Stem Cell-associated Markers Research Articles

Abstract 2452: Generation of hepatocellular carcinomas with cancer stem cell properties from primary mouse hepatocytes

Abstract BACKGROUND: Current evidence suggests that hepatocellular carcinoma (HCC), the most common adult primary liver cancer, comprises biologically distinct subgroups including the recently identified hepatic progenitor cell- and cholangiocarcinoma (CC)-like subtypes. These observations support the notion that HCCs can be derived from different cells within the lineage from adult liver stem cell to hepatocyte. AIM: We addressed two questions: (1) does the transforming potential of oncogenes depend on the stage of hepatocytic differentiation and (2) is the tumor phenotype determined by the stage of differentiation at which transformation occurs. METHODS: Fetal (ED16.5) hepatocytes were purified by magnetic sorting and adult (3-month) hepatocytes were isolated by collagenase perfusion from C57BL/6 mice. Cells were transduced with lentiviral vectors carrying oncogenic H-Ras and SV40 large T (LT) together with GFP and mCherry fluorescent reporters. Double positive cells were sorted and injected into the spleen of NOD/SCID mice. Tumors larger than 3 mm derived from the transduced fetal (n=20) and adult hepatocytes (n=20) were macrodissected and subjected to histopathology evaluation, immunohistochemistry and transcriptome profiling. Cancer stem cell traits were examined by FACS analysis of lineage specific and cancer stem cell associated markers, side population and tumor sphere formation assays using cell lines established from fetal and adult hepatocyte-derived tumors. RESULTS: Both fetal and adult hepatocytes co-transduced with oncogenic H-Ras/SV40LT produced liver tumors with similar growth characteristics and gave rise to lung metastases with 80% (8/10) and 63% (5/8) frequency, respectively. Histologically, fetal and adult hepatocyte-derived tumors were moderately-poorly differentiated HCCs with CC-like phenotype. All tumor cell lines were 100% positive for CK18, CK19, EpCam, A6, CD44, CD49f and CD29 and contained SP fraction (1-2%) as measured by FACS analysis regardless of the cell origin. CD133 was expressed at lower and variable levels in fetal (26.0±7.2%) and adult (46.1±9.1%) tumor cell lines. Both fetal and adult tumor cells possessed a sphere forming capacity which was maintained within 5 sphere generations. Unsupervised hierarchical clustering separated the fetal and adult tumors after log2 transformation by using corresponding normal samples as reference. CONCLUSION: Our results suggest that the activation of certain oncogenic pathways may have more significant effect on the tumor phenotype than the stage of hepatocytic differentiation at which the transformation occurs. Oncogenic H-Ras and SV40LT are capable of inducing a cancer stem cell-like state in mature hepatocytes that displays characteristics of hepatic stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2452. doi:10.1158/1538-7445.AM2011-2452

PC3 is a cell line characteristic of prostatic small cell carcinoma

The majority of the prostatic cancers are adenocarcinomas characterized by glandular formation and the expression of luminal differentiation markers androgen receptor (AR) and prostate-specific antigen (PSA). Most adenocarcinomas are indolent and androgen-dependent. Hormonal therapy that inhibits AR signaling produces symptomatic relief in patients with advanced and metastatic adenocarcinomas. Prostatic small cell neuroendocrine carcinoma (SCNC) is a variant form of prostate cancer (PC). In contrast to adenocarcinoma, the tumor cells of SCNC do not form glands and are negative for AR and PSA. SCNC is extremely aggressive and does not respond to hormonal therapy. The purpose of this study was to compare the important and relevant features of two most commonly used PC cell lines, LNCaP and PC3, with prostatic adenocarcinoma and SCNC. Xenograft tumors of LNCaP and PC3 were prepared and compared with human prostatic adenocarcinoma and SCNC for the expression of key signaling molecules by immunohistochemistry and Western blot analysis. LNCaP cells express AR and PSA and their growth is inhibited by androgen withdrawal, similar to human prostatic adenocarcinoma. PC3 cells do not express AR and PSA and their proliferation is independent of androgen, similar to SCNC. Adenocarcinoma cells and LNCaP cells are negative for neuroendocrine markers and stem cell-associated marker CD44 while SCNC and PC3 cells are positive. LNCaP cells have identical cytokeratin profiles to adenocarcinoma while PC3 cells have cytokeratin profiles similar to SCNC. LNCaP cells share common features with adenocarcinoma while PC3 cells are characteristic of SCNC.

