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Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit Rev Oncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001 ARTICLE IN PRESS ONCH-1372; No. of Pages 20 Critical Reviews in Oncology/Hematology xxx (2010) xxx–xxx Neoplastic stem cells: Current concepts and clinical perspectives Axel Schulenburg a,d,, Kira Brämswig b , Harald Herrmann c,d , Heidrun Karlic d , Irina Mirkina c,d , Rainer Hubmann c,d , Sylvia Laffer d , Brigitte Marian e , Medhat Shehata c,d , Clemens Krepler d,f , Hubert Pehamberger d,f , Thomas Grunt b,d , Ulrich Jäger c,d , Christoph C. Zielinski b,d , Peter Valent c,d a Bone Marrow Transplantation Unit, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria b Division of Clinical Oncology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria c Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria d Ludwig Boltzmann Cluster Oncology, Vienna, Austria e Department of Internal Medicine I, Institute for Cancer Research, Medical University of Vienna, Vienna, Austria f Department of Dermatology, Medical University of Vienna, Vienna, Austria Accepted 6 January 2010 Contents 1. Introduction ........................................................................................................... 00 1.1. Identification of putative CSC .................................................................................... 00 2. Definition of CSC ..................................................................................................... 00 3. Limitations of in vivo CSC assays ....................................................................................... 00 4. In vitro assays ......................................................................................................... 00 5. Antigens commonly expressed on CSC .................................................................................. 00 6. General problems with the so-called ‘stem cell markers’ ................................................................... 00 7. Myeloid neoplasms .................................................................................................... 00 8. Lymphoid neoplasms .................................................................................................. 00 8.1. Acute lymphoblastic leukemia (ALL) ............................................................................. 00 8.2. Multiple myeloma ............................................................................................... 00 9. Solid tumors .......................................................................................................... 00 9.1. Head and neck squamous cell cancer .............................................................................. 00 9.2. Colon CSC ..................................................................................................... 00 9.3. Liver CSC ...................................................................................................... 00 9.4. Pancreas CSC ................................................................................................... 00 9.5. Breast CSC ..................................................................................................... 00 9.6. Brain tumor stem cells ........................................................................................... 00 9.7. Prostate CSC ................................................................................................... 00 9.8. Sarcoma ........................................................................................................ 00 9.9. Other solid tumors .............................................................................................. 00 10. Melanoma stem cells (MSC) .......................................................................................... 00 11. CSC plasticity—the Hydra Model of CSC development .................................................................. 00 12. Strategies for the successful elimination of CSC ......................................................................... 00 13. Concluding remarks and future directions ............................................................................... 00 Conflict of interest .................................................................................................... 00 Reviewers ........................................................................................................... 00 Corresponding author at: Department of Medicine I, Stem Cell Transplantation Unit, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Wien, Austria. Tel.: +43 1 404006085; fax: +43 1 404005701. E-mail address: [email protected] (A. Schulenburg). 1040-8428/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2010.01.001

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ARTICLE IN PRESSNCH-1372; No. of Pages 20

Critical Reviews in Oncology/Hematology xxx (2010) xxx–xxx

Neoplastic stem cells: Current concepts and clinical perspectives

Axel Schulenburg a,d,∗, Kira Brämswig b, Harald Herrmann c,d, Heidrun Karlic d,Irina Mirkina c,d, Rainer Hubmann c,d, Sylvia Laffer d, Brigitte Marian e, Medhat Shehata c,d,

Clemens Krepler d,f, Hubert Pehamberger d,f, Thomas Grunt b,d, Ulrich Jäger c,d,Christoph C. Zielinski b,d, Peter Valent c,d

a Bone Marrow Transplantation Unit, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austriab Division of Clinical Oncology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria

c Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austriad Ludwig Boltzmann Cluster Oncology, Vienna, Austria

e Department of Internal Medicine I, Institute for Cancer Research, Medical University of Vienna, Vienna, Austriaf Department of Dermatology, Medical University of Vienna, Vienna, Austria

Accepted 6 January 2010

ontents

. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001.1. Identification of putative CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Definition of CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Limitations of in vivo CSC assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. In vitro assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Antigens commonly expressed on CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. General problems with the so-called ‘stem cell markers’ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Myeloid neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Lymphoid neoplasms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.1. Acute lymphoblastic leukemia (ALL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008.2. Multiple myeloma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Solid tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.1. Head and neck squamous cell cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.2. Colon CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.3. Liver CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.4. Pancreas CSC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.5. Breast CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.6. Brain tumor stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

9.7. Prostate CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.8. Sarcoma. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 009.9. Other solid tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: Schulenburg A, et al. Neoplastic stem cells: Current concepts and clinical perspectives. Crit RevOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

0. Melanoma stem cells (MSC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001. CSC plasticity—the Hydra Model of CSC development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Strategies for the successful elimination of CSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author at: Department of Medicine I, Stem Cell Transplantation Unit, Medical University of Vienna, Währinger Gürtel 18-20,-1090 Wien, Austria. Tel.: +43 1 404006085; fax: +43 1 404005701.

E-mail address: [email protected] (A. Schulenburg).

040-8428/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.critrevonc.2010.01.001

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

bstract

Neoplastic stem cells have initially been characterized in myeloid leukemias where NOD/SCID mouse-repopulating progenitors supposedlyeside within a CD34+/Lin− subset of the malignant clone. These progenitors are considered to be self-renewing cells responsible for the inivo long-term growth of neoplastic cells in leukemic patients. Therefore, these cells represent an attractive target of therapy. In some lymphoideukemias, NOD/SCID mouse-repopulating cells were also reported to reside within the CD34+/Lin− subfraction of the clone. More recently,everal attempts have been made to transfer the cancer stem cell concept to solid tumors and other non-hematopoietic neoplasms. In several ofhese tumors, the cell surface antigens AC133 (CD133) and CD44 are considered to indicate the potential of a cell to initiate permanent tumorormation in vivo. However, several questions concerning the phenotype, self-renewal capacity, stroma-dependence, and other properties ofancer- or leukemia-initiating cells remain to be solved. The current article provides a summary of our current knowledge on neoplasticcancer) stem cells, with special emphasis on clinical implications and therapeutic options as well as a discussion about conceptual andechnical limitations.

2010 Elsevier Ireland Ltd. All rights reserved.

wsboebc(gwsltscdptmoatip(tIalAwic

eywords: Cancer stem cells; Targeted therapy; Drug resistance

. Introduction

The principle concept of cancer stem cells (CSC) hasained acceptance in recent years [1–4]. The CSC conceptostulates the existence of subfractions of “tumor stem cells”ithin each neoplasm. These CSC exhibit the capacity for

elf-renewal and unlimited growth, and in this regard dif-er from more mature neoplastic cells (progeny) that havenly a limited capacity to divide and to survive. The con-ept of tumor stem cells may provide explanations for theailure of certain treatments to induce long-term remission.n fact, in many instances, conventional chemotherapy mayct only on more mature cells, whereas immature neoplastictem cells exhibit resistance, so that these drugs fail to targetnd eliminate CSC [1–4]. Thereby, the CSC concept points tohe need to develop new treatment strategies through whichSC can be eliminated. A prerequisite for the evaluation ofSC as “target-cell” in oncology is their identification andnowledge about target expression profiles. Therefore, sub-tantial efforts have recently been made to identify CSC inarious types of cancer and to identify molecular targets andxpression profiles in these cells.

A number of different monoclonal antibodies directedgainst various cell surface antigens have been used to iden-ify CSC-enriched cell populations in various neoplasms, ando purify these cells for molecular and functional studies [5,6].hese experiments focus on the identification and character-

zation of molecular targets, and effects of natural ligands,esponse modifiers, and targeted drugs on these cells.

The current article provides a summary of our currentnowledge about cancer- and leukemia-initiating cells, withpecial focus on clinical implications and perspectives.

