Abstract 3030: Correlation of circulating tumor cells (CTCs) expressing stemness and EMT phenotypes with immunosuppressive cells in metastatic breast cancer patients
Authors: Aggouraki Despoina , Papadaki Maria , Vetsika Eleni Kyriaki , Koutoulaki Anna , Kotsakis Athanasios , Kallergi Galatea , Agelaki Sofia , Mavroudis Dimitris , Georgoulias Vassilis 
Published: May 2015

Abstract
Background: The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of breast cancer (BC) patients is an unfavourable prognostic factor; however further characterization of these cells is required to understand their biological role. The presence of stemness and epithelial-to-mesenchymal transition (EMT) markers in CTCs could be an important marker of enhanced metastatic capacity. On the other hand, myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) play an essential role in tumor-immune evasion, by suppressing the normal function of both CD4+and CD8+ T cells. Increased levels of MDSCs and Tregs have been correlated with advanced disease and poor prognosis. Recent findings in preclinical models and primary tumours indicate that immune cells can induce EMT and stemness characteristics of cancer cells; however this has not been demonstrated in circulation. The purpose of this study was to evaluate the correlation of CTCs expressing stemness and EMT phenotypes with immunosuppressive cells in BC patients.
Patients and Methods: PB was obtained from 38 metastatic BC patients prior to treatment. Triple immunofluorescence was performed on PBMCs cytospin preparations using antibodies against putative markers for epithelial cells (anti-pancytokeratin, A45-B/B3), stemness (anti-ALDH1) and EMT (anti-TWIST) phenotypes. Quantification of ALDH1 expression levels and characterization of TWIST subcellular localization was performed using the ARIOL system. The frequency of different immune cell subpopulations was also evaluated in PB using flow cytometry and was correlated to the presence of distinct CTC phenotypes.
Results: Cytokeratin+ CTCs were detected in 16/38 (42%) patients. Patients with detectable CTCs had a higher percentage of CD4+ Tregs compared to those without CTCs (p=0.022). Moreover, elevated levels of CD4+ Tregs were associated with detectable stem cell-like CTCs (ALDH1high) (p=0.023), while in patients with non-stem CTCs (ALDH1low/-) an increased frequency of MDSCs [CD15+CD14-HLA-DR-Lin-CD33+CD11b+] occurred (p=0.020). The detection of EMT-like CTCs was not associated with the incidence of MDSCs and Tregs.
Conclusions: The detection of CTCs using ARIOL system was associated with a higher incidence of CD4+ Tregs and MDSCs in metastatic BC patients. However, different immunosuppressive cell subpopulations were correlated with phenotypically different CTCs according to EMT and stemness characteristics. These findings indicate that the immune system may be differentially involved in regulating CTCs with different biological behaviour or reversely, CTCs with different characteristics might induce specific subpopulations of immunosuppressive cells.
Citation Format: Despoina Aggouraki, Maria Papadaki, Eleni Kyriaki Vetsika, Anna Koutoulaki, Athanasios Kotsakis, Galatea Kallergi, Sofia Agelaki, Dimitris Mavroudis, Vassilis Georgoulias. Correlation of circulating tumor cells (CTCs) expressing stemness and EMT phenotypes with immunosuppressive cells in metastatic breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2014-3030