TLR4 and pSTAT3 Expression on Circulating Tumor Cells (CTCs) and Immune Cells in the Peripheral Blood of Breast Cancer Patients: Prognostic Implications

SIMPLE SUMMARY: Toll-like receptor 4 (TLR4) and phosphorylated signal transducer and activator of transcription protein 3 (pSTAT3) play a prominent role in cancer inflammation and anti-tumor immune response, and their therapeutic targeting is considered a promising strategy for the management of breast cancer (BC). We herein hypothesized that these immunomodulatory molecules may be involved in peripheral tumor-immune crosstalk and could provide valuable prognostic information. Our results provide first evidence that the expression of TLR4 and pSTAT3 on circulating tumor cells (CTCs) and immune cells of BC patients might play a role in peripheral anti-tumor response and metastatic progression, and could be associated with patient outcomes. ABSTRACT: TLR4 and pSTAT3 are key players in cancer inflammation and immune evasion; however, their role in the peripheral blood (PB) is largely unexplored. Herein we evaluated their expression in the circulating tumor cells (CTCs) and peripheral-blood mononuclear cells (PBMCs) of patients with early (n = 99) and metastatic (n = 100) breast cancer (BC). PB samples obtained prior to adjuvant and first-line therapy, were immunofluorescently stained for Cytokeratins/TLR4/pSTAT3/DAPI and analyzed via Ariol microscopy. TLR4+ CTCs were detected in 50% and 68% of early and metastatic CTC-positive patients, respectively, and pSTAT3+ CTCs in 83% and 68%, respectively. In metastatic patients, CTC detection was associated with a high risk of death (HR: 1.764, p = 0.038), while TLR4+ CTCs correlated with a high risk of disease progression (HR: 1.964, p = 0.030). Regarding PBMCs, TLR4 expression prevailed in metastatic disease (p = 0.029), while pSTAT3 expression was more frequent in early disease (p = 0.014). In early BC, TLR4 expression on PBMCs independently predicted for high risk of relapse (HR: 3.549; p = 0.009), whereas in metastatic BC, TLR4+/pSTAT3− PBMCs independently predicted for high risk of death (HR: 2.925; p = 0.012). These results suggest that TLR4/pSTAT3 signaling on tumor- and immune-cell compartments in the PB could play a role in BC progression, and may hold independent prognostic implications for BC patients.