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

.1. Identification of putative CSC

The identification of CSC is usually based on differen-ial expression of cell surface antigens (markers) through

oof

hich subpopulations with variable capacity of long-termurvival can be detected and separated using monoclonal anti-odies. Such antibodies are directed against organ-specificr/and lineage-specific antigens or so-called ‘stem cell mark-rs’, and can be employed to enrich (separate) stem cellsy fluorescence-activated cell sorting (FACS) from primaryell samples [1–4,7]. The different subsets of cancer cellsputative stem cells and more mature cells) are then investi-ated for their capacity to repopulate immunodeficient miceith the tumor/leukemia (stem cell function). In fact, CSC

hould be able to reproducibly establish the original cancer oreukemia (all or most components of the disease) in a xeno-ransplant model (also in secondary recipient mice). In mosttudies, non-obese diabetic severe combined immunodefi-ient (NOD/SCID) mice have been used [1–4,8,9]. However,epending on the type of tumor, other mouse systems mayrovide an even better engraftment [10,11]. Despite limita-ions (non-human microenvironment, slowly growing tumors

ay not establish during the lifetime of mice), immun-deficient mouse models remain a widely used approachnd are considered the best available standard-model forhe identification of cancer- and leukemia-initiating cellsn primary tissue samples. Depending on the type of neo-lasm, primary neoplastic cells are injected intravenouslyleukemias, metastatic carcinomas), subcutaneously (skinumors, solid tumors), or directly into solid organs [12–17].n case of leukemias, NOD/SCID mice are usually irradi-ted sublethally in order to provide proper engraftment ofeukemic cells in the bone marrow cavities of mice [18,19].n unresolved question is whether and what cytokines andhat human microenvironmental cells are required to facil-

tate optimal engraftment and growth/survival of neoplasticells in various disease models.

tem cells: Current concepts and clinical perspectives. Crit Rev

After injection, tumor/leukemia cell growth is monitoredver several weeks. When a tumor or leukemia has devel-ped, the mouse is sacrificed and the neoplasm examinedor histologic and molecular features [1–4]. Key questions in

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ARTICLEA. Schulenburg et al. / Critical Reviews

ach experiment are whether the xenotransplant tumor indeedesembles the original neoplasm and whether indeed most orll elements (subclones) of the original neoplasm are found inhe xenotransplant tumor [2]. To further document long-termngraftment and thus to confirm the stem cell function of CSCn xenotransplant tumors, these tumors can be recovered from

ouse tissues and can be transplanted into secondary recipi-nt mice, where self-renewing CSC should again form tumoresions and all components of the primary tumor/leukemia asell as a new CSC pool [1,2]. It is important in each project toemonstrate that the more mature cells are unable to repop-late leukemias/tumors in the same mice [20] which may beifficult to demonstrate in slowly growing/developing neo-lasms, as the time of development of the human neoplasmay exceed the lifetime of the mouse. Moreover, in such neo-

lasms, it may be difficult to delineate between engraftmentf “real” stem cells (CSC) and the persistence of more maturerogenitors that have only a limited capacity to divide. There-ore, in these neoplasms (e.g. chronic leukemias), it may bef particular importance to confirm engraftment and growthf neoplastic cells in secondary recipient mice in order toearn whether these cells exhibit or lack stem cell function.nother important question is whether engrafted cells indeed

re derived from neoplastic stem cells (CSC) or derive fromormal stem cells.

. Definition of CSC

Cancer/leukemia stem cells (CSC) are undifferentiatedells and are defined by three key features [1,21]: first, theseells can differentiate into most or all types of cells that areroduced by the original tumor. Second, CSC have the abilityo self-renew. Finally, CSC maintain the stem cell pool and

ost (or even all) mature elements of the tumor/leukemia fornlimited time periods by balancing between self-renewalproliferation without maturation) and proliferation plus dif-erentiation and maturation by asymmetrical cell division(s)1,4]. The process(es) of cell division, of self-renewal, andf differentiation of CSC are considered to be regulated bynetwork of cytokines and by the microenvironment, simi-

ar to normal stem cells [1,4]. In many instances, the sameytokine that regulates growth of normal (stem) cells in a cer-ain organ will also promote the growth and self-renewal ofeoplastic stem cells [1,4].

. Limitations of in vivo CSC assays

Despite the obvious value of an in vivo model that isufficient to demonstrate the tumor-initiating potential ofistinct subpopulations of neoplastic cells, a number of lim-tations of the xenotransplant assay have to be considered.

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

irst, neoplasms with a low growth rate (e.g. indolent neo-lasms, low-grade malignancies, preneoplasm) are difficulto analyze in a mouse xenotransplant model as in mostnstances, the development phase of the neoplasm exceeds

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PRESSology/Hematology xxx (2010) xxx–xxx 3

he lifetime of the mouse. Second, the microenvironment issually species-specific and often tumor-specific. In fact, theicroenvironment of the normal mouse may differ in several

spects from the tumor microenvironment that supports therowth of neoplastic (stem) cells in the natural (human) host.ikewise, microenvironment receptors and cytokines in theouse may not cross-interact in all cases with the respec-

ive receptors expressed on human tumor/leukemia (stem)ells. To overcome this problem, NOD/SCID mice have beenreated with human cytokines or are cotransplanted with

icroenvironmental cells in order to facilitate better growthf neoplastic (stem) cells [9]. Another important limitations that most mouse models that have been used in the past,ncluding NOD/SCID mice, harbour a residual immune sys-em through which these mice can eliminate subfractions ofnjected cancer/leukemia cells, especially when these cellsisplay numerous immunogenic antigens (more mature cells)r when cells are antibody-laden (antibody-stained) cells.he same cells may, however, grow and form tumors inice with a more severely impaired immune system [22,23].inally, most neoplasms may grow in permanently estab-

ished subclones with subclone-specific stem cells. In otherords, the stem cell pool in most neoplasms is composed of

everal (many) different subsets of stem cells with varyingiological properties and diverse growth characteristics, andt remains unknown whether all (relevant) subclones can berown in one xenotransplant model.

. In vitro assays

As stem cell research using NOD/SCID mice is expen-ive and time-consuming and may have several limitations,n vitro long-term growth assays are often used in order tocreen for stem cell fractions or CSC-regulating compounds.uch in vitro long-term growth assays have been establishedor myeloid and lymphoid neoplasms as well as various solidumors [24–28]. Interestingly, in most instances, a stromaell layer supports the long-term growth of immature neo-lastic cells in these assays, which is in line with the concepthat the (tumor) microenvironment essentially contributes torowth and survival of CSC and thus the development andiology of these neoplasms (stem cell niche). In myeloid neo-lasms, the Dexter type long-term culture system, originallystablished for normal pluripotent hematopoietic progenitorells [29] has been employed and found to support the long-erm growth of leukemias [30]. However, interestingly, somef these leukemias may even have a “growth-disadvantage”ompared to normal stem cells (long-term culture initiatingells) in these cultures [31]. In lymphoid neoplasms, includ-ng CLL and ALL, it is well known that stromal cells cannhibit apoptosis and support the growth of leukemic cells in

tem cells: Current concepts and clinical perspectives. Crit Rev

itro [32,33]. In various solid tumors and melanomas, iso-ated cells are found to form three-dimensional spheres thatresumably are composed of immature neoplastic cells andupporting/nutritive stromal cells, and thus may resemble an

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ARTICLEA. Schulenburg et al. / Critical Reviews

n vitro model of the so-called stem cell niche [34]. Suchphere formation has been described for neural cancer (stem)ells, especially glioblastomas, colon cancer, breast cancer,nd melanoma cells [24–28]. The culture system is based onhe ability of cancer cells to form three dimensional spheresn vitro, and the ability of sphere-derived cells to induce long-erm growth of tumors [24–28]. In fact, at least some ofhe tumor cells within these spheres are considered to haveelf-renewal capacity and tumor-initiating (CSC) potential.

. Antigens commonly expressed on CSC

Neoplastic stem cells are considered to express a simi-ar antigen pattern, to display similar functional properties,nd to be regulated by similar receptor ligands when com-ared to normal stem cells (derived from the same organystem). Therefore, many stem cell/progenitor cell markersre also markers of neoplastic stem cells. These antigensnclude cytokine receptors, homing receptors, and variousrug transporters (Table 1).

To identify potential surface markers of CSC, it iselpful to look for interactions of these cells with the sur-ounding microenvironment. Based on the behaviour oformal stem cells, CSC should interact with the supportingicroenvironment via several biologically relevant surface

eceptors mediating specific functions. One specific stem cellunction related to the microenvironment is “stem cell hom-ng” [35–38]. Major homing receptors discussed as beingxpressed on CSC are integrins, selectin-ligands, chemokineeceptors, other cytoadhesion molecules (CAMs), and lig-nds of matrix molecules such as L1 or the hyaluroniccid receptor CD44 [39,40]. L1 is also involved in thepithelial–mesenchymal transition (EMT) and is found onhe edge of invasive colon cancer and its metastases [41,42].ogether with L1, CD133 appears to be necessary for tumorrowth of gliomas [43]. CD133 and CD44 show overlappingxpression in various tumors and CSC [26,44,45]. CD133, alycoprotein also known as Prominin 1 (PROM1), is a mem-er of the pentaspan family of transmembrane glycoproteins5-transmembrane, 5-TM) which specifically bind to cellularrotrusions [46–48]. CD133 is expressed in hematopoietictem cells, endothelial progenitors, as well as on glioblas-oma, colon CSC, and other solid tumor CSC [46–48].he CD44 protein is a cell-surface glycoprotein involved

n cell–cell interactions, adhesion, migration, and homing49]. It is a receptor for hyaluronic acid and can also inter-ct with other ligands, such as osteopontin, collagen species,nd matrix metalloproteinases (MMPs) [49]. A specializedialofucosylated glycoform of CD44, called HCELL, is usu-lly expressed on human hematopoietic stem cells, and is aighly potent E-selectin and L-selectin ligand [50]. HCELL

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

unctions as a “bone marrow homing receptor” directingigration of human hematopoietic stem cells and mesenchy-al stem cells to the bone marrow [50]. HCELL is expressed

n breast-, prostate-, pancreatic-, and colon CSC, and is reg-

itcA

PRESSology/Hematology xxx (2010) xxx–xxx

lated by the WNT pathway [13,16,45,51]. The frequentlybserved expression of CD44 and CD133 on CSC suggestsfunctional role for these receptors in CSC biology. How-

ver, it remains unknown whether these receptors are indeedecessary for stem cell functions or are just expressed onSC without a specific function. Whatever the answer to thisuestion is, these markers are commonly found on immatureeoplastic cells (CSC) and may thereby help in the identifi-ation of CSC in various organs and tumors. More recently,t has been reported that in a colon adenoma strain, LT97,he CD44+ subpopulation exhibits a faster growth rate andxpresses higher levels of other stem cell antigens (suchs Musashi-1 and EphB2) compared to the CD44-negativeraction of adenoma cells [52]. Similarly, the CD133+ frac-ion of the HCT116 colon cancer cell line has recently beenescribed to display higher levels of stem cell antigens andfaster growth rate in vitro and in vivo compared to theD133− fraction [53].

