Cancer induction by restriction of oncogene expression to the stem cell compartment
2008; Springer Nature; Volume: 28; Issue: 1 Linguagem: Inglês
10.1038/emboj.2008.253
ISSN1460-2075
AutoresMaría Pérez-Caro, César Cobaleda, Inés González‐Herrero, Carolina Vicente‐Dueñas, Camino Bermejo‐Rodríguez, Margarita Sánchez‐Beato, Alberto Órfão, Belén Pintado, Teresa Flores, Manuel Sánchez‐Martín, Rafael Jiménez, Miguel Á. Piris, Isidro Sánchez-Garcı́a,
Tópico(s)CAR-T cell therapy research
ResumoArticle27 November 2008Open Access Cancer induction by restriction of oncogene expression to the stem cell compartment María Pérez-Caro María Pérez-Caro Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author César Cobaleda César Cobaleda Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Inés González-Herrero Inés González-Herrero Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Carolina Vicente-Dueñas Carolina Vicente-Dueñas Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Camino Bermejo-Rodríguez Camino Bermejo-Rodríguez Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Margarita Sánchez-Beato Margarita Sánchez-Beato Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain Search for more papers by this author Alberto Orfao Alberto Orfao Servicio de Citometría and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Belén Pintado Belén Pintado Genetically Engineered Mouse Facility, CNB-CSIC, Madrid, Spain Search for more papers by this author Teresa Flores Teresa Flores Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Manuel Sánchez-Martín Manuel Sánchez-Martín Departamento de Medicina, Genetically Engineered Mouse Facility, SEA, University of Salamanca, Salamanca, Spain Search for more papers by this author Rafael Jiménez Rafael Jiménez Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Miguel A Piris Miguel A Piris Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain Search for more papers by this author Isidro Sánchez-García Corresponding Author Isidro Sánchez-García Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author María Pérez-Caro María Pérez-Caro Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author César Cobaleda César Cobaleda Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Inés González-Herrero Inés González-Herrero Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Carolina Vicente-Dueñas Carolina Vicente-Dueñas Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Camino Bermejo-Rodríguez Camino Bermejo-Rodríguez Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Margarita Sánchez-Beato Margarita Sánchez-Beato Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain Search for more papers by this author Alberto Orfao Alberto Orfao Servicio de Citometría and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Belén Pintado Belén Pintado Genetically Engineered Mouse Facility, CNB-CSIC, Madrid, Spain Search for more papers by this author Teresa Flores Teresa Flores Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Manuel Sánchez-Martín Manuel Sánchez-Martín Departamento de Medicina, Genetically Engineered Mouse Facility, SEA, University of Salamanca, Salamanca, Spain Search for more papers by this author Rafael Jiménez Rafael Jiménez Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Miguel A Piris Miguel A Piris Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain Search for more papers by this author Isidro Sánchez-García Corresponding Author Isidro Sánchez-García Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain Search for more papers by this author Author Information María Pérez-Caro1, César Cobaleda2, Inés González-Herrero1, Carolina Vicente-Dueñas1, Camino Bermejo-Rodríguez1, Margarita Sánchez-Beato3, Alberto Orfao4, Belén Pintado5, Teresa Flores6, Manuel Sánchez-Martín7, Rafael Jiménez2, Miguel A Piris3 and Isidro Sánchez-García 1 1Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Salamanca, Spain 2Departamento de Fisiología y Farmacología, Universidad de Salamanca, Salamanca, Spain 3Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain 4Servicio de Citometría and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain 5Genetically Engineered Mouse Facility, CNB-CSIC, Madrid, Spain 6Departamento de Anatomía Patológica, Universidad de Salamanca, Salamanca, Spain 7Departamento de Medicina, Genetically Engineered Mouse Facility, SEA, University of Salamanca, Salamanca, Spain *Corresponding author. Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer (IBMCC), CSIC/Universidad de Salamanca, Campus Unamuno, S/N, 37007 Salamanca, Spain. Tel.: +34 923 238403; +34 923 294813; E-mail: [email protected] The EMBO Journal (2009)28:8-20https://doi.org/10.1038/emboj.2008.253 There is a Have you seen ...? (February 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info In human cancers, all cancerous cells carry the oncogenic genetic lesions. However, to elucidate whether cancer is a stem cell-driven tissue, we have developed a strategy to limit oncogene expression to the stem cell compartment in a transgenic mouse setting. Here, we focus on the effects of the BCR-ABLp210 oncogene, associated with chronic myeloid leukaemia (CML) in humans. We show that CML phenotype and biology can be established in mice by restricting BCR-ABLp210 expression to stem cell antigen 1 (Sca1)+ cells. The course of the disease in Sca1-BCR-ABLp210 mice was not modified on STI571 treatment. However, BCR-ABLp210-induced CML is reversible through the unique elimination of the cancer stem cells (CSCs). Overall, our data show that oncogene expression in Sca1+ cells is all that is required to fully reprogramme it, giving rise to a full-blown, oncogene-specified tumour with all its mature cellular diversity, and that elimination of the CSCs is enough to eradicate the whole tumour. Introduction An axiom in the treatment of tumours is that the remission is, in general, more difficult to achieve with each relapse. Despite a better understanding of the biology of tumour cells, the treatment of most cancers has not significantly changed for the past three decades and the decreasing mortality has been mostly the result of early detection and prevention rather than the consequence of effective therapeutics (Etzioni et al, 2003; Chabner and Roberts, 2005; Huff et al, 2006). A new hypothesis in cancer biology postulates that tumours are hierarchically structured as abnormal tissues, which are maintained by cancer stem cells (CSCs) (Reya et al, 2001; Perez-Caro and Sanchez-Garcia, 2006; Dalerba et al, 2007a; Sánchez-García et al, 2007). This theory provides a rationale to explain the failure of many currently used antitumoral strategies, because just a small population of CSCs resistant to the therapy would be enough to maintain the whole tumour mass (Etzioni et al, 2003; Chabner and Roberts, 2005; Huff et al, 2006). Evidences supporting this model have been recently published for human leukaemias (Bonnet and Dick, 1997; Cobaleda et al, 2000; Cox et al, 2004, 2007; Hope et al, 2004) and solid tumours (Al-Hajj et al, 2003; Singh et al, 2004; Collins et al, 2005; Kim et al, 2005; Xin et al, 2005; Bao et al, 2006a, 2006b; Dalerba et al, 2007b; Li et al, 2007; O'Brien et al, 2007; Prince et al, 2007; Ricci-Vitiani et al, 2007). However, this emerging concept has not been fully validated yet in an experimental model system. If validated, the CSC hypothesis will have far-reaching consequences for our understanding of cancer biology and for the development of new strategies to improve cancer treatment. A major obstacle to elucidate the contribution that CSCs make to the development and maintenance of cancer and their suitability as a target is the lack of a system to limit oncogene expression to the CSC compartment. Since the discovery of oncogenes in human tumours, many efforts have been made to elucidate the causal role that these oncogenes have in cancer