Contents lists available at ScienceDirect International Journal of Biochemistry and Cell Biology journal homepage: www.elsevier.com/locate/biocel Characterization of OCT3/4, Nestin, NANOG, CD44 and CD24 as stem cell markers in canine prostate cancer Camila Dorotea Costa a,1 , Andre Augusto Justo b,1 , Priscila Emiko Kobayashi b , Michelle M. Story c , Chiara Palmieri c , Renée Laufer Amorim b,2 , Carlos Eduardo Fonseca-Alves a,2, ⁎ a São Paulo State University – UNESP, Department of Veterinary Surgery and Anaesthesiology, School of Veterinary Medicine and Animal Science, Botucatu, Sao Paulo, Brazil b São Paulo State University – UNESP, Department of Veterinary Clinic, School of Veterinary Medicine and Animal Science, Botucatu, Sao Paulo, Brazil c School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, Queensland, Australia ARTICLE INFO Keywords: Dog OCT3/4 Prostate cancer NANOG CD44 ABSTRACT The cancer cell population is heterogeneous, and cancer stem cells (CSCs) are important for tumor growth and maintenance. The CSC population is associated with different neoplastic characteristics, such as cell migration, resistance to apoptosis, radiation therapy and chemotherapy. To increase the knowledge of CSCs in canine prostate cancer (PC), we characterized CSC markers in canine PC tissues and tumorspheres. We performed immunohistochemistry of OCT3/4, Nestin, NANOG, CD44 and CD24 in 10 normal canine prostatic tissue samples, 10 prostatic hyperplastic (PH) tissue samples and 28 PC tissue samples. Then, we established two canine prostate cancer cell cultures and characterized the CSC profile of tumorspheres grown from these cul- tures. Normal and PH tissues were positive for Nestin, NANOG, CD44 and CD24 only in the basal cell layer. OCT3/4 was expressed in the luminal cells of normal and PH tissues. There was no significant difference in Nestin expression among the prostatic tissues. However, we found higher expression of NANOG and CD44 in canine PC tissues than that in normal and PH tissues. Tumorspheres from canine prostate cancer cells express OCT3/4, Nestin, NANOG and CD44, indicating that these markers may be potential cancer stem cell markers in canine PC. The results obtained can be useful to better characterize the stem cell population in canine prostatic cancer and to guide future studies in comparative oncology. 1. Introduction Human prostate cancer (PC) is the most common cancer subtype in the western world and has a high mortality rate (Siegel et al., 2018). The greatest therapeutic challenge of human PC is the development of resistance to standard androgen-deprivation therapy (Cattrini et al., 2017). Studies performed on animal models of prostate cancer have increased the understanding of tumor biology, metastasis and ther- apeutic targets (Facina et al., 2018; McClurg et al., 2018), and dogs can provide an interesting spontaneous model for human PC (Fonseca- Alves, 2018). Canine PC is a multifactorial disease with a heterogeneous cancer cell population, which may promote tumor proliferation, invasion and metastasis (Keller et al., 2013). Cancer stem cells (CSCs) maintain the capacity for cellular proliferation, survival and motility and thereby contribute to carcinogenesis in many tumors, including prostate cancer in men (Kleeberger et al., 2007). The presence of CSCs could explain the long life of cancer and its immortalization since CSCs are resistant to apoptosis and chemotherapeutic agents (Jaworska et al., 2015; Jeter et al., 2015; Klarmann et al., 2009). In human PC, neoplastic cells with stem cell properties can express different CSC markers, such as OCT3/4, Nestin, NANOG, CD44 and CD24 (Miyazawa et al., 2014). However, limited information is available on the existence of CSCs in canine PC. Since canine PC can be an important model for the human counterpart, https://doi.org/10.1016/j.biocel.2019.01.002 Received 11 October 2018; Received in revised form 2 January 2019; Accepted 5 January 2019 Abbreviations: CSC, cancer stem cells; PC, prostate cancer; OCT3/4, octamer-binding protein 3/4; CD44, cluster of differentiation 44; CD24, cluster of differentiation 24; PH, prostatic hyperplastic; MELK, maternal embryonic leucine zipper kinase; WHO, World Health Organization; DAB, 3,3´-diaminobenzidine; DPBS, Dulbecco's Phosphate Buffered Saline Modified; FBS, fetal bovine serum; EDTA, 2,2',2”,2”'-(ethane-1,2-diyldinitrilo) tetraacetic acid; PBS, phosphate buffered saline; DAPI, 4′6- diamidine-2′-phenylindole dihydrochloride; DMEM, Dulbecco's Modified Eagle's Medium; EGF, epidermal growth factor; RNA, ribonucleic acid ⁎ Corresponding author. E-mail address: carlos.e.alves@unesp.br (C.E. Fonseca-Alves). 1 Both authors contributed equally to this work. 2 Both authors contributed equally to this work. International Journal of Biochemistry and Cell Biology 108 (2019) 21–28 Available online 08 January 2019 1357-2725/ © 2019 Elsevier Ltd. All rights reserved. T