Abstract P2-01-07: Effect of first-line chemotherapy in the expression of stemness and epithelial-to-mesenchymal transition markers in circulating tumor cells of patients with metastatic breast cancer.
Authors: Papadaki MA , Kallergi G , Mavroudis D , Georgoulias V , Theodoropoulos PA , Agelaki S 
Published: August 2014

Abstract
Introduction: Cancer stem cells (CSCs) are suggested to be the active source of metastatic spread. Epithelial-to-Mesenchymal transition (EMT) is considered as a crucial event in metastasis, which in addition, generates cells with stemness properties. Circulating tumor cells (CTCs) with stem cell- like and EMT characteristics could escape apoptosis and demonstrate chemoresistance. In the present study we aimed to investigate the co-expression of ALDH1, a stemness marker, and TWIST, an EMT marker, in CTCs from patients with metastatic breast cancer before and after chemotherapy, at the single cell level.
Patients and Methods: Paired PBMCs cytospins were prepared from 154 patients with metastatic breast cancer, before and after first-line chemotherapy. Triple immunofluorescence experiments were performed in samples using the pancytokeratin anti-mouse antibody (A45-B/B3), ALDH1 anti-mouse and TWIST anti-rabbit antibodies, by Zenon technology. ALDH1 expression was characterized as high or low (according to ALDH1 expression quantified in the control cell line HepG2), while TWIST expression was characterized as nuclear or cytoplasmic. 500.000 PBMCs per patient were analyzed using the ARIOL system.
Results: CTCs were identified in 81 (53%) out of 154 patients before treatment, with a total of 2.056 CTCs detected and a median of 2 CTCs per patient (range: 1–600). Up to now, in 38 out of these 81 patients, samples were also evaluated after treatment. A total of 1.232 CTCs were detected in 22 (58%) out of 38 patients, with a median of 10 CTCs per patient (range: 1–828). The great majority of CTCs expressed ALDH1 both before and after chemotherapy, however high ALDH1 expression levels in CTCs were more frequently observed after treatment (91% vs 54%). The median percentage of ALDH1high expressing CTCs per patient was also increased after chemotherapy (86% vs 64%). TWIST expression was observed in the great majority of CTCs both before and after chemotherapy, however nuclear localization of TWIST in CTCs was more often encountered after treatment (92% vs 54%). The median percentage of TWISTnuclear expressing CTCs per patient also increased after chemotherapy (89% vs 48%). High ALDH1 expression in CTCs was positively correlated with nuclear localization of TWIST, both pre- and post-chemotherapy (p = 0.0000001 and p = 0.001, respectively, Spearman analysis). Concerning the co-expression of these molecules, the phenotype ALDH1high TWISTnuclear was the most abundant at both time points, however it was more frequently observed after treatment (90% vs 41% of CTCs). In addition, the median percentage of ALDH1high TWISTnuclear expressing CTCs per patient was also increased after chemotherapy (71% vs 0%).
Conclusions: Stemness and EMT markers are co-expressed in CTCs from patients with metastatic breast cancer, both before and after the first-line chemotherapy. However chemotherapy seems to enrich these characteristics on CTCs, suggesting that CTCs possessing or acquiring these features are resistant to chemotherapy. Evaluation of the post-chemotherapy samples is ongoing and final results will be presented.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-07.