CXCR4-targeted Near-infrared Imaging Allows Detection Of Orthotopic And Metastatic Human Osteosarcoma In A Mouse Model

【Background】Osteosarcoma is one of the most common malignant diseases with high metastatic potential and high expression of CXCR4. Current anatomic imaging methods cannot detect early stage of this disease at the molecular level. Therefore, to develop a novel CXCR4-targeted specific molecular imaging modality is in critical need for early detection of primary osteosarcoma and its metastases.【Objectives】In our current study, we aim to develop a novel CXCR4-targeted NIR fluorescent imaging agent and to examine its selectivity and sensitivity in binding to in-vitro cultured osteosarcoma cells as well as human osteosarcoma xenograft and its metastases in a nude mouse model, which may provide a new molecular imaging tool for early detection of osteosarcoma and its metastases.【Methods】1. A peptide agent that target to CXCR4, CXCR4-IR-783, was synthesized and labeled with IR-783 dye. The different expressions of CXCR4 in two human osteosarcoma cells lines(F5M2 and F4) were confirmed by q RT-PCR, Western blotting assay and Confocal immunofluorescent assay. Specificity of this agent was confirmed by the suppression of CXCR4 expression with a specific sh RNA. We examined the binding ability of the agent to in-vitro cultured human osteosarcoma cells by NIR fluorescence microscopy and Kodak In-Vivo Multispectral System FX.2. Human osteosarcoma xenografts in nude mice were detected in-vivo and ex-vivo by NIR fluorescence imaging using CXCR4-IR-783 or Micro CT. The tumors at the bone in situ or other resected organs were confirmed by H&E staining. Meanwhile, the CXCR4 expression of tumor xenograft was revealed by immunohistochemistry assay.3. The presence of metastatic lesions in the lungs of mice bearing F5M2 xenografts were detected by Micro-CT scan, NIR fluorescence imaging using CXCR4-IR-783 as well as 18F-FDT PET scan. The imaging of dissected organs was also performed by NIR fluorescence imaging. The presence of osteosarcoma metastasis in the lung tissues was confirmed by histopathologic evaluation with H&E staining and by immunohistochemistry assay for CXCR4.【Results】1. We developed a novel CXCR4-targeted NIR fluorescent imaging agent,CXCR4-IR-783. By utilizing this imaging agent, we found that CXCR4-IR-783 exhibited a superior selectivity in detection of osteosarcoma cells with CXCR4 expression. Meanwhile, osteosarcoma cells with high CXCR4 expression exhibited a preferential time- and dose-dependent uptake of CXCR-IR-783. Furthermore, as few as 102 F5M2 cells could be detected upon exposure to CXCR4-IR-783.2. Human osteosarcoma xenografts with CXCR4 expression showed preferential uptake and retention of CXCR4-IR-783. In nude mice bearing the F5M2 osteosarcoma xenograft with high CXCR4 expression, the signal-noise ratio(4.87), examined by using the Kodak in-vivo multispectral system, was almost two times higher than that in mice bearing the F4 xenograft with low CXCR4 expression(2.24). CXCR4-IR-783 exhibited a time-dependent clearance from normal mouse organs.3. NIR fluorescence imaging using CXCR4-IR-783 could detect the spontaneous pulmonary metastases of primary osteosarcoma in nude mice bearing human F5M2 osteosarcoma xenograft for 4 weeks while it could not be detected by18F-FDG PET at the same time.【Conclusion】In conclusion, we detected both primary region and the early micro-metastatic nodules in the lungs of nude mice bearing CXCR4 positive human osteosarcoma xenograft by using a target-specific molecular imaging agent, CXCR4-IR-783, which will become a potential molecular imaging tool for detection of osteosarcoma and its metastases. The combination of this imaging technology and anatomical imaging can further enhance early detection of osteosarcoma.