Single-cell mass cytometry reveals T-cell exhaustion in sentinel nodes of ER+ breast cancer without histological detectable metastases

Introduction: Tumor cell infiltration in regional lymph nodes is a strong prognostic marker and guides treatment decisions in breast cancer. While the immune landscape of primary tumors has been widely studied, the composition of immune cells in sentinel (SN) and axillary lymph nodes (ALN) remains less understood. Clarifying how tumor cells shape the nodal immune microenvironment may provide insights into metastasis, cancer progression, and potential therapeutic strategies. Materials and methods: We analysed tumor draininglymph nodes from a prospective cohort of 458 treatment-naïve patients with primary operable breast cancer, representing all breast cancer subtypes. Lymph node status included negative nodes (n=357), sentinel node-positive (SN+, n=88), and axillary lymph node-positive (ALN+, n=13). Comprehensive immunophenotyping was performed by mass cytometry (CyTOF) using a 48-antibody panel. The panel included lineage markers, cell type-defining markers, and markers of activation and exhaustion.Data were processed using semi-automated FlowSOM clustering with manual clean-up to remove dead cells, doublets, and technical noise. This enabled identification of immune, tumor, apoptotic, and unclassified cell populations.After quality control, 468 lymph nodes comprising more than 45 million single cells were included in the final analysis. Results: Patients with ALN+ disease had shorter time to distant metastasis than those with SN+ or SN- nodes. Compared with SN- samples, ALN+ samples showed enrichment of exhausted T cells as well as germinal center B (GC B) cells and plasma cells, both in the full cohort and within the estrogen receptor-positive (ER+) subgroup. No immune composition differences were observed across breast cancer subtypes in SN- samples. SN+ samples from triple-negative breast cancer (TNBC) demonstrated a trend toward increased GC B and plasma cells, resembling the immune profile of ALN+ nodes, suggesting that even small SN metastases can trigger early immune activation. In SN- samples from half of the ER+ patients, a subset displayed pronounced T-cell exhaustion. These nodes were enriched for cases that appeared negative by routine pathology but contained tumor cells detectable by CyTOF. Conclusion: SN and ALN immune profiles in breast cancer are highly heterogeneous and show limited correlation with subtype, clinical variables, or outcome. Metastatic tumor cells promote T-cell exhaustion and immunosuppression, detectable in nearly half of ER+ cases. Importantly, T-cell exhaustion coincided with CyTOF-detected tumor cells that were missed by conventional pathology, indicating that even small tumor deposits can reshape the immune landscape. These findings highlight the potential of immune profiling to uncover occult tumor involvement in lymph nodes.