Another basic requirement for CSC is to protect them-elves against various external toxic stimuli and drugs, whichakes them often drug-resistant. One example for such a

rug transporter protein is ABCG2 which is able to pumput not only cell-specific substances but also exogenous tox-ns and cytotoxic drugs, and thus is responsible (in part) foresistance of CSC against various drugs. Other drug trans-orters that have been discussed as indicating the long-termepopulation-potential of malignant cells include MDR-1nd ABCB5 [54,55]. Various specific dyes like the Hoechst3342 dye, are also transported from the cell into the extra-ellular space via specific transporters, which allows theefinition of the so-called “side population” which seemslosely related to the stem cell fraction in many tumors56]. A similar distribution and association with stem cellsas been described for the enzyme aldehyde dehydrogenasealdolase) [57–61]. This enzyme has been described to benvolved in the metabolization of several cytostatic drugsncluding cyclophosphamide and thereby may be associatedith chemoresistance [60,62,63].Lastly, CSC are considered to respond to various exter-

al physiologic (sometimes paracrine or autocrine) stimulincluding various cytokines. In line with this assumption,SC express certain cytokine and chemokine receptorsn their cell surface. For example the IL-3 receptorCD123/CD131), SCF receptor (KIT) and the G-CSF-eceptor are usually expressed on leukemic CSC in AML64,65]. It has also been described that EGF receptor fam-ly members including HER2 are expressed on epithelialSC including mammary CSC [66,67]. There is also evi-ence that IGF- and FGF-receptors play an important rolen solid tumors and may be expressed on solid tumor CSC68,69]. More recently, it has been described that pancreaticSC express CXCR4 [12]. A clinically important question

tem cells: Current concepts and clinical perspectives. Crit Rev

s whether solid tumor CSC or leukemic CSC express recep-ors for erythropoietin (EPO), G-CSF, or GM-CSF, as theseytokines are often applied in cancer/leukemia patients [70].t least for AML and some tumor cell types it has been

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Table 1Cell surface molecules detectable on stem and progenitor cells.

Marker/antigen Background and function of marker/antigen

CD133 CD133 antigen is a transmembrane molecule expressed on hematopoietic stem and progenitor cells, on circulatingendothelial progenitor cells, neural stem cells, renal and prostate stem cells [215].

CD34 CD34 is an adhesion molecule expressed on human hematopoietic stem and progenitor cells, endothelial progenitorcells and vascular endothelial cells [142].

CD44 CD44 is a cell adhesion receptor, and its ligands are hyaluronate and the cytokine osteopontin. CD44 is expressed inlymphoid, mieloid and erythroid cells, mesenchymal stem cells and may be useful predictor of lymph node metastasis[1].

CD29 CD29 is a beta1 integrin involved in cell adhesion embryogenesis, tissue repair, immune response and metatastaticdiffusion of tumor cells reacting with thrombocytes, monocytes and a T and B lymphocytes and is also expressed onmesenchymal stem cells [216].

CD24 CD24 antigen is a glycosylphosphatidylinositol-linked membrane sialoglycoprotein. CD24 is present on B cells, fromthe stage pre-B to the mature B cell stage, but not on plasma cells. It is expressed on mature granulocytes and on avariety of epithelial cell types [1].

CD166 CD166 molecule, a mesenchymal stem cell marker that displayed heterogeneous expression patterns in CRC epithelialcells (15, 16) and whose increased expression levels were previously associated with poor clinical outcome in CRCpatients [45].

CD326 EpCAM (CD326) is a pan-epithelial differentiation antigen expressed on the basolateral surface of various carcinomasto varying degrees. As a homotypic cell adhesion molecule, it is intimately integrated within the Cadherin–Catenin andWNT pathways. It has recently been shown to modulate the expression of proto-oncogenes such as c-myc [217].

CD90 Thy-1 (CD90) is expressed on many cell types, including T cells, thymocytes, neurons, endothelial cells, and fibroblasts.Activation of Thy-1 can promote T cell activation, and this role of Thy-1 is reviewed elsewhere. Thy-1 also affectsnumerous nonimmunologic biological processes, including cellular adhesion, neurite outgrowth, tumor growth,migration, and cell death [218].

CD123 The specific [alpha] subunit of the interleukin-3 receptor (IL-3R-alpha, CD123) is expressed on hematopoietic cells,including monocytes, neutrophils, basophils, and megakaryocytes but not on peripheral T cells, natural killer cells,platelets, and red blood cells [219].

CD9 CD9 belongs to a tetraspanin superfamily and is expressed in a variety of blood cells including pre-B lymphocytes butnot in HSCs. It is also expressed in many types of solid tumors, and is involved in a various kinds of cell processes, suchas cell adhesion, motility, and signalling events through an association with integrin family proteins [220].

CD20 CD20 (human B-lymphocyte-restricted differentiation antigen, Bp35), is a hydrophobic transmembrane protein with amolecular weight of approximately 35 kDa located on pre-B and mature B lymphocytes. The antigen is expressed onmost B-cell non-Hodgkin’s lymphomas but is not found on stem cells, pro-B cells, normal plasma cells or other normaltissues. Plasma blasts and stimulated plasma cells may express CD20. CD20 regulates an early step(s) in the activationprocess for cell cycle initiation and differentiation, and possibly functions as a calcium ion channel. CD20 is not shedfrom the cell surface and does not internalize upon antibody binding. Free CD20 antigen is not found in the circulation;thus a drug that reacts with CD20, such as an antibody, would not be neutralized before binding to its target cell [221].

ABCB1 (MDR1) and ABCG2 ABCB1 and ABCG2 are recognized as belonging to a family of at least 48 human ABC transporters involved in avariety of essential cellular transport processes. They are the products of MDR genes and confer multidrug resistance bypumping out chemotherapy. They also pump out Hoechst dye and rhodamine. Reversal of MDR in vitro was easilyattained with a variety of inhibitors like verapamil [222].

ABCB5 ABCB5 [subfamily B (MDR/TAP)] is a novel human ABC transporter encoded on chromosome 7p15.3 [223]. ABCB5,like ABCB1, acts as an energy-dependent drug efflux transporter for the fluorescent probe rhodamine-123 [224].

CLL-1 Human CLL-1 (also known as MICL or CLEC12A), is a type II transmembrane glycoprotein and member of the largefamily of C-type lectin-like receptors involved in immune regulation. The intracellular domain of CLL-1 contains bothan immunotyrosine-based inhibition motif as well as a YXXM motif, suggesting a role for CLL-1 as a signallingreceptor [86,225].

Erythropoietin receptor The erythropoietin receptor (EpoR) consists of two peptide chains and is a member of the cytokine receptor family. Theinteraction of erythropoietin with its cell surface receptor induces a conformational change of receptor homodimers

r signand surv

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leading to the activation of intracellulaproliferation, terminal differentiation a

escribed that CSC indeed express receptors for SCF, G-CSF,nd sometimes also for GM-CSF [65].

. General problems with the so-called ‘stem cell

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

arkers’

One general problem is that the so-called stem cell mark-rs are by far not specific for stem cells or progenitor cells.

[Cet

l transduction that mediates the ability of erythropoietin to support theival [226].

ather, most of these antigens are broadly expressed on var-ous mesenchymal cells. Likewise, CD44 is expressed notnly on hematopoietic and non-hematopoietic stem cellsut also on most mature cells, including monocytes, lym-hocytes, granulocytes, epithelial cells, and melanocytes

tem cells: Current concepts and clinical perspectives. Crit Rev

50,71]. Similarly, in most leukemias and solid neoplasms,D44 is expressed on mature cells. Prominin (CD133) isxpressed on myeloid progenitor cells and on various (imma-ure and mature) mesenchymal cells, including endothelial

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ells [46–48]. Even the CD34 antigen (hematopoietic pre-ursor cell antigen-1, HPCA1) is not only expressed onematopoietic stem cells but also on more mature progen-tors and also on endothelial cells [72]. All in all, no stemell specific antigen has been identified so far. Therefore, its essential to apply combinations of markers and antibodiesnd to define stem cell-enriched fractions on the basis of typ-cal antigen combinations. Usually, one or two markers aremployed by investigators to gate for or to exclude a germayer or an organ system (e.g. CD45 as pan-hematopoietic cell

arker). In certain instances a cocktail of antibodies is usedo exclude more mature cells in a certain organ system, e.g.

ore mature hematopoietic cells by lineage-specific mark-rs (“Lin-cocktail” defining Lin-negative progenitor cells)19,73]. Here, one problem may be that in certain leukemias,SC may aberrantly express lineage-related marker antigens.

n these leukemias, application of the “Lin-cocktail” wouldead to a loss of CSC (sub)fractions.