Expression of the Stem Cell Markers Nestin and CD133 on Circulating Melanoma Cells

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells, presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study, 20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02), number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02), tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01), and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis.

FGF4-dependent stem cells derived from rat blastocysts differentiate along the trophoblast lineage

Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines not only possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.

Pituitary stem /progenitor cells: embryonic players in the adult gland?

The pituitary gland represents the endocrine core of the body, and its hormonal output governs many key physiological processes. Because endocrine demands frequently change, the pituitary has to flexibly remodel its hormone-producing cell compartment. One mechanism of pituitary plasticity may rely on the generation of new hormonal cells from resident stem/progenitor cells. Existence of such 'master' cells in the pituitary has in the past repeatedly been postulated. Only recently, however, very plausible candidates have been identified that express stem cell-associated markers and signalling factors, and display the stem/progenitor cell characteristics of multipotency, efflux capacity (side population phenotype) and niche-like organization. In other adult tissues, stem cells recapitulate the embryonic developmental path on their course towards mature specialized cells. Interestingly, the pituitary stem/progenitor cell compartment shows prominent expression of transcriptional regulators and signalling factors that play a pivotal role during pituitary embryogenesis. This review summarizes the recent progress in pituitary stem/progenitor cell identification, highlights their potential embryonic phenotype, sketches a tentative stem/progenitor cell model, and discusses further research and challenges. Recognizing and scrutinizing the pituitary stem/progenitor cells as embryonic players in the adult gland may profoundly impact on our still poor understanding of the mechanisms underlying pituitary cell turnover and plasticity.

Circulating cells and dialysis: improving cell number or increasing session number?

1. Gornall J. Stem cell renegades or pioneers? BMJ 2010; 340: c2041 2. Kaiser J, Vogel G. Embryonic stem cells. Controversial ruling throws U.S. research into a tailspin. Science 2010; 329: 1132–1133 3. Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131: 861–872 4. Park IH, Daley GQ. Human iPS cell derivation/reprogramming. Curr Protoc Stem Cell Biol 2009; Chapter 4: Unit 4A 1 5. Yamanaka S. Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors. Cell Prolif 2008; 41: 51–56 6. Le Blanc K, Ringden O. Immunomodulation by mesenchymal stem cells and clinical experience. J Intern Med 2007; 262: 509–525 7. Reinders ME, Fibbe WE, Rabelink TJ. Multipotent mesenchymal stromal cell therapy in renal disease and kidney transplantation. Nephrol Dial Transplant 2010; 25: 17–24 8. Feng Y. Fresh and cryopreserved, uncultured adipose tissue-derived stem and regenerative cells ameliorate ischemia-reperfusion induced acute kidney injury. Nephrol Dial Transplant 2010; 25: 3874–3884 9. Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001; 7: 211–228 10. Mitchell JB, McIntosh K, Zvonic S et al. Immunophenotype of human adipose-derived cells: temporal changes in stromalassociated and stem cell-associated markers. Stem Cells 2006; 24: 376–385 11. Parker AM, Katz AJ. Adipose-derived stem cells for the regeneration of damaged tissues. Expert Opin Biol Ther 2006; 6: 567–578 12. Tholpady SS, Llull R, Ogle RC et al. Adipose tissue: stem cells and beyond. Clin Plast Surg 2006; 33: 55–62 13. Bai X, Yan Y, Song YH et al. Both cultured and freshly isolated adipose tissue-derived stem cells enhance cardiac function after acute myocardial infarction. Eur Heart J 31: 489–501 14. Sanchez PL, Sanz-Ruiz R, Fernandez-Santos ME et al. Cultured and freshly isolated adipose tissue-derived cells: fat years for cardiac stem cell therapy. Eur Heart J 2010; 31: 394–397 15. WosnitzaM, Hemmrich K, Groger A et al. Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation. Differentiation 2007; 75: 12–23 16. Boquest AC, Shahdadfar A, Fronsdal K et al. Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture.Mol Biol Cell 2005; 16: 1131–1141 17. Thirabanjasak D, Tantiwongse K, Thorner PS. Angiomyeloproliferative lesions following autologous stem cell therapy. J Am Soc Nephrol 2010; 21: 1218–1222

