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Abstract

Breast cancer progression is influenced by metabolic reprogramming, immune activity, and the architecture of the surrounding tissue. However, the differences in these processes between primary tumors and metastatic sites, as well as their responses to therapy, remain poorly understood. In this thesis, I utilized spatially resolved single-cell proteomics through multiplexed ion beam imaging (MIBI) and single-cell metabolic regulome profiling (scMEP) to study HER2-negative breast cancer in both primary and metastatic clinical samples. I also established a dedicated workflow for the high-dimensional imaging of circulating tumor cells (CTCs). In primary tumors from a clinical trial testing sequential bevacizumab followed by the mitochondrial inhibitor ME-344, I found that bevacizumab induced vascular and hypoxia normalization, increasing epithelial oxidative phosphorylation and sensitizing cells to ME-344. ME-344 then triggered mitochondrial stress, DNA damage, and metabolic rewiring, accompanied by the appearance of iron-rich CD163+ macrophages. Spatial analyses showed that epithelial cells within 50 μm of these macrophages displayed the strongest metabolic alterations, highlighting the spatial dependence of treatment response. To enable future integration of circulating and tissue-resident tumor phenotypes within a unified spatial proteomics framework, I developed a cytospin-based MIBI workflow for CTCs. This protocol establishes a foundation for high-dimensional proteomic profiling of rare cell types. In metastatic breast cancer, spatial proteomics revealed distinct organ-specific metabolic and immune ecosystems: lung metastases formed proliferative, glycolytic niches enriched for cell–cell interactions, lymph node metastases exhibited immunoregulatory, exhaustion-dominated niches, and contralateral breast tissue remained comparatively quiescent. These protein-level maps refine existing transcriptomic and murine models by identifying the metabolic and immune states that are functionally abundant in situ. Together, these findings show that therapy and organ context strongly shape tumor metabolism, immune interactions, and spatial organization. By integrating clinical trial material, metastatic tissue mapping, and CTC protocol development within a single technological framework, this thesis provides mechanistic insight into therapy-induced metabolic vulnerabilities and spatially organized immune–metabolic niches, offering avenues for targeted and combination treatment strategies.