3D imaging approaches for visualization of target biomarkers in bone metastases in vivo

骨包括在骨转移过程中相互作用的多种细胞类型。在第一个资助期，我们建立了小鼠模型，用于骨转移的体内成像，导致溶骨性和骨质疏松性病变。我们开发并测试了新的成像方法，包括I）荧光二膦酸盐缀合物结合的动力学分析，其具有指示位置特异性代谢状态的摄取速率，II）用于时间推移显微CT的配准程序，其提高了体内骨矿物质和微观结构测量的长期精度，并且可以识别骨沉积或损失的局部区域，以及III）用于骨组织中的光信号衰减的校正过程。我们还IV）建立了具有osterix启动子重组的转基因小鼠品系，调节荧光蛋白Katushka的表达，从而标记祖细胞、成骨细胞和基质细胞，和V）显示Kiss 1肽的荧光缀合物定位于骨转移和多发性骨髓瘤细胞以及邻近的间充质干细胞（MSC）。基于这一医疗设备的成像生物标志物的诊断和治疗的目标，并在其他方法的发展，我们将测试的假设，分层体内多模态生物标志物成像，连接介观与显微镜的方法，将允许时空转移性疾病的严重程度和治疗效果的表征。我们在第二个资助期的具体目标包括：I）使用我们的osterix-Katushka转基因小鼠模型在介观和显微镜下监测肿瘤相关祖细胞的动态（Osx+-MSC）和成骨细胞作为骨转移性疾病进展和治疗效果的潜在生物标志物，II）光学双膦酸盐缀合物的优化和验证，在活动性骨转移部位的快速结合，和III）微CT和二次谐波生成成像方法的发展，作为溶骨性、骨转移和混合性病变中转移严重性的多模式评估的生物标志物。混合微CT和多光子/近红外荧光成像的成功建立将允许开发骨转移和多发性骨髓瘤的体内成像方法，这些方法具有在SKELMET合作项目中鉴定的生物标志物，包括Kiss 1受体和连接粘附分子2。通过本提案中概述的研究，我们的目标是临床前开发具有靶向疾病和治疗控制潜力的成像剂。

Bone comprises multiple cell types interacting with each other during skeletal metastatic processes. In the first funding period, we established mouse models for in vivo imaging of bone metastases giving rise to osteolytic and osteosclerotic lesions. We developed and tested new imaging methods including I) kinetic analyses of binding of a fluorescent bisphosphonate conjugate with uptake rates indicative of location-specific metabolic status, II) a registration procedure for time lapse micro-CT which improves the long-term precision of bone mineral and microstructure measurements in vivo and can identify local regions of bone apposition or loss, and III) correction procedures for the attenuation of optical signals in bone tissue. We also IV) established a transgenic mouse strain with recombination of the osterix promoter, regulating expression of the fluorescent protein Katushka which thus labels progenitor cells, osteoblasts and stromal cells, and V) show that a fluorescent conjugate of the Kiss1 peptide localizes to bone metastatic and multiple myeloma cells as well as neighboring mesenchymal stem cells (MSCs). Based on this armamentarium of imaging biomarkers for diagnostic and therapeutic targeting, and on additional methodological developments, we will test the hypothesis that hierarchical in vivo multimodal biomarker imaging, linking mesoscopic with microscopic methods, will allow for spatiotemporal characterization of metastatic disease severity and the effects of treatment. Our specific aims for the second funding period comprise: I) the use of our osterix-Katushka transgenic mouse model to mesoscopically and microscopically monitor the dynamics of tumor-associated progenitor cells (Osx+-MSCs) and osteoblasts in vivo as potential biomarkers for bone metastatic disease progression and therapy effects, II) the optimization and validation of optical bisphosphonate conjugates for selective, rapid binding at sites of active osteosclerotic metastases, and III) the development of micro-CT and second harmonic generation imaging methods as biomarkers for multimodal assessment of metastasis severity in osteolytic, osteosclerotic and mixed lesions. Successful establishment of hybrid micro-CT & multiphoton/near infrared fluorescent imaging will allow development of methods for in vivo imaging of bone metastases and multiple myeloma with biomarkers identified in SKELMET partner projects, including the Kiss1 receptor and junctional adhesion molecule 2. With the studies outlined in this proposal we aim at progress in the preclinical development of imaging agents that have translational potential to target disease and for therapy control.