1544)

Directed injected,Directed injected, High VEGF High VEGF (CRL1423)(CRL1423)

Low VEGF, Low VEGF, directed injected directed injected (CRL1543)(CRL1543)

Periosteal tumor Periosteal tumor growthgrowth

Lung MetastasisLung Metastasis

Lung MetastasisLung Metastasis

Lung MetastasisLung Metastasis

Lung MetastasisLung Metastasis

Lung MetastasisLung Metastasis All of the High VEGF osteosarcoma All of the High VEGF osteosarcoma

mice sacrificed at 8 weeks and 80% mice sacrificed at 8 weeks and 80% of the mice at 6 weeks developed of the mice at 6 weeks developed dispersive lung metastasis lesions. dispersive lung metastasis lesions.

Only 1 out of 7 HOS-tumor mice Only 1 out of 7 HOS-tumor mice developed a similar dispersive developed a similar dispersive pulmonary change within the same pulmonary change within the same time.time.

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SummarySummary Successfully establishment of mouse Successfully establishment of mouse

orthotopic model of osteosarcoma.orthotopic model of osteosarcoma. Pre-drilling in the bone or directed-injection Pre-drilling in the bone or directed-injection

of tumor cells resulted different tumor of tumor cells resulted different tumor formations, osteo- or soft tissue sarcomas. formations, osteo- or soft tissue sarcomas.

Initial tumor growth may not require VEGF, Initial tumor growth may not require VEGF, but expression of VEGF is correlated with but expression of VEGF is correlated with fast growing and metastasis.fast growing and metastasis.

c-fosc-fos and and c-mycc-myc expressions are elevated expressions are elevated during the tumor development.during the tumor development.

Study in ProgressStudy in Progress Investigate the correlations of other Investigate the correlations of other

cytokines/oncogenes with VEGF and cytokines/oncogenes with VEGF and the tumor growth/metastas.the tumor growth/metastas.

Evaluate the gene modification of Evaluate the gene modification of soluble Flt-1, the decoy receptor of soluble Flt-1, the decoy receptor of VEGF, as a potential therapeutic VEGF, as a potential therapeutic avenue to block/halt the growth and avenue to block/halt the growth and metastasis of osteogenic sarcoma.metastasis of osteogenic sarcoma.

AcknowledgementAcknowledgement Supported by a grant from Supported by a grant from

Sarcoma Service, Karmanos Sarcoma Service, Karmanos Cancer Institute; and FMRE, Cancer Institute; and FMRE, Orthopaedic Surgery, WSUOrthopaedic Surgery, WSU