Immunophenotypic Characterization of Human Glioblastoma Stem Cells: Correlation With Clinical Outcome Gianfranca Miconi, 1 Paola Palumbo, 1 Soheila Raysi Dehcordi, 1,2 Cristina La Torre, 1 Francesca Lombardi, 1 Zoran Evtoski, 1 Anna Maria Cimini, 1,3 Renato Galzio, 1,2 Maria Grazia Cifone, 1 and Benedetta Cinque 1 * 1 Department of Life, Health and Environmental Sciences, University of L 0 Aquila—Building Delta 6, Coppito, L’Aquila 67100, Italy 2 Operative Unit of Neurosurgery, San Salvatore Hospital, L’Aquila, Italy 3 Sbarro Institute for Cancer Research and Molecular Medicine and Center for Biotechnology, Temple University, Philadelphia, Pennsylvania ABSTRACT Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. b-tubulin III, nestin, CD133, GFAP, and SOX-2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX-2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P ¼ 0.000) and progression-free survival (PFS) (P ¼ 0.001). In addition, a very poor OS (P ¼ 0.000) and PFS (P ¼ 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX-2 may be useful to identify patients at risk of disease progression. J. Cell. Biochem. 116: 864–876, 2015. © 2015 Wiley Periodicals, Inc. KEY WORDS: GLIOBLASTOMA MULTIFORME; CANCER STEM CELL; SOX-2 EXPRESSION; OVERALL SURVIVAL; PROGRESSION FREE SURVIVAL G lioblastoma multiforme (GBM) is the most common and malignant primary brain tumor. The peak incidence is in middle adult life. In particular, the mean age for the occurrence is 56 to 60 years old [Raysi Dehcordi et al., 2012]. Despite a multimodal aggressive treatment, involving surgery, radiotherapy, and chemo- therapy, the vast majority of patients die within two years [Wen and Kesari, 2008; Clarke et al., 2010; Raysi Dehcordi et al., 2012]. According to the results of the European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC), there is a significantly increased overall survival (OS) in patients who received surgery followed by radiotherapy with concomitant and adjuvant temozolomide treatment, in whom OS could be increased from 12.1 to 14.6 months. The median progression-free survival (PFS) is about 6.9 months with radio- chemotherapy, and 5 months with radiotherapy alone [Stupp et al., 2005]. Nevertheless, 3–5% of the patients survives for more than 36 months and they are referred to as “long-term survivors” [Krex et al., 2007]. For years, parallelisms have been made between stem cell biology and oncology, especially because of the growing evidence that genes, with important roles in stem cell biology, also play a role in tumorigenesis. Cancer stem cells (CSCs) are tumor cells displaying normal neural stem cell properties including maintained prolifer- ation, self-renewal, and differentiation ability as well as tumor- initiating cell features [Clarke et al., 2006]. Since their discovery in acute myeloid leukemia in 1997 [Bonnet and Dick, 1997], CSCs have been the object of intense researches and they have been described in many tumors [Reya et al., 2001], including brain cancers [Stiles and Gianfranca Miconi and Paola Palumbo contributed equally to this work. Grant sponsor: Abruzzo Earthquake Relief Fund. *Correspondence to: Benedetta Cinque, Department of Life, Health and Environmental Sciences, University of L’Aquila —Building Delta 6, Via Pompeo Spennati, Coppito, L’Aquila 67100, Italy. E-mail: benedetta.cinque@univaq.it Manuscript Received: 11 December 2014; Manuscript Accepted: 16 December 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 5 January 2015 DOI 10.1002/jcb.25043  © 2015 Wiley Periodicals, Inc. 864 ARTICLE Journal of Cellular Biochemistry 116:864–876 (2015)