Finally, markers that are typically expressed on immatureells of a given neoplasm or organ, are applied, e.g. CD34 forymphohematopoietic cells and most myeloid leukemias. Inhese defined progenitor fractions, further subpopulations inhich CSC reside, are defined. This is performed by antibod-

es that positively or negatively identify these subfractions.ased on antibody-binding patterns and identification of sub-

ractions enriched in CSC, these cells can be isolated byulti-color flow cytometry and cell sorting. The read out that

s then used to confirm the presence of CSC is either an initro long-term culture system or – preferably – the immun-deficient mouse model. In most instances, the NOD/SCIDouse has been employed in these assays. However, there arenumber of caveats that have to be considered when using

orted cells in these mouse models and bioassays. The mostmportant caveat may be that the antibody itself may stimu-ate or may interfere with in vitro growth, engraftment, or/andong-term repopulation of progenitor cells in NOD/SCID

ice [22]. Therefore, it has to be excluded by appropriateontrol experiments, that the antibodies used to positivelyr negatively define CSC would per se induce or inhibitngraftment of CSC in NOD/SCID mice, or would interfereith in vitro CSC growth in the bioassay applied or with

n vivo engraftment [22]. Recently, Taussig et al. revealed arocedure-related problem in the description of AML CSCefined by expression of CD34 and lack of CD38 [22]. In theiraper, they were able to show that the original descriptionf leukemic stem cells as CD38-negative cells in the mouseodel may be due to the clearance of leukemic CSC togetherith the CD38 antibody by the residual immune system ofOD/SCID mice. Clearance was not observed when miceere further immunosuppressed by drugs or when the anti-ody was degraded into fab fragments which allowed CD38+eukemic cells to repopulate these mice [22]. Moreover,

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

D34+/CD38+ grew well in more severely immunodeficientL2rgamma(null) mice (NSG mice) [22]. All in all, it appearshat NOD/SCID mice may not be the most suitable strain totudy CSC growth in solid tumors and leukemias. Rather,

np

PRESSology/Hematology xxx (2010) xxx–xxx

ore severely immunodeficient mice (NOG or NSG mice)ay be required for optimal engraftment of CSC [10,11]. It

an therefore be expected that stem cell research will employOG or NSG mice in various CSC models in the future.Recently, Kim et al. found that a polyclonal anti-human

D24 rabbit antibody initiates cross-linking of CD24 onumor cells, and that this cross-link induces apoptosis inreast cancer cells in a cell culture system using MCF-7ells [74]. This may have implications for the characteriza-ion of breast cancer stem cells, since these cells supposedlyeside within the CD44+CD24low (CD24-negative) fractionf tumor cells. If CD24+ tumor cells would just be elimi-ated (faster than CD24low cells) by the antibody-inducedross-linking of CD24, the CD24-negativity could no longere regarded as a stem cell-related feature. Whether indeedD24-induced stem cell depletion occurs after antibody-inding remains to be shown.

Another major limitation may be that CSC receptors andheir ligands and homing receptors in the tissue environmentre sometimes species-specific. Therefore, CSC are oftennjected directly into a certain target organ (e.g. bone marrow,ancreas, brain, others) in these mice [12–17,44,75,76]. Nev-rtheless, it is difficult to know whether some of the CSC willot (cannot) grow in a xenotransplant model simply becausessential mouse-derived ligands are not cross-reacting withuman receptors expressed on CSC. A possible solution tohis problem may be to co-transplant human stroma cells,o humanize mice (organ-specific microenvironment), or tonject mice with all important (human) ligands in order touarantee proper engraftment of CSC.

Probably the most important problem with CSC markersnd CSC-reactive antibodies is stem cell plasticity. In fact,t has been described that leukemic and solid tumor CSCractions are usually composed of several different subclonesefined by varying profiles of molecular markers, point muta-ions in critical target genes (often causing drug resistance inubclones), and expression of cell surface receptors includingtem cell markers. This means, that CSC in a given tumor mayisplay varying combinations of cell surface antigens whichakes it difficult to define all CSC subfractions (subclones)

y monoclonal antibodies. In some instances, the phenotypiceterogeneity may also be associated with functional hetero-eneity. However, so far very little is known about cell surfacentigens defining subfractions of CSC in various solid tumorsnd leukemias. Likewise, in AML, leukemic CD34+/Lin−stem) cells are often composed of a CD133+ and a CD133−ubfraction [77].

In the following paragraphs, we review human neoplasmshere CSC have recently been identified.

. Myeloid neoplasms

tem cells: Current concepts and clinical perspectives. Crit Rev

It is generally assumed that most if not all myeloideoplasms derive from a clonal immature hematopoieticrogenitor (stem) cell. Therefore, myeloid neoplasms are

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ptimal models to study the CSC hypothesis. Normalematopoietic stem cells are considered to reside withinhe Lin-negative and CD38-negative portion of CD34-ositive progenitor cells. Leukemic stem cells have firsteen described in AML, and later in CML [19,73,78]. Inther myeloid neoplasms, neoplastic stem cells are less wellefined. The phenotype of leukemic stem cells is consideredo be similar to that of normal stem cells (Lin−, CD34+,D38− and in part CD38+) [8,19,79] (Table 2). Severalttempts have been made to identify markers that distin-uish between normal and neoplastic meyloid stem cells.his would enable therapeutic approaches that might beble to spare normal stem cells [80]. Indeed, several surfaceolecules may be expressed abundantly on CD34+/Lin−ML stem cells, whereas normal hematopoietic stem cells

ack or express only low levels of these antigens. Exam-les for antigens preferentially expressed on leukemic CSCn AML are the alpha chain of the IL-3 receptor (CD123),he Mylotarg-receptor Siglec-3 (CD33), CD96, CXCR4, andLL-1 [64,81–86]. CD33 is of special interest as CD33-

argeting drugs are available and are used to treat patientsith (refractory) AML. Fig. 1 shows the effects of Mylotargn survival of CD34+/CD38+ and CD34+/CD38− AMLells. Neoplastic stem cells have been defined in severalut not all variants of AML [8,19]. Likewise, in a groupf AML patients, leukemic blast cells and immature pro-

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

enitors are CD34-negative. In these patients, it is veryifficult to define stem cell compartments. Other examplesre promyelocytic leukemia, NPM-mutated AML variants,nd monoblastic leukemias where most of the neoplastic cells

laoi

able 2ublished cell surface phenotype of neoplastic stem cells in various malignancies.

isease Surface marker

ML CD34+, CD38−, CD44, CD123+LL Ph+ CD34+, CD38−EL-AML1–positive-ALL

CD34+/CD38−/low/CD19+CD34+/CD19+

hildhood B-ALL CD133(+)/CD19(−) and CD38(−lineage ALL CD34+/CD9+ML CD34+, CD38−, CD123+reast CD44+, CD24−/low, ESA+

ancreasCD44+CD133+

iverCD133+CD90+

olonCD133CD44

rostateCD133+/alpha 2 beta 1 integrin/CCD44+/CD24−

arcoma CD133+

elanomaCD20+CD133+

NSCD133+

PRESSology/Hematology xxx (2010) xxx–xxx 7

ay be CD34-negative cells [87]. In many of these leukemiast even remains to be demonstrated that AML stem cells resideithin the (small) CD34+ subpopulation of clonal leukemic

ells. It is also important to note that AML types in which thetem cell origin of the leukemia can be demonstrated using aOD/SCID mouse xenotransplant model are those variantshere blast cells exhibit a high proliferative potential and

esistance against chemotherapy [88].In CML, most clonal cells are CD34-negative cells. In

hese patients, a complex multi-step enrichment techniqueor the isolation of putative CD34+/Lin− stem cells has beenroposed [73]. Indeed, when these cells were injected intoOD/SCID or NOD/SCID-beta2microglobulin−/− mice,

hey were found to repopulate these mice with a CML-likeisease. By contrast, more mature CD34+ CML cells onlyroduced an early transient leukemic repopulation that waso longer detectable after 6 weeks [89]. Similar to the AMLtem cell, the CML stem cell was found to express the IL-receptor [90]. Other target antigens like CD33, CD44, orD117 were also found to be expressed on CD34+/CD38−

tem cells in CML patients [64]. CML stem cells alsoxpress ABCB1 and ABCG2, whereas the levels of OCT-expressed on CML stem cells are rather low [55,91]. BothBCG molecules are known to mediate the efflux of ima-

inib and other drugs and thus confer resistance. OCT-1,n the other hand, mediates the uptake of imatinib, and

tem cells: Current concepts and clinical perspectives. Crit Rev

ow expression of these transporter molecules is associ-ted with low drug-uptake and thus low intracellular levelsf imatinib. All these mechanisms may contribute to thentrinsic resistance of CML CSC against imatinib [55,92].

