The stability of breast cancer progenitor cells during cryopreservation: Maintenance of proliferation, self-renewal, and senescence characteristics q Feridoun Karimi-Busheri * , Victoria Zadorozhny, Daniel L. Shawler, Habib Fakhrai NovaRx Corporation, 6828 Nancy Ridge Drive, Suite 100, San Diego, CA 92121, USA article info Article history: Received 3 November 2009 Accepted 18 February 2010 Available online 21 February 2010 Keywords: Breast cancer stem cell Mammosphere Proliferation Self-renewal Senescence abstract Cancer stem cells are believed to be the driving force behind tumor progression and development. Despite extensive studies on the effects of cryopreservation on embryonic and hematopoietic stem cells there is only limited data that directly deals with in the cryopreservation of cancer stem cells. In this study, we looked at the effect of cryopreservation on breast cancer progenitor cells known as mammo- spheres, which are derived from the MCF7 breast carcinoma cell line. We focused on the effect of cryo- preservation on the cell biology and function of tumor-initiating cells using a standard method of cryopreservation with 15% dimethyl sulfoxide (Me 2 SO). Cell proliferation and survival was analyzed by alamarBlue solution on cryopreserved cells stored for 1–12 weeks and also by the expression of Ki-67. To assess self-renewal, single cells were harvested by limiting dilution procedure and wells were scored once a week. In order to investigate senescence, the activity of b-galactosidase was detected by histo- chemical staining. Our results indicate that cryopreservation of breast cancer initiating cells will not reduce the ability of the cells to proliferate following cryopreservation storage for up to 12 months. Sim- ilarly, self-renewal, a unique property of stem cells, was shown to be maintained during cryopreserva- tion. In contrast, cryopreservation of the mammospheres significantly increases the rate of senescence- mediated pathways. These data suggest that although cryopreservation of tumor-initiating cells is feasi- ble but further studies are necessary to achieve a trustable repository of tumor-initiating cells and the design of new therapeutic measures to specifically target these cells. Ó 2010 Elsevier Inc. All rights reserved. In the last decade, cancer stem cells have been identified in sev- eral human neoplasms including leukemia, brain, breast, colon, prostate, and lung tumors [1,6,9,17,25,30,36,50]. These cells are a functionally distinct subset of cells in the tumor milieu essential for the development, progression and metastasis of malignancies [39,56]. As few as 10 3 tumor stem cells can generate and maintain tumors in immunodeficient mice [1,36,44,51]. Evidence suggests that tumor stem cells have the potential for self-renewal and dif- ferentiation and resistance to radiation and chemotherapy which contribute to treatment failure and tumor progression [28,34,45]. Tumor stem cells have the ability to produce cytokines, chemo- kines and angiogenic factors [24,29] and have been shown to pos- sess up-regulated signaling cascades that are essential for cancer metastasis, including sonic hedgehog and Bmi-1 [49]. Recently it was shown that in a tumorigenic and migratory pan- creatic cell line, the presence of a small number of cells expressing the CXCR4 receptor are involved in tumor metastasis and invasion [24]. When the cells expressing the CXCR4 receptor were elimi- nated from the population the metastatic activity of pancreatic cancer cells disappeared. Similar studies in leukemic and breast stem cells have also shown that specific targeting of cancer stem cells without ablating normal stem cells may be possible [29]. Prolonged passaging of cell lines has been shown to change the cells biological behavior [5]. During cryopreservation also cells un- dergo extensive physical and biological stresses. Since its applica- tion, however, cryopreservation has played a major role in basic and clinical sciences [41]. Progress in cancer stem cell expansion, processing and banking, and any future cell-based therapy, requires an efficient method of preserving the cells at early passages in a highly regulated manner, designed to maximize the safety and clinical efficacy of the sam- ples [38]. Cryopreservation of human embryos, oocytes, ovarian tissue and hematopoietic cells, for example, are well established technol- ogies that allow banking and safe recovery of tissues or cells for as long as necessary [33,54]. There has also been significant progress in the vitrification of many tissues and whole organs including ovary and kidney [18]. Freezing and thawing of hematopoietic stem cells due to its clinical application has been the subject of intensive research and is routinely employed in all autologous settings [4,14,19]. 0011-2240/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2010.02.005 q This work was partially supported by the SBIR Grant 4425R44CA105964-04. * Corresponding author. E-mail address: fkarimi@novarx.com (F. Karimi-Busheri). Cryobiology 60 (2010) 308–314 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo