Abstract 374: Expression of the autophagosomal marker LC3 on circulating tumor cells (CTCs) of patients with early and metastatic breast cancer (BC)
Authors: Spiliotaki Maria , Koukorava Chrysa , Matikas Alexios , Georgoulias Vassilis , Mavroudis Dimitrios , Agelaki Sofia 
Published: August 2015

Abstract
Background: Autophagy is a protein and organelle degradation pathway involved in the regulation of cancer development and progression. Recent evidence suggests that autophagy serves as a cell survival mechanism under stressed conditions and it is considered to be involved in determining the response of cancer cells to anticancer therapy. Microtubule-associated protein 1 light chain 3 (LC3) has been identified as a marker for monitoring autophagy. CTCs, which have been linked to tumor progression in BC, may employ autophagy associated mechanisms to survive. Here, we investigated the expression of LC3B on CTCs isolated from patients with BC.
Methods: The expression of LC3B was first assessed by immunofluorescence using MCF7 treated with tamoxifen as a model system. LC3B expression was then evaluated in 22 and 25 CTC positive patients with early and metastatic BC before the initiation of adjuvant and first-line chemotherapy, respectively. Metastatic patients were also assessed before the administration of the second treatment cycle. Cytospins of MCF7 cells and of peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 anti-cytokeratin (CK) antibody and an LC3B anti-rabbit antibody. LC3B density was measured using the automated fluorescent microscope ARIOL system. The cutoff value for high and low LC3B expression, was set at the median value of the integrated optical density (IOD).
Results: Among 142 CTCs identified in patients with early BC, 55 (38%) presented high LC3B expression. A total of 176 CTCs were detected in metastatic patients before the initiation of first-line chemotherapy and 99 (59%) presented high LC3B expression (p = 0.0003 compared with early BC patients). After the first treatment cycle, 62 CTCs were detected with 37 (60%) of them demonstrating high LC3B expression. Nine metastatic patients remained CTC-positive both before and after the first cycle of chemotherapy. In these patients, the proportion of cells presenting high LC3B expression was 40% and 75%, pre- and post- chemotherapy, respectively (p = 0.0049).
Conclusions: The autophagosome marker LC3B is expressed in CTCs from patients with BC. High LC3B expression in CTCs was more commonly observed in metastatic, whereas low LC3B expression prevailed in early disease. Moreover, LC3B expression on CTCs was significantly increased after the administration of chemotherapy. The above observations suggest that metastatic progression in breast cancer could be linked to LC3B expression. Moreover the evaluation of LC3B on CTCs merits further investigation regarding its association with chemotherapy efficacy.
Citation Format: Maria Spiliotaki, Chrysa Koukorava, Alexios Matikas, Vassilis Georgoulias, Dimitrios Mavroudis, Sofia Agelaki. Expression of the autophagosomal marker LC3 on circulating tumor cells (CTCs) of patients with early and metastatic breast cancer (BC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2015-374