Refs.

Bonnet and Dick [19]Cobaleda et al. [105]

Hong et al. [106]Kong et al. [107]

) Cox et al. [108]Nishida et al. [109]Holyoake et al. [73]Al-Hajj et al. [13]

Li et al. [16]Hermann et al. [12]

Ma et al. [131]Yang et al. [17]

Ricci-Vitian et al. [26], O’Brien et al. [44]Dalerba et al. [45]

D44+ Collins et al. [142]Hurt et al. [51]

Suva et al. [151]

Fang et al. [24]Monzani et al. [162]

Singh et al. [15]Hemm et al. [27]

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F obtaine( at 37 ◦c he perca vital (7-

MbBIlfigalesistmgksbmstawp

nC[

tevdmsscJfmiomipb

ig. 1. Apoptosis in AML stem cells induced by Mylotarg (GO). Blast cellsupper graphs) or with gemtuzumab/ozogamicin (GO = Mylotarg), 1 �g/ml,ytometry using antibodies against CD34 and CD38, and AnnexinV-FITC. Tnd in the CD34+/CD38− fraction of the clone (left panel) after gating for

ore recently, imatinib-resistance of CML stem cells haseen employed to screen for stem cell-related markers inCR/ABL transformed (imatinib-exposed) murine cells [93].

n these studies, several antigens potentially involved ineukemic stem cell growth and survival have been identi-ed. One of these CML stem cell-related genes is the Alox5ene that encodes the 5-lipoxygenase (5-LO) [87]. In thebsence of Alox5/5-LO, BCR/ABL failed to induce a CML-ike disease in mice, whereas Alox5-deficiency showed noffects on growth of normal stem cells [93]. These datauggest that growth and survival of leukemic stem cellsn CML are regulated by specific gene products, and theame may hold true for AML. Also, recent data suggesthat neoplastic stem cells in various myeloid neoplasms

ay use similar if not identical signalling pathways forrowth and survival in vivo. These pathways include the PI3inase-mTOR pathway, WNT-�-catenin pathway, and Notchignalling-pathway [94]. However, all these pathways maye shared by normal and leukemic stem cells, and thereforeay not be optimal targets, at least when treatment should

pare normal (stem) cells. Also, Hedgehog signalling seems

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

o be important for the development of myeloid leukemiasnd may be a promising target pathway [95]. Other path-ays found in leukemic stem cells are the JAK2-STAT5athway and NF-kappa-B pathway. In most instances, sig-

pct[

d from a patient with AML (FAB M0) were incubated with control mediumC for 48 h (lower graphs). Then, apoptosis was analyzed by combined flowentage of apoptotic cells was analyzed in CD34+/CD38+ cells (right panel)AAD negative) cells.

alling is initiated by specific oncoproteins, like BCR-ABL inML, or PML-RAR-alpha in acute promyelocytic leukemia

96–100].In other myeloid neoplasms, very little is known about

he biology and phenotype of neoplastic stem cells. Floriant al. examined the phenotype of CD34+/CD38− cells inarious myeloid neoplasms including myelodysplastic syn-romes, CML, and systemic mastocytosis [64]. In all theseyeloid neoplasms, the phenotype, determined by antibody-

taining, appeared to be very similar, and included majorurface targets such as CD123 and CD33 [64]. In classi-al JAK2 V617F-mutated myeloproliferative disorders, theAK2 mutant is usually detectable in the CD34+/CD38−raction of clonal cells [101,102]. However, JAK2 V617Fay not be detectable in all neoplastic stem cell subclones

n these patients [103], and leukemic progression (sec-ndary AML) is often accompanied by a loss of the JAK2utant, which may be explained by expansion of a more

mmature JAK2 V617F-negative subclone during diseaserogression [103]. Otherwise, very little is known about theiology, target expression profiles, and phenotypes of neo-

tem cells: Current concepts and clinical perspectives. Crit Rev

lastic stem cells in JAK2+ MPN. Recently, a defective stemell niche has been discussed as an important factor con-ributing to the pathogenesis of JAK2-mutated neoplasms104].

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. Lymphoid neoplasms

.1. Acute lymphoblastic leukemia (ALL)

Little is known about subpopulations of CD34+ ALLells that display stem cell function. In 2000, Cobaleda etl. showed that in Ph+ (BCR/ABL+) ALL, the NOD/SCIDouse-repopulating ALL stem cell resides within theD34+/CD38− fraction of the clone, similar to AML

tem cells [105]. When ≥100 CD34-positive/CD38-negativeells were injected intravenously, leukemias developed after–6 weeks in these mice [105]. Hong et al. recentlyescribed that the CD34+/CD38−/low/CD19+ cells in TEL-ML1–positive c-ALL (0.002% of total mononuclear cells)ropagate leukemias in NOD/SCID mice [106]. In contrast,ong et al. found that CD38 is not useful for the identi-cation of a c-ALL stem cell [107]. They observed thatD34+/CD38−/CD19+ as well as CD34+/CD38+/CD19+ells are able to establish leukemias after 4–15 weekshen at least 5 × 103 cells were intravenously injected intoOD/SCID/IL2r null mice [107]. Recently, Cox et al. found

vidence that the pediatric B-ALL stem cell resides withinCD133+/CD19− subpopulation [108]. After injection of

43–50,000 CD133+/CD19− cells into NOD/SCID mice,ngraftment was observed after 8–10 weeks [108]. Anotherotential B-ALL stem cell marker may be CD9. Nishida etl. injected at least 1 × 104 CD9+ (cultured) ALL cells intra-enously into a total of 20 NOG mice, and all mice diedithin 45 days due to leukemia [109]. Despite the com-on notion that the ALL stem cell should be CD34+ cells,

his may not hold true for all B-lineage ALL variants andlso not for T ALL [110,111]. Recent data suggest that theotch pathway may be involved in CSC function in patientsith T-ALL [112]. Notch is already known to play a role inormal T-cell development and T-ALL [113–116]. Whereasn ALL, at least first attempts have been made to identify

leukemic stem cells, there is very limited if any infor-ation about NOD/SCID mouse-repopulating cells derived

rom other lymphoproliferative neoplasms, such as chronicymphocytic leukemia (CLL) or other Non-Hodgkin’s Lym-homas (NHL) [117]. In one study, Nowakowski et al. found amall CD5+/CD19+/ABCG+ population of CLL cells [111].rom gene expression profiling data and an increase of thisubpopulation of CLL cells after therapy, the authors con-luded that these cells may have stem cell like properties118]. More recent data suggest that the CLL cell with stemell-like properties may also reside within a small CD34+ubset of leukemic cells that co-express CD19 and CD5unpublished observation).

.2. Multiple myeloma

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

Matsui et al. were the first to report on the existence ofyeloma-initiating stem cell fractions in multiple myeloma

atients [119]. They showed that that CD138+ fraction ofyeloma cells cannot undergo long-term proliferation but

wamd

PRESSology/Hematology xxx (2010) xxx–xxx 9

rise from clonogenic CD138-negative B cells [112]. In par-icular, CD138− cells were found to act clonogenic in vitrond to produce myelomas in NOD/SCID mice [119]. In008 it was found that a combination of dexamethasone,enalidomide, bortezomib, and 4-hydroxycyclophosphamidenhibits the growth of CD138+ multiple myeloma cells butot the growth of CD138-negative myeloma precursor cellsn vitro. Several lines of evidence suggest that the phenotypef myeloma stem cells is similar to that of normal memorycells (CD138−/CD20+/CD27+) [119–121].

. Solid tumors

.1. Head and neck squamous cell cancer

Despite combination therapy head and neck squamous cellancer (HNSCC) remains one of the most difficult challengesn oncology. HNSCC resistance to various drugs has limitedhe usefulness of chemotherapy in this disease. Recently,D44 has been identified as a potential marker of CSC inNSCC [122]. When 5 × 103 CD44-positive poorly to wellifferentiated primary HNSCC cells were injected subcuta-eously into NOD/SCID mice or Rag2�DKO mice, theseells gave rise to tumors within 10–16 weeks [122]. Thetem cell-related gene BMI-1 was demonstrated to be overex-ressed in the CD44+ subpopulation of HNSCC tumor cellsompared to CD44− cells [122]. In vitro experiments per-ormed with various types of cancer cells suggest that CD133ay also be a marker for long-term proliferating cells, but

t remains unknown whether CD133 is indeed a stem cellarker for HNSCC [123].