development. These previous studies have shown that oncogene expression is not only required for initiation of cancer but also for the maintenance of the disease (Chin et al, 1999; Huettner et al, 2000; Boxer et al, 2004). In mouse models where oncogene expression is driven by tissue-specific promoters, tumours arise at high frequency, but disappear again when the inducing stimulus is switched off (Chin et al, 1999; Huettner et al, 2000; Boxer et al, 2004). However, it has not been biologically proven that cancer is a hierarchically organized tissue that can be created and maintained similar to a normal stem cell-based tissue. If that is indeed the case, then cancer should be created and maintained similarly to any other normal stem cell-driven tissue, similar to the haematopoietic system. In a normal stem cell-driven tissue, genetic programming of stem cells is all that is required to (re)constitute all differentiated cells forming the tissue and the genetic information responsible for the stem cell programming is not present anymore within the differentiated cells that form the tissue. Thus, we reasoned that a similar organization could be happening for cancer formation. To address these biological questions, we have used the BCR-ABLp210 oncogene associated with chronic myeloid leukaemia (CML) in humans as a model (Koeffler and Golde, 1981; Melo and Barnes, 2007). CML is a paradigmatic stem cell disorder that begins as a protracted chronic phase, characterized by high leukocyte counts and enlarged spleen and liver. In all patients, the chronic phase of CML eventually converts to a blast crisis that is indistinguishable from acute leukaemia. The specific BCR-ABL inhibitor STI571 can prolong the remission times of CML patients because it is able to eliminate the BCR-ABL-expressing differentiated cells that constitute the bulk of the tumour (Druker et al, 2001). However, it cannot eliminate BCR-ABL-expressing CSCs, even though it can penetrate into the cells, and these CSCs eventually repopulate the tumour with STI571-resistant mature cells (Graham et al, 2002; Hu et al, 2006; Primo et al, 2006; Jiang et al, 2007). Here, we show that CML phenotype and biology can be established in mice in which we limited BCR-ABLp210 expression to stem cell antigen 1 (Sca1)+ cells. These model mice mimic CML human pathology. The course of the CML disease in the Sca1-BCR-ABLp210 mice was not modified on STI571 treatment. However, we show that BCR-ABLp210-induced CML is reversible through the unique elimination of the CSCs. To our knowledge, this study demonstrates for the first time that limited oncogene expression in CSCs is all that is required to fully reprogramme it, giving rise to a full-blown, oncogene-specified tumour with all its mature cellular diversity, and that CSC elimination is enough to eradicate the whole tumour. Results Derivation of Sca1-BCR-ABLp210 mice The mouse Ly-6E.1 promoter (Miles et al, 1997) was used to drive Sca1-directed expression of a human BCR-ABLp210 (Sca1-BCR-ABLp210) or TK-IRES-BCR-ABLp210 transgene (Sca1-TK-IRES-BCR-ABLp210) in C57BL/6 × CBA mice (Figure 1). This promoter is known to have heterologous expression in haematopoietic stem cells (HSCs) (Miles et al, 1997), hence transgene expression is restricted to a limited percentage of Sca1-expressing cells. The BCR-ABLp210-overexpressing animals had normal gestation and were viable. Three independent lines were obtained and used to examine the phenotype further (Table I). As c-kit is known from earlier studies to be downregulated in leukaemia stem cells (Blair and Sutherland, 2000; Neering et al, 2007), our functional definition of stem cell in this study does not include c-kit as a surface marker. Quantitative RT–PCR of BCR-ABLp210 and/or TK messenger mRNA confirmed that expression was largely confined to Sca1+ cells with little or no ectopic expression in Sca1− cells (Figure 1). In line with these