Potential localization of putative stem/progenitor cells in human bulbar conjunctival epithelium

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, it is likely that pockets of conjunctival epithelial stem cells may also exist throughout the conjunctival epithelium. This study was to investigate the potential localization of putative stem/progenitor cells in the human bulbar conjunctival epithelium by evaluating 6 keratins and 13 molecules that have been previously proposed stem cell associated or differentiation markers. We found that cornea specific cytokeratin (CK) 3 was not expressed by the bulbar conjunctival epithelial cells. In contrast, CK4 and CK7 were expressed by the superficial cells of bulbar conjunctival epithelium. CK14 and CK15 were confined to the basal cell layer. CK19 was strongly expressed by all layers of the bulbar conjunctival epithelium. The expression patterns of molecular markers in the basal cells of human bulbar conjunctival epithelium were found to be similar to the corneal epithelium. Basal conjunctival epithelial cells strongly expressed stem cell associated markers, including ABCG2, p63, nerve growth factor (NGF) with its receptors tyrosine kinase receptor A (TrkA) and neurotrophin low-affinity receptor p75NTR, glial cell-derived neurotrophic factor (GDNF) with its receptor GDNF family receptor alpha 1 (GFRalpha-1), integrin beta1, alpha-enolase, and epidermal growth factor receptor (EGFR). The differentiation associated markers nestin, E-cadherin and involucrin were not expressed by these cells. These findings indicate that the basal cells of bulbar conjunctival epithelium shares a similar expression pattern of stem cell associated markers to the corneal epithelium, but has a unique pattern of differentiation associated cytokeratin expression.

Sin3a Is Required by Sertoli Cells to Establish a Niche for Undifferentiated Spermatogonia, Germ Cell Tumors, and Spermatid Elongation

Microenvironments support the maintenance of stem cells and the growth of tumors through largely unknown mechanisms. While cell-autonomous chromatin modifications have emerged as important determinants for self-renewal and differentiation of stem cells, a role for non-cell autonomous epigenetic contributions is not well established. Here, we genetically ablated the chromatin modifier Swi-independent 3a (Sin3a) in fetal Sertoli cells, which partly comprise the niche for male germline stem cells, and investigated its impact on spermatogenic cell fate and teratoma formation in vivo. Sertoli cell-specific Sin3a deletion resulted in the formation of few undifferentiated spermatogonia after birth while initially maintaining spermatogenic differentiation. Stem cell-associated markers Plzf, Gfra1, and Oct4 were downregulated in the mutant fetal gonad, while Sertoli cell markers Steel and Gdnf, which support germ cells, were not diminished. Following birth, markers of differentiating spermatogonia, Kit and Sohlh2, exhibited normal levels, but chemokine-signaling molecules chemokine (C-X-C motif) ligand 12 (CXCL12)/stromal cell-derived factor 1 (SDF1) and chemokine (C-X-C motif) receptor 4 (CXCR4), expressed in Sertoli cells and germ cells, respectively, were not detected. In the juvenile, mutant testes exhibited a progressive loss of differentiating spermatogonia and a block in spermatid elongation, followed by extensive germ cell degeneration. Sertoli cell-specific Sin3a deletion also suppressed teratoma formation by fetal germ cells in an in vivo transplantation assay. We conclude that the epigenome of Sertoli cells influences the establishment of a niche for germline stem cells as well as for tumor initiating cells.

Correlation of stem cell marker nestin expression on circulating melanoma cells with extension of disease and survival.

8551 Background: Within circulating melanoma cells (CMCs) there may be a subset of cells with stem cell characteristics able to develop distant metastasis. We evaluated the presence of melanoma cells bearing stem cell phenotype in the bloodstream of patients (pts) with metastatic melanoma. Stem cell marker assessment included nestin (NES) and CD133. Methods: Twenty-five ml blood were collected after informed consent from 32 consecutive pts affected by metastatic uveal (n = 14) or cutaneous (n = 18) melanoma and from (n = 6) healthy volunteers according to ethically approved protocols. Blood was enriched for tumour cells by CD45 depletion of the non-melanoma cell fraction using a magnetic bead separation technique. Potential melanoma cells were identified using flow cytometry with the markers MELAN-A and HMB45. Multiparameter cytometry was carried out to co-stain with the combinations of CD133 and NES. Five tissue samples from metastatic lesions of five different pts were stained with the same antibodies by immunohistochemistry. Results: No cells positive for the melanocyte markers were detected in blood from volunteers. In pts MELANA and/or HMB45 positive cells were detected in 28 samples (87.5%). The absolute number of melanoma cells ranged from 0 to 1233 (median 53) per 10 mL of blood. NES expressing cells were more represented compared to CD133 expressing cells (median 18.0% [0.3%-85.1%] vs. median 0.1% [0%-23.4%]). Percentage of NES-positive cells correlated significantly with tumor burden (p = 0.01) and number of metastatic sites (p = 0.03), whereas percentage of CD133-positive cells and absolute total number of cells did not. Cox regression analysis revealed levels of LDH (HR: 12.8 [1.35-121.5]; p = 0.02), number of metastatic sites (HR 3.87 [1.66-9.03]; p = 0.02), tumor burden (HR 5.72 [1.57- 20.9]; p = 0.01) and high level of NES expression (HR 3.5 [0.92-13.6]; p = 0.04) to be factors related to shorter overall survival. CD133 and NES expression profiles on CMCs and in matched metastatic tissue were similar. Conclusions: Melanoma cells in peripheral blood expressed stem cell associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. No significant financial relationships to disclose.

Molecular signatures and biological pathway profiles of human corneal epithelial progenitor cells

Identification and isolation of adult stem cells are still challenging for stem cell biologists. For example, no consensus exists yet regarding definitive markers for corneal epithelial stem cells, which have been identified to reside in the limbus for two decades. This study characterized the molecular signatures and biological pathways of limbal epithelial progenitors, the rapid adherent cells (RAC) isolated by adhesion on collagen IV, using human genome microarrays, real-time PCR and immunofluorescent staining. The microarrays produced highly reproducible data not only for all gene transcripts, but also for significantly changed genes, although the total 12 samples of 3 cell populations in 2 arrays were isolated from 4 separate experiments at different time period. The hierarchical clustering heatmap visually revealed that RAC progenitor population displayed distinguishably characteristic gene expression profile. With verification of 27 important genes by quantitative real-time PCR, the microarray data not only confirm the expression patterns of 15 known genes as stem cell associated markers representing limbal stem cell phenotype, but also identified many significantly regulated genes expressed by limbal progenitor cells. Transcription factor TCF4 and cell surface protein SPRRs were identified as potentially positive or negative markers, respectively, for corneal epithelial progenitor cells. Using GenMAPP and MAPPFinder, we have identified three patterns of biological pathway profiles, overexpressed, underexpressed and balanced, by RAC progenitors based on gene ontology categories. These genes and related pathways are interesting targets for further identification and isolation of limbal stem cells as well as other tissue-specific adult stem cells.

Abstract A29: High aldehyde dehydrogenase activity is general marker for normal hematopoietic stem cells but not leukemic stem cells in acute myeloid leukemia

Abstract Only a minority of cells, the leukemic stem cells (LSCs), within Acute Myeloid Leukemia (AML) are typically responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which originates mainly from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure leukemia. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukaemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high aldehyde dehydrogenase (ALDH) activity compartment contains the LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. High ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry. Here we have analyzed ALDH activity in normal HSCs and LSCs, both present within the same BM, in 23 AML patient samples. In 7 AML cases, a high SSCloALDHbr cell population was identified (>5%). We show that in 9 BM AML samples, defined as CD34min (ie <1% CD34+ cells, in which the CD34+CD38-cells are all normal HSC), the ALDH activity is lower in the LSC then in the HSC. The activity of the normal HSC within this AML BM is identical as the ALDH activity of normal HSC in NBM of healthy donors. In 9 BM AML patients, defined as CD34+ and with both normal and leukemic CD34+CD38-present, we show that the marker positive CD34+CD38-LSCs have lower ALDH activity then the marker negative CD34+CD38-HSCs. Altogether, we show that, although malignant AML blast cells have varying ALDH activity, a common feature of all AML cases is that the normal HSCs that co-exist with leukemic (stem) cells in the BM of AML patients have a higher ALDH activity as compared to their malignant counterparts. In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as mafosfamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A29

Distribution and analysis of stem and progenitor cell populations in large mammal and human kidneys

Zonal identification of stem cell‐associated marker distribution within adult kidneys may facilitate development of assays to monitor efficacy of regenerative medicine approaches to treating renal disease. In this study, we used quantitative RT‐PCR to analyze stem and progenitor cell marker distribution (e.g., CD117, CD146, CD133, CD271, CD34, Oct‐4A, and SOX2) within discrete compartments of canine kidneys and found that adipose tissue associated with the major calyx and renal pedicle contains potential stem and progenitor cell populations. Primary canine renal cultures also contained cells with multi‐lineage differentiation potential, as determined by growth‐factor mediated expression of epithelial, endothelial, smooth muscle, adipocytic, and osteogenic markers. Extending this analysis to primary human renal cells isolated from cadaveric donors with and without kidney disease suggested that human kidneys may also contain cells capable of multi‐lineage differentiation. Multiple stem and progenitor markers, including CD117, CD146, CD133, and CD271, were up‐regulated in cells isolated from diseased human tissue compared to healthy, consistent with the hypothesis that stem and progenitor populations are activated and/or mobilized in response to renal disease. Taken together, these data may serve as a foundation for the development of molecular assays to monitor the regenerative response.

Breast Cancer Stem Cells and the Epithelial-Mesenchymal Transition.

Abstract The acquisition of highly malignant traits by carcinoma cells is often associated with the activation of a transdifferentiation program termed the epithelial-mesenchymal transition (EMT). The EMT program is deployed in a variety of steps of embryonic morphogenesis, enabling the interconversion of epithelial and mesenchymal cell types. When both normal and neoplastic epithelial cells undergo an EMT, they acquire addition changes in addition to expressing characteristic mesenchymal cell markers. In particular, they acquire motility, an increased resistance to apoptosis and, in the case of carcinoma cells, invasiveness. The motility and invasiveness components associated with the EMT would appear to empower many types of cancer cells to metastasize. An interesting question is how the resulting disseminated cancer cells are able to spawn a macroscopic tumor mass at distant anatomical sites, since this clonal expansion would appear to require an ability to self-renew. Recently, we have examined the cellular products of an EMT. Contrary to our expectations, the resulting cells acquired many of the attributes of stem cells, including the expression of stem cell-associated cell-surface markers. Conversely, mammary epithelial cells that naturally express these markers also express greatly elevated levels of at least four pleiotropically acting transcription factors that have been found able to induce an EMT. More recent studies have confirmed that the product of an EMT in mouse mammary epithelial cells is a cell that has a greatly increased ability to generate an entire mammary ductal epithelial tree. Moreover, we find that cells with the attributes of stem cells exist naturally as minor subpopulations in cultured human mammary epithelial cells. These cells can differentiate spontaneously into non-stem cells and, conversely, the non-stem cells appear capable of dedifferentiating spontaneously into cells with progenitor or stem-cell phenotypes. Hence, the study of certain aspects of mammary epithelial cell biology, including the biology of mammary carcinoma stem cells, may be feasible with cultured cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr A1-1.

High Aldehyde Dehydrogenase Activity (ALDH) Is a General Marker for Normal Hematopoietic Stem Cells but Not Leukemic Stem Cells in Acute Myeloid Leukemia (AML). .

