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

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.

Investigating the Role of CTCs with Stem/EMT-like Features in Metastatic Breast Cancer Patients Treated with Eribulin Mesylate

“…These limitations preclude any valid conclusions about the clinical relevance of specific phenotypic changes in CTCs. In addition, CD45 was not included in the immunofluorescence panel for CTC analysis due to the inability of our method to combine more than four markers (cytokeratins, ALDH1, TWIST1, DAPI); consequently, the identification of CTCs was based on the expression of cytokeratins and morphologic criteria as described in numerous reports from our group and others [ 23 , 24 , 36 , 37 ]. However, our previous studies in independent cohorts of patients with BC and healthy volunteers [ 11 , 13 , 38 , 39 , 40 , 41 ] revealed that there were no cytokeratin+/CD45+ cells evident in the peripheral blood of patients or healthy donors, thus indicating the high specificity of our method.…”

“…A total of 2 × 10 6 PBMCs (two slides) per patient were analyzed using the automated image analysis Ariol microscopy system and the Ariol system CTCs software (Genetix, Cambridge, UK) [ 23 , 24 , 25 ]. As described in our previous studies, CK expression was used to define CTCs, whereas high ALDH1 expression and nuclear TWIST1 localization were used to define the CSC+ and partial-EMT phenotypes, respectively, on CK+ CTCs.…”

“…The detection of at least one CTC was used to define patient positivity for CTCs; in accordance, the detection of at least one CTC of a particular phenotype was used to define patient positivity for this phenotype [ 12 , 23 , 24 , 25 ].…”

“…The median follow-up time was estimated by reverse Kaplan–Meier analysis. The Cox regression analysis was used for the prediction of PFS and OS; the variables with statistical significance in univariate analysis were included in a multivariate regression analysis, following the “one in ten” rule [ 24 ]. P -values were considered significant at the p < 0.05 level.…”

Single-Cell RNA Sequencing Uncovers Heterogeneous Circulating Tumor Cell Subsets in Breast Cancer

“…Taken together, these findings imply that CTCs show enhanced expression of genes that activate survival signals and, in parallel, modulate their interactions with circulating immune cells. It is critical to note here that the mechanisms promoting the immune evasion capacities of CTCs and the tumor–immune crosstalk in the periphery represent a promising field in cancer research, with potential prognostic and therapeutic implications for BC patients [ 14 , 20 , 21 , 22 , 23 ].…”