Single-cell immune profiling of regional lymph nodes during early-stage breast cancer progression

INTRODUCIONTumor cell infiltration in regional lymph nodes is a strong prognostic marker, guiding treatment decisions in breast cancer. While the immune cell composition in primary tumors has been more widely explored in later years, the immune cell composition of the sentinel node (SN) and axillary lymph nodes (ALN) remains understudied. A better understanding of how primary tumor and metastatic tumor cells alter the nodal immune microenvironment can shed light on metastasis and cancer progression to unveil new treatment strategies. MATERIALS AND METHODSFrom a prospective clinical cohort of 458 treatment-naive patients with primary operable breast cancer, we performed comprehensive immunophenotypic analysis using mass cytometry analysis of non-metastatic (SN-) and metastatic (SN+) and ALN (ALN+) lymph nodes. RESULTSAs expected, patients with ALN+ cases had a shorter time to distant metastases than SN+ and SN- cases. We identified an exhausted T-cell phenotype and an increase in Germinal Center B (GC B) cells and plasma cells in ALN+ samples compared to SN- samples, both in the whole cohort as well as when investigating estrogen-receptor positive (ER+) patients only. There were no differences in immune cell composition across breast cancer (BC) subtypes within SN-samples. SN+ samples from triple negative BC (TNBC) showed a trend towards increased abundance of GC B and plasma cells, similar to more advanced ALN+, suggesting that smaller TN metastases may trigger an immune activation at an early stage of dissemination. Further analysis of SN- samples from ER+ patients revealed a subset of patients where the immune response had a more exhausted T-cell phenotype. This group was enriched for lymph nodes that were deemed negative by ordinary pathology examination (microscopy) but had detectable tumor cells by CyTOF analysis. CONCLUSIONThe immune profiles of SN and ALN samples from breast cancer patients are highly diverse, showing limited associations to BC subtype, clinical parameters or patient outcome. Metastatic tumor cells play a significant role in driving T-cell exhaustion and immunosuppression. Notably, in approximately 50% of the ER+ samples, T-cell exhaustion was detectable. This coincides with the presence of tumor cells identified by CyTOF, which were likely missed by conventional pathological examination. These findings suggest that small tumor deposits alter the immune composition, and the immune profile reveals the presence of tumor cells.