Project Summary:This A1 proposal focuses on impaired pathological fracture healing in the presence of breast cancer cells, not on the entire complex sequence of cancer spread from the breast to bone. Metastatic cancer cells can settle in anywhere in the bone including bone marrow, cortical bone, or outside of the bone. Cancer cells are spilled in and out of bone at the time of fracture. Pathological fracture calluses are deficient and very often fail to ossify despite adjuvant therapies such radiation therapy and anti-resorptive agents including Rank:Fc antibody (denosumab) or bisphosphonates (zoledronate). Failed fracture healing is not solely a result of bone resorption. There is no mechanistic understanding as to why fractures do not heal well in the presence of certain types of breast cancer cells. Unraveling the mechanisms underlying impaired fracture healing will enable patients to benefit from future scientific endeavors employing mechanism-based treatments. If there is a way of addressing impaired fracture repair while also inhibiting cancer growth in bone, the clinical care for pathological fractures will be immensely impacted. We conducted whole transcriptome bulk RNAsequencing of several different types of breast cancer cells that are grown in breast vs. bone. We observed that breast cancer cells that inhibit fracture repair are pro-inflammatory with heightened MEK1-pERK1/2-cytokine- hyperinflammation signaling in the bone microenvironment. Temporal expression of pro-Inflammatory cytokines and bone-acting proteins such as TNF, interleukins, and sclerostin are completely deranged and prolonged following femur fractures in the presence of breast cancer cells such as MDA231, HC1806, and 4T1 However, there is a critical knowledge gap as to how these different breast cancer cell types affect chondro-progenitors, osteoblasts, osteoclasts, and osteocytes within the intact fracture callus architecture as suggested by the Reviewer. Two notable changes in this A1 proposal include the refinement of the animal models and a new set of compelling spatial transcriptomic biology data incorporating the Reviewers’ critiques and suggestions. We posit a central hypothesis that highly inflammatory pERK1/2-high breast cancer cells inhibit the normal fracture callus formation and maturation by causing prolonged hyper-inflammation and subsequent derangements of callus spatial transcriptomics. We propose 3 Specific Aims. Aim 1. To define temporal and spatial transcriptomic changes of inflammatory, osteogenic, and chondrogenic lineage cells in structurally intact fracture callus in vivo. Aim 2. To rescue impaired osteogenesis of osteoprogenitors, chondroprogenitors, and periosteal cells in the presence breast cancer cells by inhibition of pERK1/2-induced inflammation ex vivo. Aim 3. In Vivo Pre-clinical Therapeutic Translation: To establish a mechanism-based rescue of impaired pathological fracture healing by targeting specific pro-inflammatory kinases in vivo....