Liquid biopsy reveals the immune status and protein profiles linked to CTC burden and clinical outcomes in metastatic breast cancer

Background
Metastatic breast cancer (mBC) remains a major therapeutic challenge. Increasing evidence suggests that metastatic dissemination and resistance to treatment are sustained by systemic immune dysfunction and vascular remodeling. Yet, the underpinning mechanisms remain incompletely understood. Therefore, we applied a multilayered liquid biopsy strategy to characterize the systemic immunovascular landscape of mBC and identify clinically actionable circulating biomarkers of disease progression and risk stratification.

Methods
In this prospective study, peripheral blood samples were collected from 60 patients at diagnosis of stage IV mBC before treatment initiation. Circulating tumor cells (CTCs) were enumerated with the CellSearch® system. Immune profiling was performed by flow cytometry, and plasma proteomic analysis using the Olink® 96 Immuno-Oncology panel. Progression-free survival (PFS) was analyzed using Kaplan–Meier and Cox proportional hazards models, including multivariate analysis to identify independent prognostic factors.

Results
Tumors were mainly HR⁺/HER2⁻ (83.3%), and the median follow-up and progression-free survival (PFS) were 31.5 months and 19.9 months, respectively. CTCs were detected in 58.3% of patients (range: 1–962 CTCs/7.5 mL blood). Comprehensive immune profiling revealed that total leukocytes and CD3⁺ T cells were strongly reduced and regulatory T cells (Tregs; CD4⁺CD25highCD127low/–), which expressed various inhibitory receptors (PD-1, CTLA-4, TIGIT, LAG-3), were increased in patient’s vs. age-matched healthy donors. The co-expression of PD-1, CTLA-4, LAG-3 and TIGIT in effector T cells (CD4⁺, CD8⁺) indicated exhaustion. Circulating monocytes/macrophages exhibited a shift toward the immunosuppressive CD163⁺CD206⁺ phenotype. Plasma proteomic profiling identified an inflammatory and angiogenic signature (elevated IL-6, IL-8, HGF, ANGPT2, NOS3, CSF1 and galectin-9, and reduced FASLG and TWEAK expression). Notably, Treg proportion and IL-8, HGF, galectin-9 and TNFRSF12A levels were higher in CTC-positive than CTC-negative patients. Liver metastases, triple-negative receptor status, and high CTC count predicted shorter PFS. Multivariate Cox analysis identified IL-8 (HR = 1.49, p = 0.004) and NOS3 (HR = 4.72, p = 0.003) as independent PFS predictors.

Conclusion
This integrated cellular and proteomic liquid biopsy approach showed that mBC is a systemic immunovascular disease characterized by T-cell exhaustion, Treg expansion, TIGIT-mediated cytotoxic restraint, monocyte polarization, pro-angiogenic cytokine milieu and CTC-mediated immune escape. Circulating IL-8 and NOS3 may represent blood-based prognostic biomarkers and therapeutic targets in mBC.