Tumor imaging with multicolor fluorescent protein expression |
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Authors: | Norio Yamamoto Hiroyuki Tsuchiya Robert M Hoffman |
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Institution: | (1) Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa Ishikawa, 920-8641, Japan;(2) AntiCancer, Inc., 7917 Ostrow Street, San Diego, CA 92111-3604, USA;(3) Department of Surgery, University of California, 200 W. Arbor Dr., San Diego, CA 92103-8220, USA |
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Abstract: | Imaging with fluorescent proteins has been revolutionary and has led to the new field of in vivo cell biology. Many new applications
of this technology have been developed. Green fluorescent protein (GFP)-labeled or red fluorescent protein (RFP)-labeled HT-1080
human fibrosarcoma cells were used to determine clonality of metastasis by imaging of metastatic colonies after mixed implantation
of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow
colonies were scored as nonclonal. Dual-color fluorescent cancer cells expressing GFP in the nucleus and RFP in the cytoplasm
were engineered. The dual-color cancer cells enable real-time nuclear–cytoplasmic dynamics to be visualized in living cells
in vivo, including mitosis and apoptosis. The nuclear and cytoplasmic behavior of dual-color cancer cells in real time in
blood vessels was observed as they trafficked by various means or extravasated in an abdominal skin flap. Dual-color cancer
cells were also visualized trafficking through lymphatic vessels where they were imaged via a skin flap. Seeding and arresting
of single dual-color cancer cells in the lung, accumulation of cancer-cell emboli, cancer-cell viability, and metastatic colony
formation were imaged in real time in an open-chest nude mouse model using assisted ventilation. Novel treatment was evaluated
in these imageable models. UVC irradiation killed approximately 70% of the dual-color cancer cells in a nude mouse model.
An RFP-expressing glioma was transplanted to the spinal cord of transgenic nude mice expressing nestin-driven green fluorescent
protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. ND-GFP cells staining positively
for neuronal class III-β-tubulin or CD31 surrounded the tumor, suggesting that the tumor stimulated both neurogenesis and
angiogenesis. The tumor caused paralysis and also metastasized to the brain. The Salmonella typhimurium A1-R tumor-targeting bacterial strain was administered in the orthotopic spinal cord glioma model. The treated animals had
a significant increase in survival and decrease in paralysis. S. typhimurium A1-R was effective against primary bone tumor and lung metastasis expressing RFP in a nude mouse model. S. typhimurium A1-R was effective against both axillary lymph and popliteal lymph node metastases of human dual-color pancreatic cancer
and fibrosarcoma cells, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. Imaging with fluorescent
proteins will reveal mechanisms of cancer progression and provide visual targets for novel therapeutics. |
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