Abstract 4035Poster Board III-971Only a minority of cells, the leukemic stem cells (LSCs), within AML are responsible for tumor growth and maintenance. Many patients experience a relapse after therapy which is thought to originate from the outgrowth of therapy resistant LSC. Therefore, eradication of the LSCs is likely necessary to cure AML. Both the normal hematopoietic stem cells (HSCs) and LSCs co-exist in the bone marrow (BM) of leukemia patients and therefore success of an anti-stem-cell strategy relies on specific induction of LSC death while sparing the normal HSC. In AML, apart from the CD34+CD38- and the side population (SP) compartment, the high ALDH activity compartment contains the LSCs. The SP and ALDH defined compartments may include both CD34+ and CD34- HSCs and LSCs. ALDH is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes and high ALDH activity results in resistance to alkylating agents such as the active derivatives of cyclophosphamide. Recent data has shown that ALDH is highly expressed in both normal progenitor and stem cells and in AML blast cells. In view of the applicability of LSC specific therapies the detoxification by ALDH might be of importance. Therefore, a difference in ALDH activity between the normal HSC and the malignant LSC might be used to preferentially kill the LSC and spare the HSC.We have previously shown that CD34+CD38- and SP LSCs can be identified and discriminated from HSCs using stem cell-associated cell surface markers, including C-type lectin-like molecules (CLL-1), lineage markers, such as CD7, CD19, and CD56 and recently cell size characteristics as measured by flow cytometry (Terwijn, Blood 111: 487,2008). Here we have analyzed ALDH activity in 23 AML cases. In 7 AML cases, a high SSCloALDHbr cell population was identified (median: 10,9%, range 5,24-15,29%). In 16 cases there were rare (<5%) SSCloALDHbr cells.We have analyzed ALDH activity in aberrant marker defined HSCs and LSCs, both present within the same BM samples in 18 AML patients (summarized in Figure 1). In 9 BM AML samples, defined as CD34-, the CD34+ compartment contained only normal CD34+CD38- HSCs. The ALDH activity in the CD34- cells, which includes by definition in this AML subgroup the LSC, is a factor 4,4 (range 1,7-18,9) lower than in the HSC (Figure 1, panel 1). The ALDHbrSSClo cells present in these CD34- AML cases contained both normal CD34+ and CD34- cells. The activity of the normal HSC within this AML BM is similar to that of the normal HSC in NBM of healthy donors (Figure 1, panel 3).In addition, 9 BM AML patients, defined as CD34+ AML and with both marker negative, normal HSCs and marker positive LSCs present, were analyzed for ALDH activity. We show that the marker positive CD34+CD38- LSCs have 7,7 fold (range 1,73-29,2 fold) lower ALDH activity than the marker negative CD34+CD38- HSCs (Figure 1, panel 2). Altogether, we show that, although malignant AML blast cells have varying ALDH activity, a common feature of all AML cases is that the normal HSCs that co-exist with leukemic (stem) cells in the BM of AML patients have a higher ALDH activity as compared to their malignant counterparts (summarized in figure 1).In conclusion, high ALDH activity is an unique marker of normal HSC within the AML BM (irrespective of AML phenotype, CD34+ or CD34-) at diagnosis. Consequently, AML patients with high ALDH activity in the normal HSC might benefit from treatment with alkylating agents such as cyclophosphamide, whereby the difference between the ALDH activity in LSC and HSC defines the therapeutic window. At present, drugs, known to be dependent on low ALDH for proper activity, are tested for their LSC specific killing while sparing the normal HSC. Additionally, transcriptional profiles are obtained from purified ALDH+ HSC and ALDH- LSC. This will enable us to use this general discriminating property to identify molecules that differ between the LSC and HSC and can function as LSC specific therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Early T-Cell Precursor-ALL in Adult T-ALL.

