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CCR Practice of Translational Oncology Pancreatic Cancer Stem Cells: Implications for theTreatment of Pancreatic Cancer Diane M. Simeone Abstract Pancreatic cancer is a highly lethal disease that is usually diagnosed at a late stage for which there are few effective therapies. Emerging evidence has suggested that malignant tumors are quite heterogeneous and that they are composed of a small subset of distinct cancer cells (usually defined by cell surface marker expression) that are responsible for tumor initiation and propagation, termed cancer stem cells.These cells are termed cancer stem cells because, like normal stem cells, they possess the ability to self-renew and make differentiated progeny. Recent studies of human pancreatic cancers have shown a population of pancreatic cancer stem cells that have aberrantly activated developmental signaling pathways, are resistant to standard chemotherapy and radiation, and have up-regulated signaling cascades that are integral for tumor metastasis. An improved understanding of the biological behavior of these cells may lead to more effective therapies to treat pancreatic cancer. In this review, approaches to develop and test therapeutics targeting pancreatic cancer stem cells are discussed. Pancreatic cancer is a highly lethal disease that is usually diagnosed at a late stage for which there are few effective therapies. Approximately 37,000 patients will be diagnosed with pancreatic adenocarcinoma in the year 2008 and most of these patients will die in the 1st year, making it the fourth leading cause of cancer death in the United States (1). Attempts to better understand the molecular characteristics of pancreatic adenocarcinoma have focused on studying the gene and protein expression profiling of pancreatic cancer. These studies, however, have not accounted for the heterogeneity of the cells that exist within a tumor. Emerging data suggest that malignant tumors are quite heterogeneous and that tumors are composed of a small set of distinct cells termed cancer stem cells, which are responsible for tumor initiation and propagation, and a much larger set of more differentiated cancer cells, which have very limited proliferative potential. These cells have been termed cancer stem cells because like their normal stem cell counterparts, they possess the ability to self-renew and produce differentiated progeny. Much of the groundwork in isolating cancer stem cells in solid organs arose from studies in hematopoietic malignancies. Dick and colleagues in 1997 isolated the first cancer stem cell in myeloid leukemia using cell surface marker expression and the ability of human leukemia cells to engraft in nonobese, diabetic, severe combined deficiency mice and be passaged serially (2). Two important tools integral to the isolation and study of cancer stem cells, that is, fluorescence-activated cell Authors’Affiliation: University of Michigan, Ann Arbor, Michigan Received 6/26/08; accepted 6/30/08. Requests for reprints: Diane M. Simeone, University of Michigan, 2210B Taubman Center, 5343, 1500 East Medical Center Drive, Ann Arbor, MI 48109. Phone: 734-615-1600; Fax: 734-232-6188; E-mail: [email protected]. F 2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-0584 Clin Cancer Res 2008;14(18) September 15, 2008 sorting and establishment of human tumor xenograft models in immunocompromised mice, were highlighted in this study and subsequently adapted by researchers studying solid organ malignancies. With the use of these tools, the first epithelial solid organ cancer stem cell was identified in breast cancer by Al-Hajj et al. (3). This study reported a phenotypically distinct and relatively rare population of tumor cells with the cell surface marker expression of CD44+CD24-/low ESA+ that were highly tumorigenic and possessed the ability to form tumors that recapitulated the patient’s tumor in immunodeficient mice (3). Cancer stem cells have now been identified in several tumor types, including colon, prostate, head and neck, brain, liver, melanoma, and multiple myeloma (4 – 10). We recently reported the identification of human pancreatic cancer stem cells (11). Pancreatic cancer stem cells, defined by expression of the cell surface markers CD44+CD24+ESA+ (0.2-0.8% of all pancreatic cancer cells), were highly tumorigenic and possessed the ability to both self-renew and produce differentiated progeny that reflected the heterogeneity of the patient’s primary tumor. We also observed the upregulation of the developmental signaling molecules sonic hedgehog and Bmi-1 in pancreatic cancer stem cells. Recently, Hermann and colleagues found that CD133+ cells in primary pancreatic cancers and pancreatic cancer cell lines also discriminate for cells with enhanced proliferative capacity (12). CD133 had been previously used to identify a cancer stem cell population in brain and colon cancers (4, 7). Interestingly, their report states that there was an approximately 14% overlap between CD44+CD24+ESA+ and CD133+ cells (12). These findings suggest that more than one set of specific cell surface markers may enrich for pancreatic cancer stem cell populations and that a more distinguishing expression marker or set of markers to identify pancreatic cancer stem cells may yet to be discovered. From a clinical standpoint, the identification of cancer stem cells within human pancreatic cancers has important implications for treatment. In several types of cancer, cancer stem cells 5646 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 2008 American Association for Cancer Research. Cancer Stem Cells in theTreatment of Pancreatic Cancer Fig. 1. Picture of tumor spheres derived from CD44+CD24+ESA+ human pancreatic cancer stem cells. have been shown to be resistant to conventional chemotherapy and radiation therapy and are thought to be the culprit behind cancer metastasis and recurrence after clinical remission. In hematopoietic malignancies, Michor and colleagues showed that a subpopulation of human chronic myeloid leukemia stem cells were resistant to the Abl tyrosine kinase inhibitor imatinib, an agent with proven effectiveness against differentiated chronic myeloid leukemia cells (13). The human leukemic stem cells that survived imatinib treatment regenerated the tumor, providing further evidence in support of the important role played by cancer stem cells in tumorigenesis. Evidence of the resistance of brain cancer stem cells to standard therapies was shown in a study of glioblastoma cancer stem cells, where the cancer stem cell population expressing CD133+ in both