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

Anticancer Res. 2021 Feb;41(2):661-670. doi: 10.21873/anticanres.14817.

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+CD25high; 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+CD25high 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.

Keywords: CTC; EMT; MDSCs; Tregs; breast cancer; cancer stem cells; immune cells.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers, Tumor / blood
  • Breast Neoplasms / blood
  • Breast Neoplasms / immunology*
  • Breast Neoplasms / pathology
  • Case-Control Studies
  • Epithelial-Mesenchymal Transition*
  • Female
  • Flow Cytometry
  • Humans
  • Immunophenotyping
  • Middle Aged
  • Myeloid-Derived Suppressor Cells / immunology*
  • Myeloid-Derived Suppressor Cells / metabolism
  • Neoplastic Cells, Circulating / immunology*
  • Neoplastic Cells, Circulating / metabolism
  • Neoplastic Cells, Circulating / pathology
  • Neoplastic Stem Cells / immunology*
  • Neoplastic Stem Cells / metabolism
  • Neoplastic Stem Cells / pathology
  • Phenotype
  • T-Lymphocytes, Regulatory / immunology*
  • T-Lymphocytes, Regulatory / metabolism
  • Tumor Escape*

Substances

  • Biomarkers, Tumor