Abstract 911▪▪This icon denotes an abstract that is clinically relevant. Introduction:Recently, a small subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was described, which is closely associated with the gene expression profile of early T-cell precursors (ETPs). This subtype, termed ETP-ALL, showed a highly unfavorable outcome compared to non-ETP(='typical')-ALL. Based on the results of Coustan-Smith et al. (Lancet Oncology, 2009), the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) and St-Jude Children's hospital modified their treatment in children with ETP-ALL to a more intensive regime including stem cell transplantation. ETP-ALL is characterized by a specific immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers). Here we explored the existence of ETP-ALL in adults and further studied the molecular characteristics of this specific T-ALL subtype. Patients and methods:We examined the gene expression profiles of 86 adult T-ALL patients obtained from the Microarray Innovations in LEukemia (MILE) multicenter study (HG-U133 Plus 2.0, Affymetrix, Haferlach et al., JCO in press). In addition, bone marrow of 296 patients from the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) were analyzed by flow cytometry and expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by real-time-PCR. Results:Using the published list of differentially expressed genes in ETPs (Coustan-Smith et al. 2009) we performed unsupervised clustering analyses of the 86 T-ALL samples. A cluster of 17 samples (19.8%) displayed an ETP-associated gene expression profile and were defined as ETP-ALL. Comparing the gene expression profiles of ETP-ALL and typical T-ALL, 2065 probe sets were differentially expressed in ETP-ALL (FDR 0.05). In addition to genes used for classification, we also identified genes known to be involved in the pathogenesis of T-ALL (e.g. PROM1, BCL2, LMO2, LYL1). In particular, stem cell associated genes such as, BAALC (2.52-fold, p=0.003), IGFBP7 (2.76-fold, p=0.002) or MN1 (3.41-fold, p<0.001) were upregulated in ETP-ALL, whereas HOX11 (45-fold, p=0.004), a marker for thymic T-ALL, was downregulated. An independent cohort of 297 patient samples from the GMALL study group was examined by flow cytometry and real-time PCR. 19 (6.4%) samples revealed the ETP-ALL immunophenotype. As expected, all patient samples were found in the group of early T-ALL, representing 23.5% of all early T-ALLs. There was a significant correlation between a lower leukocyte count at first diagnosis and the classification of ETP-ALL (p=0.001). Gene expression measured by real-time-PCR was performed for genes associated with poor outcome in T-ALL: BAALC (2.11-fold, p<0.001) and IGFBP7 (3.59-fold, p=0.003) were significantly upregulated in the group of ETP-ALL. Similarly, the genes MN1 (4.52-fold, p<0.001) and WT1 (2.76-fold, p=0.036), described as poor prognostic markers in cytogenetically normal AML, were also upregulated in ETP-ALL. Conclusion:In adult T-ALL, a subset of patients shares the gene expression profil and immunophenotype of ETP-ALL, which is in line with recent findings in pediatric patients. The gene expression profile of this subset is significantly correlated to stem cell associated markers predictive for inferior outcome in T-ALL. Interestingly, adverse factors in CN-AML are also aberrantly expressed in ETP-ALL suggesting a myeloid origin of ETPs and indicating a closer relationship between ETP-ALL and AML. The prognostic impact and the determination of the most appropiate set of markers needs to be further investigated. These results support the GMALL strategy to regard early T-ALL patients as high risk with assignment to stem cell transplantation. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Cells with Characteristics of Cancer Stem/Progenitor Cells Express the CD133 Antigen in Human Endometrial Tumors

Cancer stem cells represent an attractive therapeutic target for tumor eradication. The present study aimed to determine whether CD133 expression may identify cells with characteristics of cancer stem/progenitor cells in human endometrial tumors. We analyzed 113 tumor samples for CD133/1 expression by flow cytometry, immunohistochemistry, and semiquantitative reverse transcription-PCR. CD133(+) cells were isolated and used to assess phenotypic characteristics, self-renewal capacity, ability to maintain CD133 expression and form sphere-like structures in long-term cultures, sensitivity to chemotherapeutic agents, gene expression profile, and ability to initiate tumors in NOD/SCID mice. Primary tumor samples exhibited a variable degree of immunoreactivity for CD133/1, ranging from 1.3% to 62.6%, but stained negatively for other endothelial and stem cell-associated markers. Isolated CD133(+) cells expanded up to 4.6-fold in serum-replenished cultures and coexpressed the GalNAcalpha1-O-Ser/Thr MUC-1 glycoform, a well-characterized tumor-associated antigen. Dissociated bulk tumors formed sphere-like structures; cells grown as tumor spheres maintained CD133 expression and could be propagated for up to 12 weeks. CD133(+) cells purified from endometrioid adenocarcinomas were resistant to cisplatin-induced and paclitaxel-induced cytotoxicity and expressed a peculiar gene signature consisting of high levels of matrix metalloproteases, interleukin-8, CD44, and CXCR4. When serially transplanted into NOD/SCID mice, CD133(+) cells were capable of initiating tumor formation and recapitulating the phenotype of the original tumor. CD133 is expressed by human endometrial cancers and might represent a valuable tool to identify cells with cancer stem cell characteristics.