results, neither BCR-ABL protein nor downstream signalling was detected in Sca1−Lin+ cells of Sca1-BCR-ABLp210 mice (Supplementary Figure 1 online). As reported earlier in transgenic reporter lines with the same promoter (Miles et al, 1997; Ma et al, 2002a, 2002b), BCR-ABLp210 was expressed in a limited percentage of Sca1+ cells (25–50%) as measured by single-cell RT–PCR (Supplementary Figure 2 online). This enables cell transformation to occur in a similar manner as it happens in humans, where cells expressing the oncogene are present along with non-expressing cells. The phenotype described here is therefore due primarily to the expression of BCR-ABLp210 in Sca1+ cells. Figure 1.Sca1-BCR-ABLp210 and Sca1-TK-IRES-BCR-ABLp210: transgene constructs, expression and survival. (A) Schematic representation of the genomic structure of the mouse Sca1 locus and the Sca1-BCR-ABLp210 and Sca1-TK-IRES-BCR-ABLp210 transgenic vectors used in this study. The HSV-TK/BCR-ABLp210 fusion gene construct is a bicistronic construct consisting of the herpes simplex thymidine kinase (TK) cDNA separated from BCR-ABLp210 by the picornaviral internal ribosome entry site (IRES) sequence. NotI sites used to excise the transgene fragments and EcoRI sites used to examine Southern blots are indicated. (B) Identification of the transgenic mice by Southern blot analysis of tail snip DNA after EcoRI digestion. Human ABL cDNA was used for the detection of the transgene. Sca1-BCR-ABLp210 and the endogenous c-Abl are indicated. Lines IS1A and IS1B are two different Sca1-BCR-ABLp210 transgenic lines. (C) Quantification of BCR-ABLp210 expression in Sca1-BCR-ABLp210 mice and quantification of thymidine kinase (TK) and BCR-ABLp210 expression in Sca1-TK-IRES-BCR-ABLp210 mice by real-time PCR in Sca1+Lin− and Sca1−Lin+ cells. Percentage of TK and BCR-ABLp210 transcripts with reference to β-actin is shown. (D) Quantification of BCR-ABLp210 expression by real-time PCR in Sca1+Lin− cells of Sca1-BCR-ABLp210 mice (line IS1A or IS1B) and of double Sca1-BCR-ABLp210 mice (line IS1A × IS1B). (E) Kaplan–Meier survival plots of Sca1-BCR-ABLp210 mice (line IS1A or IS1B), double Sca1-BCR-ABLp210 mice (line IS1A × IS1B) and Sca1-TK-IRES-BCR-ABLp210 mice (line IS9A). The total number of mice analysed in each group is indicated. Statistical analysis was performed using the χ2 test, and the corresponding P-values are given in parentheses. Download figure Download PowerPoint Table 1. Incidence and age of CML onset in Sca1-BCR-ABLp210 and Sca1-TK-IRES-BCR-ABLp210 mice Transgenic line Mice autopsieda Mice with tumour (%)b Age in months at tumour onset Haematopoietic tumour type (%) WBC/μl, neutrophils (%) Hgb (g/100 ml) Spleen weight (mg) IS1A (Sca1-BCR-ABLp210) 34 34 (100) 8–12 CML–BC (100) 12 900±1750 (43±4) 11.5±1.3 285±36 IS1B (Sca1-BCR-ABLp210) 25 25 (100) 4–10 CML–BC (100) 11 850±1650 (38±5) 12.7±1.1 217±29 IS1A+IS1B 10 10 (100) 1–2 Leukaemia ND 13.2±1.3 ND IS9A (Sca1-TK-IRES-BCR-ABLp210) 23 23 (100) 6–10 CML–BC (100) 13 300±230 (41±8) 11.1±1.2 261±23 ND, not determined. a Number of mice during or after the period of cancer. b Number of mice with CML and percentage of tumour incidence. Normal range of WBC counts was 4000–10 000/μl. Mean neutrophil percentage of control animals was 10±4% WBCs, and the neutrophil percentage in control animals did not exceed 20%. Thus, neutrophilia is defined as more than 20% neutrophils. Hgb indicates haemoglobin (normal range, 12–16 g/100 ml). Normal spleen weight in control animals is 78–92 mg (n=25). The WCC estimations were made when the mice were premorbid. CML–BC indicates mice spontaneously progress to blast crisis. P<0.001 for Sca1-BCR-ABLp210 mice versus control mice. CML development in Sca1-BCR-ABLp210 mice The expression of BCR-ABLp210 in Sca1+ cells led to significant increases in the numbers of white blood cells (WBCs) and in the percentages of neutrophils (Table I) in the peripheral blood (PB) of