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Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients

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Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients Published OnlineFirst June 18, 2013; DOI: 10.1158/1535-7163.MCT-12-1167 Molecular Cancer Companion Diagnostics and Biomarkers Therapeutics Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients Galatea Kallergi1, Georgios Konstantinidis2, Harris Markomanolaki1, Maria A. Papadaki1, Dimitris Mavroudis1,3, Christos Stournaras2, Vassilis Georgoulias1,3, and Sofia Agelaki1,3 Abstract The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (poly- caspase kit: 8.1% vs. 47.4% of the total CTC number; P ¼ 0.0001; M30-antibody: 32.1% vs. 76.63%; P ¼ 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics. Mol Cancer Ther; 12(9); 1886–95. Ó2013 AACR. Introduction hypothesis, the survival of these cells depends on their Circulating tumor cells (CTC) are increasingly consid- distinctive biologic characteristics as well as on the micro- ered as a "liquid biopsy" that allows the assessment of environment at the secondary site (10). Only rare subsets tumor changes over time simply by repeated blood draws of cells finally succeed in establishing a cross-talk with (1). The presence of CTCs in the blood of patients with stromal cells in secondary organs that promote tumor cell breast cancer has emerged as a poor prognostic indicator survival, angiogenesis, and metastatic outgrowth. (2–4). Indeed, metastasis is associated with the presence of Characterizing the viability of CTCs in each patient disseminated (DTC) or circulating (CTC) tumor cells in might be important to improve prognostication and indi- the bone marrow and peripheral blood, respectively (5, 6). vidualize treatment. Thus, the presence of exclusively It has been shown that these cells present significant apoptotic CTCs in a patient may represent a favorable heterogeneity (7–9) and the delineation of their specific prognostic factor, whereas the preponderance of prolif- molecular characteristics could enhance the understand- erating cells could be related to poor patient’s outcome. ing of their biology and clinical relevance. Several studies have focused on the apoptotic status of Although many CTCs migrate early from the primary CTCs. Larson and colleagues (11) evaluating the presence tumor into the circulation, 97% of them will be cleared of apoptotic and nonapoptotic CTCs in metastatic prostate within a few days (6). According to the seed and soil cancer reported that the number of apoptotic or intact CTCs differs among patients. Similarly, both apoptotic and nonapoptotic CTCs could be detected in a patient Authors' Affiliations: Laboratories of 1Tumor Biology and 2Biochemistry, with metastatic breast cancer (12). Viable CTCs and DTCs University of Crete School of Medicine and Department of Medical Oncol- ogy, 3University General Hospital of Heraklion, Heraklion, Greece were also evaluated in patients with ovarian cancer where it was shown that patients with viable DTCs after treat- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). ment had significantly reduced progression-free survival (13). The detection of DTCs in patients with breast cancer Corresponding Author: Galatea Kallergi, Laboratory of Tumor Cell Biol- ogy, School of Medicine, University of Crete, Heraklion 71110, Crete, after the completion of neoadjuvant treatment was asso- Greece. Phone: 302-8103-92783; Fax: 302-8103-92857; E-mail: ciated with progressive disease (14). Moreover, apoptotic [email protected] DTCs were mainly identified in patients with disease doi: 10.1158/1535-7163.MCT-12-1167 remission (14). In addition, serologic cell death biomar- Ó2013 American Association for Cancer Research. kers such as M30 and M65 have been detected in the serum 1886 Mol Cancer Ther; 12(9) September 2013 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst June 18, 2013; DOI: 10.1158/1535-7163.MCT-12-1167 Apoptotic CTCs in Breast Cancer of patients with prostate, colorectal, and lung cancer and sample collection. All patients gave their informed con- this has been associated with response to treatment and sent to participate in the study, which has been approved the status of CTCs or DTCs (15–17). by the Ethics and Scientific Committees of our Institution. Evaluation of the prognostic significance of CTCs in PBMCs were isolated by Ficoll–Hypaque density gra- early breast cancer revealed that a substantial number of dient (d ¼ 1,077 g/mL) centrifugation at 1,800 rpm for 30 patients with detectable CTCs either before the initiation minutes. Aliquots of 5 Â 105 PBMCs were cytocentrifuged of any systemic treatment or during adjuvant hormone at 2,000 rpm for 2 minutes on glass slides. Cytospins were treatment do not relapse even many years after their dried and stored at À80C until use. At least two slides detection (3, 18). This observation clearly supports the with a total of 5 Â 105 PBMCs were analyzed for each hypothesis that not all CTCs are capable of metastatic examined molecule in individual patients. seeding. This could be related to the induction of senes- cence in CTCs or to a low proliferative potential or, even, Cell cultures to the presence of apoptotic cells. On the other hand, we MCF7 and SKBR3 breast cancer cells (obtained from have reported that activated kinases such as phospho- American Type Culture Collection). Authentication was AKT and phospho-PI3K that are involved in survival done by staining experiments with antibodies for estro- pathways are expressed in CTCs from patients with early gen, progesterone, and HER2 and EGFR receptors, just and metastatic breast cancer (19, 20). There are also con- before the beginning of the study and spontaneously flicting studies concerning the proliferation status of these during the research. No other authentication test was cells. In 2005, Muller and colleagues (21) reported that done. All cell lines revealed the expected phenotype. CTCs from patients with breast cancer do not express MCF7 and SKBR3 cell lines were centrifuged on cytos- Ki67. However, other studies in patients with prostate and pins according to the procedure followed for patients’ small cell lung revealed Ki67 expression in many patients’ samples and they were used as controls for apoptotic and CTCs but in different frequencies per patient (22, 23). Ki67 staining experiments, respectively. The aims of the current study were to assess the apo- MCF7 cells (mammary adenocarcinoma cell line) were ptotic status of CTCs in patients with early and metastatic incubated with 2 mmol/L staurosporine for 2 hours induce breast cancer using two different methods as well as their apoptosis (24). MCF7 cells were cultured in 1:1 (v/v) proliferative potential. In addition, the apoptotic and Dulbecco’s modified Eagle medium (DMEM)/Ham’s proliferative status of CTCs before and after adjuvant F12 medium (GIBCO-BRL Co) supplemented with 10% treatment was also evaluated. FBS (GIBCO-BRL), 2 mmol/L L-glutamine (GIBCO-BRL), 30 mmol/L NaHCOB3B, 16 ng/mL insulin, and 50 mg/ Materials and Methods mL penicillin/streptomycin (GIBCO-BRL). SKBR3 cells Patient samples and cytospin preparation were cultured in RPMI supplemented with 10% FBS. Cells n ¼ Fifty-six patients with early ( 29) and metastatic were maintained in a humidified atmosphere of 5% CO2 in (n ¼ 27) breast cancer were enrolled in the study. These air. Subcultivation was conducted with 0.25% trypsin and patients harvested cytokeratin-positive CTCs when rou- 5 mmol/L EDTA (GIBCO-BRL). All experiments were tinely screened in the context of the initial evaluation of carried out during the logarithmic growth phase. Twenty their disease. CK-positive CTCs were detected by double to 24 hours before the experiments, cells were transferred immunofluorescence using anti-cytokeratin and anti- in serum-free medium containing only L-glutamine, CD45 antibodies. Patients were enrolled in this study if NaHCO3 and penicillin/streptomycin. After incubation, they had at least two CTCs per 106 peripheral blood cells were centrifuged on cytospins according to the same mononuclear cells (PBMC).
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