Isolation of tumour stem-like cells from benign tumours

Background:Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours.Methods:Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice.Results:In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments.Conclusion:This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.

Natural killer cells kill human melanoma cells with characteristics of cancer stem cells

Experimental and clinical data suggest that tumours harbour a cell population retaining stem cell characteristics that can drive tumorigenesis. CD133 is considered an important cancer stem cells (CSC)-associated marker. In a large variety of human malignancies, including melanoma, CD133(+) cells have been reported to comprise CSC. In this study, we show that melanoma cell lines are highly heterogeneous for the expression of several stem cell-associated markers including CD133, c-kit/CD117 and p75 neurotrophin receptor/CD271. Since no information is available on the ability of NK cells to recognize and lyse melanoma stem cells, we assessed whether melanoma cell lines, characterized by stem cell-like features, were susceptible to lysis by IL-2-activated NK cells. We show that activated NK cells efficiently kill malignant melanoma cell lines that were enriched in putative CSC by the use of different selection methods (i.e. CD133 expression, radioresistance or the ability to form melanospheres in stem cell-supportive medium). NK cell-mediated recognition and lysis of melanoma cells involved different combinations of activating NK receptors. Since CSC have been reported to be both drug resistant and radioresistant, our present data suggest that NK-based adoptive immunotherapy could represent a novel therapeutic approach to possibly eradicate metastatic melanoma.

Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer.

Tumor contains small population of cancer stem cells (CSC) that are responsible for its maintenance and relapse. Analysis of these CSCs may lead to effective prognostic and therapeutic strategies for the treatment of cancer patients. We report here the identification of CSCs from human lung cancer cells using Aldefluor assay followed by fluorescence-activated cell sorting analysis. Isolated cancer cells with relatively high aldehyde dehydrogenase 1 (ALDH1) activity display in vitro features of CSCs, including capacities for proliferation, self-renewal, and differentiation, resistance to chemotherapy, and expressing CSC surface marker CD133. In vivo experiments show that the ALDH1-positive cells could generate tumors that recapitulate the heterogeneity of the parental cancer cells. Immunohistochemical analysis of 303 clinical specimens from three independent cohorts of lung cancer patients and controls show that expression of ALDH1 is positively correlated with the stage and grade of lung tumors and related to a poor prognosis for the patients with early-stage lung cancer. ALDH1 is therefore a lung tumor stem cell-associated marker. These findings offer an important new tool for the study of lung CSCs and provide a potential prognostic factor and therapeutic target for treatment of the patients with lung cancer.

Selective expression of CD44, a putative prostate cancer stem cell marker, in neuroendocrine tumor cells of human prostate cancer

Hormonal therapy is effective for advanced prostate cancer (PC) but the disease often recurs and becomes hormone-refractory. It is hypothesized that a subpopulation of cancer cells, that is, cancer stem cells (CSCs), survives hormonal therapy and leads to tumor recurrence. CD44 expression was shown to identify tumor cells with CSC features. PC contains secretory type epithelial cells and a minor population of neuroendocrine cells. Neuroendocrine cells do not express androgen receptor and are quiescent, features associated with CSCs. The purpose of the study was to determine the expression of CD44 in human PC and its relationship to neuroendocrine tumor cells. Immunohistochemistry and immunofluorescence were performed to study CD44 expression in PC cell lines, single cells from fresh PC tissue and archival tissue sections of PC. We then determined if CD44+ cells represent neuroendocrine tumor cells. In human PC cell lines, expression of CD44 is associated with cells of NE phenotype. In human PC tissues, NE tumor cells are virtually all positive for CD44 and CD44+ cells, excluding lymphocytes, are all NE tumor cells. Selective expression of the stem cell-associated marker CD44 in NE tumor cells of PC, in combination with their other known features, further supports the significance of such cells in therapy resistance and tumor recurrence.