.2. Colon CSC

Since the gastrointestinal tract is an organ-system withhigh turnover of cells where proliferation and cell self-

enewal take place, it was of great interest to learn abouthe location, biology, and phenotype of colon CSC. Sev-ral lines of evidence suggest that immature colon cells (andresumably also CSC) are located in colon crypt bottoms.ith regard to the phenotype, first reports pointed to CD133

s a potential CSC marker antigen colon cancer [26,44]Table 2). When CD133-positive cells were injected intohe kidney capsule or subcutaneously into NOD/SCID mice,olon tumors developed after several weeks [26,44]. Later,alerba et al. found that CD44 is another potential marker forSC in colon cancer patients [45]. When CD44+/EpCAM+ells were injected subcutaneously into NOD/SCID miceumors developed after 20 weeks in these mice [45]. Fur-hermore, they found that the CSC population is furtherharacterized by coexpression of CD166 [45]. Recently, it

tem cells: Current concepts and clinical perspectives. Crit Rev

as reported that CD133+ cells derived from colon spheresre clonogenic cells and can form adenocarcinomas in aouse xenotransplant model [28]. These CD133+ sphere-

erived cells in part co-expressed CD24, CD29, CD44, and

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D166. Interestingly, of all markers tested, only CD24 wasound to enrich for cells with even higher clonogenic activ-ty. All in all, several surface markers, including CD44 andD133 have been discussed as potential markers of colonSC (Table 2). With regard to CD44, this may also hold true

or colon adenomas. In fact, it has been shown that the colondenoma cell strain LT97 consists of a CD44+ and a CD44−ortion, and that both subfractions differ in their growthinetics and expression of stem cell markers [52]. In particu-ar, CD44-positive LT97 cells attach and grow for unlimitedime periods, whereas CD44− cells are slowly growing cellsFig. 2) [52]. Moreover, in contrast to CD44− LT97 cells,D44+ LT97 cells display nuclear beta-catenin and expresseta-catenin target genes, such as ephrin B receptor (ephB2)nd the musashi1 antigen (msi1) [52]. These stem cell anti-ens are also detectable in colon CSC [124]. All in all, CD44ay not only be a CSC marker for established colon ade-

ocarcinomas, but also for CSC in preneoplastic lesions, i.e.olon adenomas which may have implications for the biol-gy of tumor development. In fact, colon carcinoma CSCay develop from adenoma CSC by subclone selection after

ccumulation of further hits [125,126].

.3. Liver CSC

A number of previous studies suggest that hepatic can-er cells derive from immature progenitor cells in theiver [127]. There are four candidates for liver CSC: bone

arrow derived cells, oval cells, hepatocytes, and hep-topancreatic stem cells [128]. Recent data suggest thathe so-called small oval cells in hepatocellular carcinomas

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HCC) are involved in the carcinogenic process and dis-lay stem cell markers [129,130]. Since these small ovalells are also found in the normal liver, the hypothesisas raised that HCC arise from normal liver progenitor

ig. 2. Correlation between expression of CD44 and proliferative poten-ial of LT97 cells. The adenoma cell strain LT97 was cultured in complete

edium and passaged once every 10–14 days. Expression of CD44 on LT97ells was determined by flow cytometry in early (passage up to 20), inter-ediate (passage 20–30), and late (passage above 30) cultures. The figure

hows the percentage of CD44+ cells (black bars) and the plating efficiencyhatched bars) over time (early, intermediate, late passage).

reBIClipCsep[msemEcean

PRESSology/Hematology xxx (2010) xxx–xxx

ells by maturation arrest. Functional analysis confirmedhat distinct cell subsets present in HCC exhibit stemell-like properties including long-term survival (immor-ality), transplantability, and resistance to (chemo)therapy.

ore recently, several markers that may indicate stem cellunction in HCC cells have been identified. These mark-rs include CD133 and CD90 [131,132]. When 5 × 103

D45−/CD90+ cells, isolated from tumor specimens or bloodamples of HCC patients, were injected into the liver ofCID/Beige mice, tumors developed within 12–16 weeks in

hese mice.

.4. Pancreas CSC

In pancreatic cancer, CSC have also been described.hese pancreatic cancer stem cells express the cell sur-

ace markers CD44, CD24, and EpCAM, and represent.5–1.0% of all pancreatic cancer cells [16]. When ≥100D44+/CD24+/EpCAM+ cells were injected into the pan-reas of NOD/SCID mice, tumors developed within 4 weeksn these mice [16]. Another report suggested that CD133s expressed on pancreatic CSC [12]. When 500 CD133-ositive pancreas tumor cells were injected into the pancreasf NMRI-nu/nu mice, tumors developed within 3 weeks [12].ancreatic CSC display high levels of genes involved in self-enewal, such as the Sonic Hedgehog (SHH) antigen andMI-1 [133,134]. In addition, CD133+/CXCR4+ cells haveeen described to be responsible for metastasis formation inancreatic cancer in a model employing the L3.6pl cell line12].

.5. Breast CSC

In 2003, Al-Hajj et al. identified a NOD/SCID mouse-epopulating breast cancer cell subpopulation. These cellsxpress CD44, the breast/ovarian cancer-specific antigen38.1, and epithelial-specific antigen ESA (= EpCAM) [13].

n contrast to non-repopulating breast cancer cells, breastSC coexpress CD44 but display only low amounts of or

ack CD24. When ≥100 CD44+/B38.1+/CD24− cells werenjected into the mammary fat pad of NOD/SCID mice, pal-able tumors developed within 12 weeks, whereas none of theD44−/B38.1− cells developed tumors in these mice. Other

tudies have suggested that high aldehyde dehydrogenase lev-ls may be indicative for enhanced malignant and metastaticotential and thus may help in the identification of breast CSC60]. Most consecutive studies were performed in cell lineodels, whereas only very little is known about expression of

urface antigens or targets in primary breast CSC. One inter-sting question will be to learn whether breast CSC expressembers of the ERBB family of oncogenic receptors. In fact,RBB receptors are major targets of therapy in this type of

tem cells: Current concepts and clinical perspectives. Crit Rev

ancer. Data published so far suggest that mammary CSCxpress EGFR and ERBB2/Her2 but lack estrogen receptorlpha [135,136]. This may be due to several different mecha-isms such as gene silencing by epigenetic events [137,138].

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hether this holds true for all breast cancer variants remainst present unknown.

.6. Brain tumor stem cells

Hemmati et al. characterized and isolated stem cells fromuman paediatric brain tumors of different pathologic sub-ypes, including glioblastoma and medulloblastoma [27]. Theuthors reported that a minority of the brain tumor cellsere able to form neurospheres and to grow to tumors whenenotransplanted into rat brain. These neurosphere-derivedells were found to be Lin-negative and expressed CD133s well as nestin [27]. More recently, Singh et al. confirmedhat brain CSC reside within the CD133+/nestin+ fractionf brain tumor cells in a NOD/SCID mouse model [15]. Inact, when as few as 100 CD133+ glioblastoma cells werenjected into the brain of NOD/SCID mice, tumors devel-ped after 12 to 14 weeks in most animals. Bao et al. foundhat L1 CAM may also serve as a marker for brain CSCince L1 CAM is overexpressed on CD133+ cells, and tar-eting of L1 CAM reduced the tumorigenicity of CD133+ells [43]. Ehtesham et al. showed that glioblastoma pro-enitors in glioma-derived spheres may express CXCR4nd that binding of its ligand CXCL12 results in increasedrowth of neurosphere cells [139]. Recently, Read et al.ound that CD15+ cells are enriched for CSC in medulloblas-omas [140]. After intracranial implantation of 3 × 105 cells,tc+/− mice develop tumors within 50 days.

.7. Prostate CSC

The isolation of CSC in prostate cancer is difficult dueo the heterogeneity of prostate tumors and the small sam-le size. It is unclear whether prostate CSC derive fromhe basal or luminal layer [141]. However, selection ofells with a CD133+/alpha 2 beta 1 integrin+/CD44+ phe-otype resulted in enrichment for prostate cancer-initiatingells [142–145]. Moreover, CD44+ prostate cancer cellsave recently been described as more invasive and showno have increased Hedgehog signalling and activation of theI3K/AKT signalling pathway compared to CD44− cells146–148]. In 2008, Hurt et al. observed stem cell-like prop-rties of CD44+/CD24− cells derived from prostate cell linesLNCaP and DU145) [51]. The expression of androgen recep-ors on prostate CSC is controversial [142,147,149]. The genexpression profile of prostate cancer stem cells can also beelated to Gleason grade and patient outcome [150].

.8. Sarcoma

Recently, Suva et al. identified for the first time a Ewingarcoma cancer stem cell by sorting with magnetic beads for

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

D133+ cells [151]. After cell isolation from primary Ewingarcoma tissue, these CD133+ cells were found to establishwing sarcomas with parental tumor phenotype and hierar-hical organization in NOD/SCID mice [151]. The authors

mtbb

PRESSology/Hematology xxx (2010) xxx–xxx 11

ere also able to show that these tumors again containedwing sarcoma-initiating stem cells when re-transplanted

nto secondary recipient mice [151].

