CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells Jian Wang 1 , Per Ø. Sakariassen 1 , Oleg Tsinkalovsky 2 , Heike Immervoll 2,3 , Stig Ove Bøe 1 , Agnete Svendsen 1 , Lars Prestegarden 1 , Gro Røsland 1 , Frits Thorsen 1 , Linda Stuhr 1 , Anders Molven 2,3 , Rolf Bjerkvig 1,4 and Per Ø. Enger 1,4,5 * 1 Department of Biomedicine, University of Bergen, Bergen, Norway 2 Section for Pathology, The Gade Institute, University of Bergen, Bergen, Norway 3 Department of Pathology, Haukeland University Hospital, Bergen, Norway 4 NorLux Neuro-Oncology, Centre Recherch e de Public Sant  e, Luxembourg 5 Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway CD133 is a cell surface marker expressed on progenitors of haema- topoietic and endothelial cell lineages. Moreover, several studies have identified CD133 as a marker of brain tumor-initiating cells. In this study, human glioblastoma multiforme biopsies were engrafted intracerebrally into nude rats. The resulting tumors were serially passaged in vivo, and monitored by magnetic resonance imaging. CD133 expression was analyzed at various passages. Tumors initi- ated directly from the biopsies expressed little or no CD133, and showed no contrast enhancement suggesting an intact blood-brain barrier. During passaging, the tumors gradually displayed more contrast enhancement, increased angiogenesis and a shorter sur- vival. Real-time qPCR and immunoblots showed that this was accompanied by increased CD133 expression. Primary biopsy sphe- roids and xenograft tumors were subsequently dissociated and flow sorted into CD133 negative and CD133 positive cell populations. Both populations incorporated BrdU in cell culture, and expressed the neural precursor marker nestin. Notably, CD133 negative cells derived from 6 different patients were tumorgenic when implanted into the rat brains. For 3 of these patients, analysis showed that the resulting tumors contained CD133 positive cells. In conclusion, we show that CD133 negative glioma cells are tumorgenic in nude rats, and that CD133 positive cells can be obtained from these tumors. Upon passaging of the tumors in vivo, CD133 expression is upregu- lated, coinciding with the onset of angiogenesis and a shorter sur- vival. Thus, our findings do not suggest that CD133 expression is required for brain tumor initiation, but that it may be involved dur- ing brain tumor progression. ' 2007 Wiley-Liss, Inc. Key words: CD133; brain cancer; angiogenesis; cancer stem cell; xenograft At present, there is a search for tumor cell subpopulations that may be responsible for tumor initiation and progression. Such cells have been termed cancer stem cells and are defined by their capacity to self-renew, express stem cell markers and to initiate tumors in vivo. 1,2 Potential cancer stem cells have been identified in leukae- mias, 3–5 breast, 6 prostate, 7 bone, 8 colon and brain cancer. 9–13 In some cases, these tumor-initiating cells have been distinguished from the non-tumor-initiating ones based on expression of cell sur- face markers. For instance, it has been shown that only CD44 1 / CD24 2 /Lineage 2 breast cancer cells are tumorgenic in animals. 6 In malignant brain tumors, CD133 has been suggested to be a cancer stem cell marker 11,14 since only CD133 positive cells from brain tumor biopsy material were able to initiate brain cancer in a mouse model. 14 Prominin-1 (PROM-1), also called CD133, is a protein with several isoforms of unknown physiological or patho- logical function, and is localized both in the cytoplasm and at the cell surface. 15,16 It is expressed by human neural stem cells and has been proposed to have a function in central nervous system (CNS) development. 17 It is also expressed by CD341, VEGFR- 21 endothelial precursor cells, which may home to sites of neo- vascularization, where they differentiate into endothelial cells and contribute to tumor angiogenesis. 18 Thus, CD133 expression seems also to be linked to angiogenesis. However, data regarding the nature of these brain tumor-initiat- ing cells are diverging, and experimental studies have identified nestin-expressing cells as the cell population susceptible to trans- forming events in the CNS, 19 while stem cell assays conducted on brain tumor tissue reliably produce nestin expressing tumor spheres. 11 Moreover, tumors within the same histological group often contain subgroups with different gene expression profiles that predispose the patients to clinically different outcomes. 20 In ependymomas, data suggest that such molecularly distinct sub- groups are derived from different populations of progenitor cells. 21 Within the C6 glioma cell line, even CD133 negative cells possess clonogenic, self-renewal and tumorgenic capacities. 22 Fur- thermore, it has recently been demonstrated that CD133 negative cells from human glioblastoma multiforme (GBM) exhibit cancer stem cell properties as well, although their proliferation index was lower than for CD133 positive cells. 23 Thus, the role of CD133 positive cells versus other cell populations in brain tumor initia- tion and progression is still uncertain. We have previously developed a human glioma model by implanting human GBM biopsy spheroids into the brains of nude rats, 24,25 and showed that these tumors express a number of stem cell markers. 24 The establishment of naive human GBM biopsy material into a host of a different species (rat), and the expression of primitive cell markers, indicate a high cellular adaptability. In the present work, we used this model to assess the expression of CD133 in GBM patient biopsies as well as in the xenograft tumors during passaging. To assess the tumorgenic potential of CD133 negative cells, these cells were isolated by FACS and implanted separately. Our results clearly show that CD133 negative cells give rise to tumors in vivo as well as CD133 positive tumor cells. Material and methods Tissue collection The biopsy material was obtained from the Department of Neu- rosurgery, Haukeland University Hospital, Bergen, Norway. All biopsies were primary GBMs. The Regional Ethical Committee at Haukeland University Hospital, University of Bergen, Norway, approved the collection of tumor tissue. In vitro cell culture Biopsy spheroids were prepared as previously described. 26 Briefly, tissue samples were minced into 0.5 mm fragments and Grant sponsor: EU (6th Framework Program); Grant number: 504743; Grant sponsors: Norwegian Cancer Society, the Norwegian Research Council, Innovest AS, Helse Vest, Haukeland University Hospital, the Ber- gen Translational Research Program, the Centre Recherche de Public Sant e Luxembourg. The first two authors contributed equally to this work. *Correspondence to: Department of Biomedicine, University of Ber- gen, Jonas Lies vei 91, 5009 Bergen, Norway. Fax: 14755586347. E-mail: per.enger@biomed.uib.no Received 25 May 2007; Accepted after revision 7 August 2007 DOI 10.1002/ijc.23130 Published online 22 October 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 761–768 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer