Altered local and systemic immune profiles underlie lymph node metastasis in breast cancer patients

Abstract

Cancer-mediated immune dysfunction contributes to tumor progression and correlates with patient outcome. Metastasis to tumor draining lymph nodes (TDLNs) is an important step in breast cancer progression and is used to predict patient outcome and survival. Although lymph nodes are important immune organs, the role of immune cells in TDLNs has not been thoroughly investigated. We hypothesized that the host immune response in node negative (NN) patients is more intact and thereby can resist tumor invasion compared to node positive (NP) patients. As such, lymph node metastasis requires breakdown of the host immune response in addition to escape of cancer cells from the tumor. To investigate the immunological differences between NN and NP breast cancer patients, we purified and profiled immune cells from the three major compartments where cancer and immune cells interact: tumor, TDLNs and peripheral blood. Significant down-regulation of genes associated with immune-related pathways and up-regulation of genes associated with tumor-promoting pathways was consistently observed in NP patients' TDLNs compared to NN patients. Importantly, these signatures were seen even in NP patients' tumor-free TDLNs, suggesting that such immune changes are not driven solely by local tumor invasion. Furthermore, similar patterns were also observed in NP patients' tumor and blood immune cells, suggesting that immunological differences between NN and NP patients are systemic. Together, these findings suggest that alterations in overall immune function may underlie risk for LN metastasis in breast cancer patients.

Figure 1. Comparison between immune cells from NN and NP patient LNs

Heatmaps depicting significantly differentiated genes with fold-change differences larger than 1.5 between NN and NP patients’ LNs. All three comparisons show clustering of the NN and NP patient groups. The NN (green) and NP (red) group clusters are shown on the top and the gene clusters are shown on the left. Gene expression values are mean-centered. (A) comparison between immune cells from NN and NP patients’ tumor-invaded SLNs; (B) comparison between immune cells from NN and NP patients’ tumor-free SLNs; (C) comparison between immune cells from NN and NP patients’ tumor-free NSLNs.

Figure 2. Fold-change differences between genes associated with immune-related pathways in NN and NP patients

Patient specific fold change differences between genes associated with immune-related pathways resulting from NN vs. NP patient comparison in each of the comparisons performed. (A) NN vs. NP patients’ purified immune cell genes from tumor-invaded SLNs (T+SLN), tumor-free SLN (T-SLN) and tumor-free NSLNs (T-NSLN). (B) NN vs. NP patients’ purified immune and tumor cells from the primary tumor and purified immune cells from the peripheral blood. (C) NN vs. NP patients’ heterogeneous cell population from the primary tumor in two publically available datasets – van de Vijver et al. (295 patients: 151 NN, 144 NP) and Sotiriou et al. (99 patients: 46 NN, 53 NP). The numbers above the comparison represent significant P-values obtained in Student’s t-test. NN = Node negative patients; NP = Node positive patients. Similar results were obtained using the gene expression values rather than the fold change differences (Supplementary Figure 1).