.9. Other solid tumors

In gastric cancer up to now only CD44 is described aspotential CSC marker [152]. Another group postulated

hat some gastric tumors may even arise from bone mar-ow cells [153]. Lung CSC are not well characterized soar. In one paper, Kim et al. describe bronchioalveolar stemells (BASCs) as potential lung CSC, whereas in anotheraper, lung CSC are identified as CD133+ cells [154,155].hen 1 × 104 EpCAM+/CD133+ cells from small and non-

mall-cell lung cancers were injected subcutaneously, tumorseveloped within 3 weeks in SCID mice [155]. This obser-ation has recently been confirmed for non-small-cell lungancer cells by Tirino et al. [156]. As in many other organs,here is also evidence for the expression of CD44 as well asD133 on the surface of ovarian CSC [157–159]. Baba etl. injected 4–5-week-old BALB/cAnNCr-nu/nu mice withorted CD133− (left flank) and CD133+ (right flank) A2780ells. They observed that CD133+ cells formed tumors thatrew to a larger size and appeared within shorter time thanumors from CD133− cells isolated from the same parentalell line [158]. On the other hand, Kusumbe et al. showedhat CD133+ cells in ovarian cancer are only responsible forhe development of the tumor vasculature by giving rise tondothelial cells. By contrast, CD133+ ovarian cells were notumorigenic in this study [160]. In one report CD133+ cellsrom anaplastic thyroid cancer showed reconstitution of theumor in NOD/SCID mice after injection of at least 10,000D133+ cells [161].

0. Melanoma stem cells (MSC)

So far, little is known about tumor-initiating cells in skinancer patients. In fact, CSC have only been investigatednd partly characterized in malignant melanomas. Fang et al.escribed that melanoma spheres can be grown from pri-ary melanoma cells, and that melanoma sphere-derived

ells exhibit long-term growth, multilineage potential, andumor-initiating potential in SCID mice [24]. They found thatsubpopulation of cells in these spheres is CD20+/CD45−

ells that co-express melanoma antigens. This CD20+ frac-ion of melanoma cells, when cultured separately, was foundo form larger spheres than CD20− cells, and showedong-term proliferation in vitro. Based on these data, its tempting to speculate that melanoma stem cells resideithin the CD20+ fraction of cells, although this has not

ormally been proven by mouse experiments using primary

tem cells: Current concepts and clinical perspectives. Crit Rev

elanoma cells sorted for CD20+ and CD20− cells. In addi-ion, the expression of CD20 on melanoma cells has noteen confirmed in other studies. Another marker that haseen discussed as a potential stem cell marker for human

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ARTICLE2 A. Schulenburg et al. / Critical Reviews

elanoma is CD133 [162]. It has been described that only.2–0.8% of primary metastatic melanoma cells expressD133 [162]. Monzani et al. observed that 40–50 daysfter subcutaneous injection of 3.5 × 105 CD133-positiveells into NOD/SCID mice, these mice develop detectableelanoma lesions, whereas the CD133-negative fraction did

ot form tumors in NOD/SCID mice [162]. These data sug-est that melanoma stem cells may reside within a CD133+raction of the clone, at least in metastatic melanomas [163].n line with this hypothesis, several melanoma cells linesisplay CD133 [164,165]. Other studies have shown that cer-ain drug-transporters that (when expressed) are indicativef chemoresistance, may be expressed on melanoma stemells (and stem cells in other tumors). Among these trans-orters are MDR1, ABCG2 and ABCB5 [166]. Schatton et al.escribed that the ABCB5+ fraction of primary (freshly iso-ated) melanoma cells is enriched for melanoma-repopulatingtem cells, whereas ABCB5-negative cells are less capablef initiating melanomas in NOD/SCID mice [54]. ABCB5+elanoma cells were found to co-express other potential

tem cell markers including ABCB1, TIE1, nestin, and CD4454,167].

However, the melanoma-initiating potential of singlestem) cells may greatly depend on the microenvironmentnd the mouse strain employed to demonstrate repopulation.uintana et al. have recently shown that up to 30% of allelanoma cells are melanoma-repopulating cells (indepen-

ent of their phenotype) when interleukin-2 receptor gammaull NOD/SCID mice are used, whereas tumorigenicity ofhe same cells is much lower when NOD/SCID mice withn intact interleukin-2 receptor are employed [23]. Theseata suggest that the frequency of melanoma-initiating cellsay be relatively high, at least in a severely immunocom-

romised host. Whether the same cells (all these cells) arelso able to repopulate melanomas in an immunocompetentost or in patients remains unclear. In fact, the stem cellunction of a given tumor/melanoma cell may not only beredetermined by intrinsic factors (genetic and epigenetictem cell programs) but also be microenvironmental factorsfactors defining the stem cell niche) and also the immuneystem (immunosurveillance, apoptosis-induction by killerells, tumor cell phagocytosis).

The observation that up to 30% of melanoma cells mayave the potential to repopulate melanomas in NOG micend thus have stem cell function, has changed our views onhat types and subpopulations of melanoma cells indeed are

epopulating and non-repopulating cells. Unfortunately, onlyfew markers can clearly discriminate larger subpopulationsf melanoma cells with different potential to metastasize ando form new tumor lesions in patients. One such marker ishe erythropoietin (EPO) receptor. In fact, the EPO receptors only expressed in trace amounts in normal melanocytes,

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

ut is expressed in melanoma cells [168–171]. Whereas inrimary melanomas, only a small subpopulation of cells dis-lay the EPO receptor, in metastatic melanomas up to 30% ofll cells express this antigen [168]. Moreover, it has recently

pta

PRESSology/Hematology xxx (2010) xxx–xxx

een described that EPO receptor expression is associatedith disease progression and the metastasizing potential ofelanomas [169,172]. Finally, EPO has been described to

nitiate signalling and promote survival in human melanomas171]. Whether indeed the EPO receptor is a functionally rev-lant antigen expressed specifically on melanoma initiatingells is currently under investigation.

1. CSC plasticity—the Hydra Model of CSCevelopment

A number of previous and more recent data suggest thateoplastic stem cell clones display substantial plasticity andre often composed of several different subclones. In manyases, subclone formation may precede the development offrank neoplasm, and only a few (or even only one) of

hese subclones may progress to an overt malignancy. Thisssumption is consistent with the multi-hit theory of cancerevelopment [173–176] and would predict that neoplastictem cells detectable in the NOD/SCID or NOG (NSG)ouse assay would not all be able to repopulate all com-

onents (cell subclones) in a given neoplasm. Rather, thisodel predicts early formation of subclones with different

ransformation potential, and each subclone must be expectedo contain subclone-specific stem cells. A good exampleor the presence of multiple subclones in one neoplasm ishe coexistence of two histologically different hematopoi-tic disorders in one patient. Another example is CML,here during treatment with imatinib, one or more different

ubclones exhibiting imatinib-resistant BCR/ABL mutantsay be selected (by treatment), and each of these subclonesay progress into a clinically relevant leukemia (subclone-

pecific progress) [91,177,178]. Whereas many of theseubclones bearing (drug-resistant) BCR/ABL mutations maye present (as small clones) before imatinib is started, somef these subclones may progress to leukemia during imatinibherapy which may point to clonal instability and a potentialffect of the drug on subclone formation. Similar observationsf stem cell plasticity have been made in other hematopoi-tic malignancies and may also apply to non-hematopoieticeoplasms [179].

Recent data suggest the stem cell plasticity may evennvolve the supportive stroma or tumor/leukemia-associatedngiogenesis. In particular, it has been described thatndothelial cells in lymphomas and other neoplasms are ofonoclonal origin [180]. Other studies suggest that cultured

one marrow stroma cells and mesenchymal progenitors inatients with Ph+ CML are of clonal origin [181]. However,ther studies suggest that stromal cells and mesenchymal pro-enitor cells in patients with CML are non-clonal cells andot derived from the Ph+ clone [182,183].

tem cells: Current concepts and clinical perspectives. Crit Rev

The above described heterogeneity and plasticity of CSCopulations in various neoplasms may be one reason forhe complexity of CSC evolution and function, and maylso explain why it is difficult to design effective treatment

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pproaches sufficient to eliminate all these cells and sub-lones in cancer/leukemia patients [184–186].

2. Strategies for the successful elimination of CSC

Several different strategies have been considered to inhibitrowth and/or survival of CSC, with the ultimate goal to elim-nate all CSC in these malignancies. These concepts includeelevant surface targets, signal transduction molecules, andertain survival molecules expressed in CSC (Fig. 3). Manyoncepts still relate to myeloid or lymphoid neoplasms,hereas so far, only a few treatment strategies have been pre-

ented for solid tumors and melanomas. Moreover, whereasuch is known about the expression of various surface and

ytoplasmic drug targets in solid tumors and leukemias, onlyery little is known about expression of the same targets ineoplastic stem cells (Fig. 3).

In myeloid neoplasms, potential surface targets includehe IL-3R alpha chain CD123, the Mylotarg-receptor Siglec-

(CD33), CD44, and the tyrosine kinase receptor KITCD117). Target expression can be exploited by selectinglocking antibodies or by constructing antibody–toxin con-ugates, cytokine–ligand–toxin conjugates, or antibody–drugonjugates. A good example for a ligand–toxin con-ugate is the IL-3-diphteria toxin fusion protein [187].n example for an antibody–drug conjugate is theD33-targeting fusion molecule gemtuzumab–ozogamcin

Mylotarg) [81–83] (Fig. 1). It has also been reported thatn unconjugated blocking CD123 antibody can effectivity

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

educe leukemia cell growth in mice [188] and even in a fewatients with AML [189]. So far, only a few studies havettempted to demonstrate functional significance of targetxpression in leukemic stem cells. In one study, Mylotarg was

aw

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ig. 3. Expression of molecular targets in and on neoplastic cells and their progenitoeoplastic cells (left part of panel), little is known about expression of molecular transduction; TK, tyrosine kinase(s); TF, transcription factor(s); TSG, tumor suppre

PRESSology/Hematology xxx (2010) xxx–xxx 13

ound to induce apoptosis in CD34+/CD38−/CD33+ stemells in AML [65] (Fig. 1). A novel and promising markeror stem cell targeting in AML may be CLL-1 that is ableo discriminate between normal and leukemic stem cells inhe bone marrow [86]. Another promising marker is CD96ince Hosen et al. showed that CD96 is specific for AMLtem cells. He showed that CD96+ AML cells can engraftrradiated Rag2(−/−) gamma(c)(−/−) mice [85]. However,o far no targeted drugs specific for CD96 or CLL-1 haveeen developed. Several different CD antigens are employeds targets of therapy in B-cell Non-Hodgkin’s lymphomas,ncluding CD20, CD22, CD23, CD25, or CD52. Clinical tri-ls have shown that antibodies directed against some of thesentigens (especially CD20 and CD52) can improve treat-ent and prognosis in these patients. It is therefore tempting

o speculate that some of these antigens are also expressedn immature lymphoma-initiating tumor cells. Studies arengoing to define the CD antigen profile in lymphoid stemells in various NHLs. An interesting aspect is that somef the lymphoid markers, like CD20, are also expressed onon-hematopoietic CSC. In solid tumors, cell surface targetsnclude members of the ErbB receptor family, IGF receptors,GF� receptors, and FGF receptors [190–193]. A generalroblem with surface targets is that CSC subclones oftenxhibit or develop resistance against antibody-based or otherrugs. Several different mechanisms of drug resistance haveeen discussed, including expression of multi-drug resistanceene products like MDR1 or reduced antibody-binding. Inddition, subclones that do not express the surface CD anti-en may be selected by antibody-based therapy. Therefore,

tem cells: Current concepts and clinical perspectives. Crit Rev

ntibody-therapy is usually combined with conventional orith other targeted drugs.Apart from surface markers, also intracellular targets

ave been identified in CSC. Among those, signal trans-

rs. Whereas much is known about target expression profiles in more matureargets in leukemic (neoplastic) stem cells (right panel part “?”). ST, signalssor gene(s); DR, death receptors.

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uction molecules and survival molecules may representost promising target antigens. These drugs include tyro-

ine kinase inhibitors, farnesyl transferase inhibitors, andther kinase inhibitors including PI3 kinase and mTORlockers [194–196]. In some myeloid neoplasms like CML,CR/ABL tyrosine kinase inhibitors have been used withonsiderable success [197]. More recently tyrosine kinasenhibitors have also been employed in clinical trials in solidumors and melanomas [98,196,198–209]. Among survival-elated targets, most promising molecules may be membersf the BCL-2 family (BCL-2, MCL-1, BCL-xL, others) andarious Heat shock proteins (HSP32, HSP70, HSP90, others)210–212]. Studies are ongoing to define whether these tar-ets are expressed in CSC in various hematopoietic or solidumors. Likewise, it has been described that the HSP32, alsonown as heme oxygenase 1, is expressed in CSC in variouseukemias and solid tumor models [213,214].

Despite intensive research and some progress in targetedherapy in solid tumors, only a few targeted drugs have pro-uced convincing results in clinical trials, which may be dueo resistance and the complexity of the signalling cascadesn cancer cells and CSC. This is mainly due to (the variousorms of) resistance as well as CSC plasticity with target-egative subclone formation. Therefore, it seems reasonableo combine targeted drugs (antibodies, small molecules) withach other or with conventional therapy (chemotherapy) inrder to overcome drug resistance in CSC.

Finally, provided that specific antigens of CSC are iden-ified, an attractive approach would be to induce specificmmune responses and to establish vaccination therapypproaches in these malignancies.

3. Concluding remarks and future directions

The emerging concept of neoplastic stem cells and CSC-elated targets may offer new insights into the biology and theathogenesis of various malignant disorders and new possi-ilities for the design of targeted drug therapies. Since CSCisplay considerable heterogeneity and plasticity, eradicationf these cells and thus cure may only be reached when combi-ations of anticancer drugs (therapies) are applied. Therefore,reatment designs aiming at cancer/leukemia stem cell elim-nation need to take all potential targets and all relevant stemell subclones into account. There is hope for the futurehat such novel treatment approaches will improve therapyn cancer patients.

onflict of interest

All the authors declare that they have no proprietary, finan-ial, professional or other personal interest of any nature or

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

ind in any product, service and/or company that could beonstrued as influencing the position presented in the reviewntitled “Neoplastic stem cells: Current concepts and clinicalerspectives”.

PRESSology/Hematology xxx (2010) xxx–xxx

eviewers

Dr. Dominique Bonnet, Cancer Research UK, Londonesearch Institute, Haematopoietic Stem Cell Laboratory, 44incoln’s Inn Fields, London WC2A 3PX, United Kingdom.

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iographies

Axel Schulenburg graduated at and is now working at theedical University of Vienna, Bone Marrow Transplantationnit. His research focus is Cancer stem cells.

Kira Brämswig studied medicine in Würzburg and Viennand has M.D. and Ph.D. degrees. She is now workingt the Oncology Department of the Medical Universityf Vienna. Scientifically she is focused on tumorangio-enesis.

Harald Herrmann obtained his M.D. in 2008 and is nowPh.D. student at the Medical University of Vienna. He is

nrolled in the Ph.D.-program malignant diseases and focusesis research on stem cells in myeloid leukemias.

Heidrun Karlic studied biology and biochemistry athe University of Vienna with a Ph.D. graduation ands since July 1987 senior scientist at the Ludwig Boltz-

ann Institute for Leukemia Research and Hematology,anusch Hospital, Vienna Austria. Her research focus is

pigenetics.

Irina Mirkina studied molecular biology at the Russiancademy of Sciences (RAS), Moscow, Russia from 1996

o 1999 with a Ph.D. She is now working on melanomatem cells as a postdoctoral scientist in the laboratory of the

edical University of Vienna.

Rainer Hubmann studied molecular biology at the Uni-ersity of Vienna and holds a Ph.D. He is now a postdoct the Medical University of Vienna and is working on theegulation and function of NOTCH2 in B-CLL.

Sylvia Laffer studied biology at the University of Viennarom 1986 to 1992 and has a Ph.D. She is now working onancer stem cells for the Ludwig Boltzmann Cluster Oncol-gy.

Brigitte Marian studied pharmacy from 1972 to 1977 athe University of Vienna and has a M.Sc. and a Ph.D. incience. She was a research fellow at the Memorial Sloanettering Cancer Center, New York, USA and is now ansc. Prof. at the Institute of Cancer Research at the Med-

cal University Vienna. Her research is focused on Colonancer.

Medhat Shehata is a postdoctoral scientist at the Medicalniversity of Vienna and is involved in research of Chronic

ymphocytic leukemia.

Clemens Krepler obtained his M.D. in 2000 and is nowspecialist degree in dermatology and venereology at theedical University of Vienna. He is enrolled in the Ph.D.-

rogram malignant diseases and focuses his research on stem

tem cells: Current concepts and clinical perspectives. Crit Rev

is research focus is melanoma.

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Peter Valent is Asc. Professor for experimental hematol-

ARTICLE0 A. Schulenburg et al. / Critical Reviews

Thomas Grunt studied biology from 1976 to 1982 athe University of Salzburg. He has Ph.D. and masteregrees. He is now professor at the Oncology Laboratory in

Please cite this article in press as: Schulenburg A, et al. Neoplastic sOncol/Hematol (2010), doi:10.1016/j.critrevonc.2010.01.001

ienna.

Ulrich Jäger is full professor and head of the Hematologyepartment of the Medical University of Vienna. His research

nterests are lymphatic malignancies.

otm

PRESSology/Hematology xxx (2010) xxx–xxx

Christoph Zielinski is full professor and head of the Oncol-gy Department at the Medical University of Vienna.

tem cells: Current concepts and clinical perspectives. Crit Rev

gy at the Medical University of Vienna. He is working inhe research of stem cells in hematologic malignancies and

ast cells.