Project Summary/Abstract Building upon the parent CSBC U01 project that investigates the role of bone mesenchymal stem/stromal cells in fostering the early outgrowth of disseminated tumor cells, this supplement project expands to unravel tumor- immune cell interactions in clinically advanced bone metastases, particularly focusing on HER2-low breast cancer resistance to T-DXd treatment. HER2-low breast cancer accounts for 45–55% of all breast cancer cases, and bone metastasis is more common in this subtype than others. Novel HER2-directed antibody-drug conjugate (ADC) agent, trastuzumab deruxtecan (T-DXd), has shown antitumor activity in HER2-low tumors. However, a significant portion of patients showed de novo resistance. Surprisingly, 25% of the HER2-zero cohort showed measurable objective response. These findings suggest the involvement of additional factors beyond HER2 expression levels in determining T-DXd response and/or resistance. Our preliminary study, using NanoString GeoMX spatial proteomics, in investigating eleven bone metastasis patients’ samples (4 responders vs. 7 non-responders to the T-DXd treatment), highlighted a complicated correlation between various immune features and T-DXd treatment responsiveness. In this project, we will employ the CosMX single-cell 6,000-plex mRNA and 64-plex protein platform to profile the bone metastasis niche, leverage the MD-SPACER computational tool developed from the parent U01 project to analyze cell-cell/cell-niche interactions and the Wong lab's S2C2 tool to model spatial cell-cell crosstalk intracellular and intercellular signaling. This project marks a new collaboration between Wong’s team and Dr. Bill Li, a clinical pathologist from the Emory Winship Cancer Institute, and Dr. Wenjuan Dong, an immunologist at the Houston Methodist Cancer Center. Neither Li nor Dong is funded by any CSBC program. In addition, this project will involve two MD/PhD trainees who will gain hands-on experience in spatial omics experimentation and analysis.