Sca1-BCR-ABLp210 animals (Figure 2A). Flow cytometry of bone marrow (BM) cells demonstrated significantly increased numbers of cells staining positively for the myeloid markers Gr1 and Mac-1, comprising up to 75–90% of the cells in the BM of transgenic animals (Figure 2A). However, the percentage of Sca1+Lin− cells in BM did not increase (data not shown), similarly as it happens in human CML (Jamieson et al, 2004). Macroscopic analysis of these animals showed splenomegaly (Table I) and hepatomegaly (79% of the animals). The histologic examination of the spleen and liver demonstrated the presence of megakaryocytes as markers of myeloid metaplasia (Figure 2B) and expansion of the splenic red pulp by predominantly granulocytic myeloid cells (Figure 2C). This was confirmed by flow cytometric analysis (Figure 2A). In addition, infiltration of myeloid cells was also seen in the liver, lymph nodes and lung (Figure 2C). The major cause of death in animals that had progressive disease was related to myeloid infiltration of extramedullary organs and kidney failure (30%), or the evidence for progression to blast crisis (70%). In addition, targeting Sca1+ cells in non-haematopoietic tissues gave rise to carcinoma development (lung adenocarcinoma 10%, hepatocarcinoma 3%, GIST 2%, osteogenic sarcoma 2% and Sertoli cell tumour 2%; data not shown). Altogether, the survival time of mice expressing BCR-ABLp210 in Sca1+ cells ranged from 4 to 18 months (Figure 1E; Table I). Mice surviving myeloid infiltration of extramedullary organs progressed spontaneously to a blast crisis characterized by the appearance of blasts (myeloid or lymphoid) in PB, BM, spleen and liver (Figure 2D and E). Similar to the human disease, this secondary disorder appeared in the context of a myeloproliferative disease (Figure 2F). The time of transition to blast crisis has been described to be reduced with increased levels of BCR-ABL in human CML cells (Melo and Barnes, 2007; Modi et al, 2007). Similarly, when compound Sca1-BCR-ABLp210 transgenic mice were generated (Figure 1), leukaemia was detectable at 1–2 months of age (Figure 1; Table I). Thus, the limited expression of BCR-ABLp210 to Sca1+ cells is able to mimic human CML, characterized also by a progression from chronic towards an acute phase (blast crisis), which is also invariably fatal in Sca1-BCR-ABLp210 mice and that is dependent on the dose of the oncogene expression in the Sca1+ cells. Figure 2.Sca1-driven BCR-ABLp210 expression induces CML. (A) Cells from PB of Sca1-BCR-ABLp210 mice were analysed by flow cytometry. Representative FACS analysis demonstrating accumulation of mature myeloid cells in PB and spleen, and an increase in the myeloid fraction within BM. PB, peripheral blood; BM, bone marrow. (B) Representative histologic appearance of liver and spleen of diseased Sca1-BCR-ABLp210 mice after haematoxylin–eosin staining. Megakaryocytes in spleen and liver define myeloid metaplasia and are indicated by arrows. (C) Organ infiltration by myeloid cells. Haematoxylin–eosin-stained sections of the spleen (megakaryocytes, myeloid blasts and mature myeloid cells), liver (perivascular infiltration of the liver by blasts and mature myeloid cells), peritoneal lymph node (with myeloid metaplasia) and lung (infiltration of the lung by blasts and mature myeloid cells). Download figure Download PowerPoint Figure 3.