Integrative plasma-to-spatial proteomics reveals fibroblast-associated signatures in liver metastatic breast cancer

Liver metastases from breast cancer (BCLM) are associated with aggressive disease and poor survival outcomes. Liver-directed locoregional therapies (LRT) based on radiation are limited by resistance and relapse. While liquid biopsy enables systemic monitoring, it provides limited insight into the tumor microenvironment and mechanisms of resistance. In this hypothesis-generating study, high-throughput plasma proteomics alongside digital spatial profiling of matched tumor tissues was employed to link systemic signals with local tumor biology, aiming to elucidate mechanisms underlying therapy response.

Plasma and tissue samples were collected from BCLM (n = 14) patients receiving LRT. High-throughput proteomic analysis (Olink Proteomics, Uppsala, Sweden) was run to detect and quantify circulating proteins in plasma. Matched tissue samples were obtained before LRT and analyzed using Digital Spatial Profiling (GeoMX, NanoString Technologies, Seattle, WA) to assess the protein expression within defined tissue compartments. Results were then correlated with clinical outcome.

Systemic proteomic analysis showed significant high levels of CD8A, CX3CL1, FGF-21 before treatment in non-survivors suggesting a hyperactivated yet ineffective immune state. In contrast, survivors exhibited elevated plasma MMP1 (p = 0.035, AUC = 0.896) alongside reduced intratumoral levels. Spatial analysis did not identify significant differences in the distribution of immune cell signaling within the TME. However, compartment-specific profiling illuminated critical patterns: Ki-67 in immune stroma, not tumor cells, correlated with response, while fibronectin and α-SMA were abnormally enriched in tumor epithelial cells of non-survivors. These findings were consistent with a mesenchymal-like signature associated with poor survival, highlighting epithelial fibronectin as a candidate biomarker that warrants further investigation.

Integrating plasma and spatial proteomics reveals key resistance mechanisms to LRT in BCLM with the opposing roles of MMP1 and fibronectin in blood versus tumor. Cancer-associated fibroblasts, linked to the epithelial compartment of the tumor, may represent potential targets for future investigation, particularly through MMP1 inhibition, potentially acting as adjuvants to radiation therapy.