Epithelial-to-mesenchymal Transition Heterogeneity of Circulating Tumor Cells and Their Correlation With MDSCs and Tregs in HER2-negative Metastatic Breast Cancer Patients

Abstract

Background: To investigate the correlation between circulating tumor cells (CTCs) bearing cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) phenotypes and the different immunosuppressive cells in peripheral blood of patients with metastatic breast cancer (mBC). Materials and Methods: Blood was obtained from 38 pre-treated patients with mBC before a new line of treatment. CTC detection and characterization was performed by triple immunofluorescent staining, while Myeloid-derived Suppressor Cells (MDSCs) and T regulatory cells (Tregs) were analyzed by multi-flow cytometry. Results: CTCs were detected in 16 (42.1%) of patients. Based on the co-expression of ALDH1, TWIST and CK, CTCs revealed an important heterogeneity: CTCs with a CSC/partial-EMT, CSC/Epithelial-like, non-CSC/partial-EMT and non-CSC/Epithelial-like phenotype were detected in 7 (18.4%), 7 (18.4%), 1 (1.4%) and 9 (23.7%) of patients, respectively. Immunophenotyping of MDSCs identified 2 monocytic [M-MDSCs; CD14⁺CD15⁺CD11b⁺CD33⁺HLA-DR⁻Lin⁻ (CD14⁺CD15⁺) and CD14⁺CD15^–CD11b⁺CD33⁺ HLA-DR⁻Lin⁻ (CD14⁺CD15^–)] and one granulocytic [G-MDSCs; CD14⁻CD15⁺CD11b⁺CD33⁺HLA-DR⁻Lin⁻ (CD14^– CD15⁺)] subpopulations, expressing inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS), respectively. Patients with detectable CTCs had a higher frequency of Tregs (CD3⁺CD4⁺CD25^high; p=0.022) whereas a positive correlation was found between CTC counts and the percentage of Tregs (p=0.005) and CD14⁺CD15⁺ M-MDSCs (p=0.024). Patients with a partial-EMT phenotype had a higher frequency of CD14⁺CD15⁺ M-MDSCs (p=0.023). Patients harboring the non-CSC/epithelial-like CTC subpopulation had an increased frequency of CD14-CD15⁺ G-MDSCs (p=0.020), along with decreased levels of CD3⁺CD4⁺CD25^high FoxP3+ Tregs (p=0.020). Conclusion: These findings provide evidence that CTCs in ER⁺/HER2^– mBC patients may be under the control of the immune system and various immune escape mechanisms might be involved during the different stages of their biological evolution.