(D) Phenotypic characteristics of cells from peripheral blood (PB) as determined by flow cytometry. Blast cells but not mature granulocytes are present in PB from two different leukaemic Sca1-BCR-ABLp210 mice. Note that the B-cell leukaemia is characterized by the presence of blast cells co-expressing Mac-1 and B220. (E) Liver haematoxylin–eosin-stained sections showing blast infiltration and histologic appearance of blood smears (Giemsa staining) in Sca1-BCR-ABLp210 mice in blast crisis. Blast cells infiltrate both liver and PB. (F) Representative histologic appearance of liver and spleen tissue sections stained by Masson's trichrome of Sca1-BCR-ABLp210 mice in blast crisis. The presence of fibrosis (green colour) and blast infiltration in the liver and the spleen of Sca1-BCR-ABLp210 mice demonstrate that the blast crisis takes place in the context of a myeloproliferative disease. Download figure Download PowerPoint The fusion protein responsible for CML has been shown to cause genome instability in humans (Giehl et al, 2005; Melo and Barnes, 2007). We examined Sca1-BCR-ABLp210 mice for evidence of secondary genetic changes. First, we examined the overall DNA methylation status in tumour cells of Sca1-BCR-ABLp210 mice by immunolocalization of 5-methylcytosine to give qualitative information on nuclear distribution. We performed similar analyses in human CD34+ CML cells. Although control cells present unmethylated DNA, an aberrant DNA methylation pattern was observed in tumour cells of human CML (n=3) and Sca1-BCR-ABLp210 mice (n=8) (see Supplementary Figure 3 online). As genetic instability and centrosome defects are common and early detectable features in CML (Melo and Barnes, 2007), we sought to investigate whether centrosome aberrations occur in Sca1-BCR-ABLp210 mice. We examined 14 CML samples including Sca1+ cells of 9 newly diagnosed mice (chronic phase) and 5 blast crisis specimens by using a centrosome-specific antibody to pericentrin. Centrosome abnormalities were detected in 23.6±5.1% of chronic phase cells and in 61.6±5.3% of blast crisis cells, but in only 3.1±0.9% of controls (see Supplementary Figure 3 online). The similarities between the secondary genetic changes observed in humans and Sca1-BCR-ABLp210 mice suggest that similar mechanisms contribute to CML progression in both species. Nature of the leukaemogenic cell in the Sca1-BCR-ABLp210 model We next examined the nature of the leukaemogenic cell in the Sca1-BCR-ABLp210 model. To determine whether the Sca1+Lin− population contains CSC, we sorted Sca1+Lin− and Sca1−Lin+ cells from mice that developed CML. Transplantation of purified fractions of cells into sublethally irradiated syngeneic recipient mice was used to assess leukaemogenesis in vivo. Each of the mice transplanted with Sca1+ Lin− cells developed CML that was phenotypically identical to the primary disease (Table II). Importantly, Sca1−Lin+ cells did not show leukaemic engraftment into secondary recipients or induced leukaemia, even at 10 times higher concentrations. Overall, these data indicate that the CSCs reside in the Sca1+ cell compartment. All these facts indicate that restricting oncogene expression to the stem cell compartment is sufficient to generate all the CML cell types, including the most differentiated ones, which do not express the oncogene and are unable to propagate the disease. Table 2. CML disease is readily transplantable to secondary recipients Sorted cells Number of transplanted cells Transplanted animals Incidence of CML (%) Latency of disease (days±s.d.) Sca1+Lin− (BM, Sca1-BCR-ABLp210 mice) 10.000 8 100 69±14 1.000 8 100 85±11 Sca1+Lin− (BM, control mice) 10.000 8 0 NA 1.000 8 0 NA Sca1−Lin+ (BM, Sca1-BCR-ABLp210 mice) 1 × 105 8 0 NA 1 × 106 8 0 NA Sca1−Lin+ (BM, control mice) 1 × 105 8 0 NA 1 × 106 8 0 NA NA, not applicable. Eight irradiated syngenic recipients mice per cohort were transplanted with the indicated number of cells. The diagnosis of CML was confirmed by histological and immunophenotypic examination of the ill recipient mice. Characterization of CSCs in Sca1-BCR-ABLp210 mice Having prospectively purified a population highly enriched for CSC, we used gene expression to identify the genes that are associated with BCR-ABLp210 reprogramming of stem cells (Supplementary Table I online; Figure 3). We performed a supervised analysis of the transcriptional profiles of CSCs purified from Sca1-BCR-ABLp210 versus those from control mice. The data showed that a