Project Summary For breast cancer, skeletal metastases are a major source of the pain, disability and mortality associated with disease progression. As with other solid tumors metastasizing to bone, breast cancer displays a striking tropism for vertebral bone, with approximately 2-5 vertebral metastases for every one long bone metastasis, implying that vertebral-specific factors are key drivers of metastasis. Passive blood flow cannot explain the increased metastatic tropism for vertebral versus long bones, indicating that unrecognized biologic factors are likely to drive the high rates of vertebral metastasis observed clinically. We have here identified a new skeletal stem cell responsible for generating the osteoblasts that mineralize and remodel the vertebrae (vertebral skeletal stem cells, vSSCs) and provide evidence that this cell is a major determinant of vertebral-specific pathology, including the high rates of vertebral metastases observed clinically. Even after normalizing all anatomic factors through the use of in vivo bone organoids, vSSC-derived bone tissue recruited tumor cells more efficiently than comparable stem cells from long bones. Targeting loss- of-function to vSSCs using a mouse cre line developed for this project selectively reduces vertebral metastasis rates. In both organoid systems and native vertebrae, vSSCs drive high rates of initial vertebral seeding with tumor cells, indicating that vSSC-driven tropism is a major contributing factor behind the high vertebral metastatic rates observed in breast cancer. Building upon this observation, we have identified candidate trophic factors expressed by vSSCs and have generated evidence implicating specific vSSC-derived factors in vertebral metastatic tropism and outgrowth. Here, we will perform key studies needed to advance the rigor, underlying mechanism, clinical relevance and translational therapeutic impact of this discovery. First, (Aim 1), we will establish the metastatic functions of this vSSC in the native vertebral environment, including investigating how vSSCs may organize the entire vertebral metastatic niche and which vSSC-derived cell types contribute to metastasis. Next (Aim 2), we have identified a candidate vSSC-derived mediator driving metastatic tropism and will here determine how secretion of this factor is regulated and how it signals to tumor cells to impact metastasis. Lastly (Aim 3), we will develop the therapeutic impact of this discovery by determining if the human counterparts of vSSCs display a conserved metastatic function in xenograft systems and by conducting proof-of-concept therapeutic studies of blocking candidate vSSC-derived metastatic mediators. Altogether, this study will establish that a new stem cell responsible for forming the vertebrae drives vertebral metastases, thereby offering a new model for the site specificity of skeletal metastases, an explanation for the high rates of vertebral metastases observed clinically and new therapeutic...