Mafalda Laranjo1, Maria João Carvalho2, Tânia Costa3, André Alves3, Rui Caetano Oliveira4, João Casalta-Lopes5, Patrícia Cordeiro3, Filomena Botas3, Ana Margarida Abrantes6, Artur Paiva7, Carlos Oliveira8, Maria Filomena Botelho6. 1. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CIMAGO, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CNC.IBILI, University of Coimbra, Coimbra, Portugal. Electronic address: mafaldalaranjo@fmed.uc.pt. 2. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CIMAGO, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Gynecology A Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal. 3. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal. 4. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Pathology Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal. 5. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CIMAGO, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Radiation Oncology Department, Coimbra Hospital and Universitary Centre, Coimbra, Portugal. 6. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CIMAGO, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; CNC.IBILI, University of Coimbra, Coimbra, Portugal. 7. Cytometry Operational Management Unit, Clinical Pathology Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal. 8. CIMAGO, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
Abstract
OBJECTIVES: This study aimed to characterize mammospheres from hormonal receptor (HR) positive and triple-negative breast cancer (TNBC), hypothesizing a differential profile of CSC and differentiation markers, and a stemness enrichment when successive sphere forming-protocols are performed. METHODS: Breast cancer cells MCF-7 and HCC1806 were submitted to sphere-forming protocols. The first sphere generation (MS1) was cultured in adherent conditions (G1). This procedure was repeated and generations of mammospheres (MS1, MS2, and MS3) and sphere-derived cells in adherent conditions (G1, G2, and G3) were obtained. The mammosphere forming capacity, self-renewal, area and doubling time were evaluated. Flow cytometry regarding CD133, CD24, and CD44 and western-blot regarding aldehyde dehydrogenase (ALDH), hormonal receptors and P53 expression was performed. RESULTS: Breast cancer cell lines harboured the capacity to form spheres, which originated derived adherent populations. The sphere-forming capacity was enhanced in HCC1806-MS3 compared to MS1. Self-renewal was higher in MCF-7 mammospheres, which also had an increased area. The putative CSC markers CD133 showed tendency to be enhanced in mammospheres but the CD44+/CD24-/low phenotype was not identified. The expression of ALDH was greater in mammospheres from MCF-7 and HCC1806 than in the respectively derived adherent cells. The expression of oestrogen receptor (ER)-α, progesterone receptor (PR) and P53 decreased in MCF-7 spheres. ER-β expression was lower in mammospheres from both cell lines compared with parental and derived adherent populations. CONCLUSIONS: Loss of HR and P53 expression in HR-positive mammospheres evidences the minor population of CSC which shares characteristics with the TNBC phenotype.
OBJECTIVES: This study aimed to characterize mammospheres from hormonal receptor (HR) positive and triple-negative breast cancer (TNBC), hypothesizing a differential profile of CSC and differentiation markers, and a stemness enrichment when successive sphere forming-protocols are performed. METHODS:Breast cancer cells MCF-7 and HCC1806 were submitted to sphere-forming protocols. The first sphere generation (MS1) was cultured in adherent conditions (G1). This procedure was repeated and generations of mammospheres (MS1, MS2, and MS3) and sphere-derived cells in adherent conditions (G1, G2, and G3) were obtained. The mammosphere forming capacity, self-renewal, area and doubling time were evaluated. Flow cytometry regarding CD133, CD24, and CD44 and western-blot regarding aldehyde dehydrogenase (ALDH), hormonal receptors and P53 expression was performed. RESULTS:Breast cancer cell lines harboured the capacity to form spheres, which originated derived adherent populations. The sphere-forming capacity was enhanced in HCC1806-MS3 compared to MS1. Self-renewal was higher in MCF-7 mammospheres, which also had an increased area. The putative CSC markers CD133 showed tendency to be enhanced in mammospheres but the CD44+/CD24-/low phenotype was not identified. The expression of ALDH was greater in mammospheres from MCF-7 and HCC1806 than in the respectively derived adherent cells. The expression of oestrogen receptor (ER)-α, progesterone receptor (PR) and P53 decreased in MCF-7 spheres. ER-β expression was lower in mammospheres from both cell lines compared with parental and derived adherent populations. CONCLUSIONS: Loss of HR and P53 expression in HR-positive mammospheres evidences the minor population of CSC which shares characteristics with the TNBC phenotype.