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

骨包括多种细胞类型，在骨骼转移过程中相互作用。在第一个资助期，我们建立了小鼠模型，用于骨转移引起的溶骨和骨硬化病变的体内成像。我们开发并测试了新的成像方法，包括I)荧光双膦酸盐缀合物结合的动力学分析与指示位置特定代谢状态的摄取率，II)时间推移微ct的注册程序，它提高了体内骨矿物质和微观结构测量的长期精度，可以识别局部骨偏移或丢失区域，以及III)骨组织中光信号衰减的校正程序。我们还建立了一个重组骨启动子的转基因小鼠品系，调节荧光蛋白Katushka的表达，从而标记祖细胞、成骨细胞和间质细胞；V)表明Kiss1肽的荧光偶联物定位于骨转移细胞和多发性骨髓瘤细胞以及邻近的间充质干细胞（MSCs）。基于这些用于诊断和治疗靶向的成像生物标志物，以及其他方法的发展，我们将测试一个假设，即分层体内多模态生物标志物成像，将介观和微观方法联系起来，将允许转移性疾病严重程度和治疗效果的时空表征。我们在第二个资助期的具体目标包括：I)使用我们的osterix-Katushka转基因小鼠模型在体内介观和显微镜下监测肿瘤相关祖细胞（Osx+-MSCs）和成骨细胞的动态，作为骨转移疾病进展和治疗效果的潜在生物标志物；II)优化和验证光学双膦酸盐偶联物在活性骨硬化转移位点的选择性快速结合；III)微型ct和二次谐波成像方法的发展，作为多模式评估溶骨性、骨硬化性和混合性病变转移严重程度的生物标志物。微型ct和多光子/近红外荧光混合成像技术的成功建立，将有助于利用SKELMET合作项目中确定的生物标志物（包括Kiss1受体和连接粘附分子2）开发骨转移和多发性骨髓瘤的体内成像方法。通过本提案中概述的研究，我们的目标是在具有靶向疾病和治疗控制的转化潜力的显像剂的临床前开发方面取得进展。

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.