total of 293 genes are reproducibly regulated in Sca1-BCR-ABLp210 versus control HSC with a false discovery rate (FDR) ⩽0.07% (Supplementary Table I online). Human CML has been characterized earlier according to gene expression profile (Kronenwett et al, 2005). Remarkably, the gene expression patterns of Sca1+Lin− cells from Sca1-BCR-ABLp210 mice were similar to those from the human CML CD34+ cells (Figure 3A), thereby further validating the model by reflecting the similarities in the transcriptome between the two populations. Thus, BCR-ABLp210-dependent, stem cell-driven murine CML shares the molecular features of human CML. Figure 4.Identity of cancer stem cells in Sca1-BCR-ABLp210 mice. To identify genes associated with BCR-ABLp210-induced reprogramming of stem cells, we have compared the gene expression profiles of purified CSC populations versus normal HSCs. Both CSCs and HSCs were isolated as Sca1+Lin− cells. (A) Comparison of the mouse and human CSCs at the molecular level. Graphical description of the expression pattern in CSCs (Sca1+Lin− cells) from BM (n=10) and PB (n=7) of Sca1-BCR-ABLp210 mice of genes that have been previously published to be significantly regulated in the human CML CD34+ fraction (Kronenwett et al, 2005). The genes were similarly regulated in CSCs of Sca1-BCR-ABLp210 mice. We referred the ratios of the CSCs to the control haematopoietic stem cells (Sca1+Lin− cells purified from control mice). Green indicates that the genes are downregulated in CSCs versus HSCs and red indicates upregulation. Green asterisks mark genes that are downregulated in human CD34+ CML. The remaining genes behave similarly in both human and mouse stem cells. (B) Embryonic surface proteins in CSCs of Scal-BCR-ABLp210 mice. Graphical description of the expression pattern in CSCs (Sca1+Lin− cells) from BM (n=12) of Sca1-BCR-ABLp210 mice of previously identified upregulated cell surface markers in undifferentiated mouse embryonic stem cells (Nunomura et al, 2005). We identified genes for which signal intensities were upregulated (threshold±2) in CSCs and in at least seven CSC samples. We referred the ratios of the CSCs to the control haematopoietic stem cells (Sca1+Lin− cells purified from control mice). Each gene (identified at right) is represented by a single row of coloured boxes; each experimental mouse is represented by a single column. Data are displayed by a colour code. Red fields indicate higher values than the median, green fields indicate lower values than the median. Download figure Download PowerPoint The CSC hypothesis represents a modern-day interpretation of the proposal made by Rudolph Virchow and Julius Cohnheim that cancer results from the activation of dormant embryonal-rest cells (Virchow, 1855; Cohnheim, 1867). Accordingly, we next proceeded to examine, in CSCs from Scal-BCR-ABLp210 mice, the expression of embryonic surface markers, the presence of which has been identified earlier in undifferentiated mouse embryonic stem cells (Nunomura et al, 2005) (Figure 3B). We could identify reproducible upregulation of gene signals corresponding to 55 embryonic stem cell surface proteins in the CSCs from Sca1-BCR-ABLp210 mice (FDR ⩽1%). These results show that CSCs in Sca1-BCR-ABLp210 present embryonic figures that could represent attractive targets for selective CSC removal. STI571 treatment does not modify the survival of Sca1-BCR-ABLp210 mice We also examined the effect of STI571 treatment in Sca1-BCR-ABLp210 mice. STI571 treatment began 1 day after leukaemia was confirmed by PB analysis. Mice were monitored clinically and by serial PB count for evidence of leukaemia. STI571 did not prolong the survival of these mice (Figure 4A) and Sca1-BCR-ABLp210 mice treated with STI571 did not demonstrate a marked reduction in WBC and spleen weight (Figure 4B). This idea is in agreement with the insensitivity of the human
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