Dual-Color Imaging of Angiogenesis and Its Inhibition in Bone and Soft Tissue Sarcoma

Hayashi, Katsuhiro; Yamauchi, Kensuke; Yamamoto, Norio; Tsuchiya, Hiroyuki; Tomita, Katsuro; Amoh, Yasuyuki; Hoffman, Robert M.; Bouvet, Michael

doi:10.1016/j.jss.2006.11.018

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Volume 140, Issue 2 , Pages 165-170, 15 June 2007

Dual-Color Imaging of Angiogenesis and Its Inhibition in Bone and Soft Tissue Sarcoma

Katsuhiro Hayashi, M.D.
- Department of Surgery, University of California San Diego, San Diego, California
- AntiCancer, Inc., San Diego, California
- Department of Orthopedic Surgery, School of Medicine, Kanazawa University, Kanazawa, Japan
Kensuke Yamauchi, M.D.
- Department of Orthopedic Surgery, School of Medicine, Kanazawa University, Kanazawa, Japan
Norio Yamamoto, M.D.
- Department of Orthopedic Surgery, School of Medicine, Kanazawa University, Kanazawa, Japan
Hiroyuki Tsuchiya, M.D.
- Department of Orthopedic Surgery, School of Medicine, Kanazawa University, Kanazawa, Japan
Katsuro Tomita, M.D.
- Department of Orthopedic Surgery, School of Medicine, Kanazawa University, Kanazawa, Japan
Yasuyuki Amoh, M.D.
- AntiCancer, Inc., San Diego, California
Robert M. Hoffman, Ph.D.
- Department of Surgery, University of California San Diego, San Diego, California
- AntiCancer, Inc., San Diego, California
Michael Bouvet, M.D.
- Department of Surgery, University of California San Diego, San Diego, California
- To whom correspondence and reprint requests should be addressed at Moores UCSD Cancer Center, 3855 Health Sciences Drive, La Jolla, CA 92093-0987.

Received 20 August 2006 published online 11 April 2007.

Background

Angiogenesis is a critical step in tumor growth, progression, and metastasis. Soft tissue and bone sarcoma are resistant to most therapeutic approaches. Angiogenesis of these tumors may be an effective target. We hypothesized that we could inhibit tumor growth by targeting angiogenesis in a mouse model of sarcoma. We demonstrate in this report, using powerful color-coded fluorescent imageable tumor-host models, the onset of angiogenesis of these sarcomas and its inhibition.

Materials and methods

Transgenic mice were used as the host in which green fluorescent protein (GFP) is driven by a regulatory element of the stem cell marker nestin (ND-GFP). Nascent blood vessels express ND-GFP in this model. We visualized, by dual-color fluorescence imaging, angiogenesis of sarcoma formed by the HT-1080 human fibrosarcoma cell line expressing red fluorescent protein (RFP) in the ND-GFP mice. Tumor cells were injected into either the muscle or the bone.

Results

Nestin was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, including the surrounding tissues. Immunohistochemical staining showed that CD31 colocalized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. The mice were given daily i.p. injections of 5 mg/kg of doxorubicin after implantation of tumor cells. Doxorubicin significantly decreased the mean nascent blood vessel density in the tumors as well as decreased tumor volume.

Conclusion

The dual-color model of the ND-GFP nude mouse and RFP sarcoma cells is useful for the visualization and quantitation of bone and soft tissue tumor angiogenesis and evaluation of angiogenic inhibitors for such tumors. These data suggest targeting angiogenesis of sarcomas as a promising clinical approach.

Key Words: GFP, RFP, nestin, nascent blood vessels, HT1080, doxorubicin