primary tumors and xenografts increased 2- to 4-fold following ionizing radiation (14). This enrichment of CD133+ cancer stem cells was due to a preferential activation of DNA damage response, rendering these cells resistant to the DNA-damaging effects of radiation. Todaro and colleagues showed that CD133+ colon cancer stem cells were resistant to cell death induced by the chemotherapeutic agents oxaliplatin and 5fluorouracil and that this resistance was mediated by the expression of interleukin-4 by the CD133+ colon cancer stem cells. Treatment with an interleukin-4 receptor antagonist or an anti – interleukin-4 neutralizing antibody strongly enhanced the antitumor efficacy of these chemotherapeutic drugs through the selective sensitization of CD133+ cells (15). Some recently published data suggest that pancreatic cancer stem cells may also be resistant to chemotherapy and radiation. In a study by Hermann and colleagues, they found that CD133+ populations in the L3.6p pancreatic cancer cell line were enriched after exposure to gemcitabine (12). We have observed that treatment with ionizing radiation and the chemotherapeutic agent gemcitabine results in the enrichment of the CD44+CD24+ESA+ www.aacrjournals.org population in human primary pancreatic cancer xenografts,1 indicating that novel therapies that target cancer stem cells may improve survival in this deadly disease. Thus, if cancer stem cells seem to be the drivers in tumor initiation and maintenance, how should we proceed in determining the best way to target them? A major next step would be to perform more detailed studies to understand the biological properties of pancreatic cancer stem cells from primary human pancreatic cancers. A good place to start is to perform global gene profiling of pancreatic cancer stem cells compared with nontumorigenic pancreatic cancer cells and normal pancreatic epithelial cells to understand the signaling pathways important in pancreatic cancer stem cell function and determine which pathway or combination of pathways should be targeted. This work is currently under way in a number of laboratories. An alternative approach is to perform a small interfering RNA library screen to identify genes that are important in pancreatic cancer stem cell self-renewal. A useful in vitro assay to perform this type of work is the ‘‘sphere’’ assay. In addition to the gold standard in vivo dilutional tumor propagation assays used to identify cancer stem cells, cancer stem cells have also been identified based on in vitro sphereforming assays. It has been shown in both normal and cancerous neural tissue that the ability of cells to form colonies in spherical aggregates in nonadherent culture conditions is reflective of cells with self-renewal capacity (7, 16). Using a version of this assay with a slightly modified culture medium, we have found that single, plated CD44+CD24+ESA+ cells form such spheres that we term ‘‘tumorspheres,’’ whereas CD44-CD24-ESA- cells do not (Fig. 1). These CD44+CD24+ESA+ tumorspheres can be passaged multiple times without loss of 1 5647 Unpublished observations. Clin Cancer Res 2008;14(18) September 15, 2008 Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 2008 American Association for Cancer Research. CCR Practice of Translational Oncology tumorsphere-forming capability, and in so showing self-renewal capacity in vitro. Such a tumorsphere assay could be used to screen for other potential cell surface markers to identify pancreatic cancer stem cells, or to perform high-throughput drug or small interfering RNA screening, as the tumorsphere assays can be done quickly and more cheaply than the in vivo tumor xenograft studies, reserving the in vivo tumor xenograft studies to validate findings observed in the tumorsphere assays. It will be important to use primary pancreatic cancer cells for such screens, as in our experience pancreatic cancer cell lines with both the presence and absence of cancer stem cell markers possess the ability to form tumorspheres, suggesting that the long-term passaging of primary cells in serum-containing culture medium alters the properties of cell lines such that they no longer reflect what is observed in primary pancreatic cancer cells. This has been observed in brain cancers (17), where unlike traditionally grown tumor cell lines, brain cancer cells perpetuated in serum-free, nonadherent conditions (tumorsphere assays) recapitulated the genotype, gene expression patterns, and in vivo biology of human glioblastomas. An important aspect of testing potential cancer stem cell therapeutics will be the utilization of an optimal preclinical model system. We consider the primary pancreatic cancer orthotopic xenograft model system as optimal for testing potential therapeutics, as this model system best reflects the tumor heterogeneity that is observed in actual patients. The validity of this model system may be strengthened by the cotransfer of appropriate human pancreatic stromal cells, because pancreatic cancer produces a dense desmoplastic reaction, and it has been shown that pancreatic cancer cells are less responsive to therapeutic agents when in the presence of an active pancreatic stroma (18). As there is much yet to be learned about the function of pancreatic cancer stem cells, clinical trials with agents designed to target cancer stem cells will soon be upon us. In fact, the first clinical trial to target cancer stem cells in human cancer, specifically breast cancer, using a g secretase inhibitor to block Notch signaling is under way. Many pharmaceutical companies are investing heavily in developing new therapeutics to target cancer stem cells, and more efficient inhibitors of several developmental signaling pathways are currently being developed or in the early phases of testing (19). An important issue that will need to be sorted out before embarking on clinical trials to target pancreatic cancer stem cells will be determining the best way to measure the efficacy of these new therapies. Traditionally, the effectiveness of cancer agents is measured by tumor shrinkage. Tumor response is usually defined as tumor shrinkage by at least 50%. If cancer stem cells are resistant to therapy and make up a very small percentage of cells within the tumor, the effect of therapeutics may reflect the effect on the differentiated, nontumorigenic cancer cells, rather than cancer stem cells. For clinical trials testing pancreatic cancer stem cell therapeutics, new measures of efficacy will need to be devised. What will be the best cell surface markers to use to measure cancer stem cell burden? Can we use the immunohistochemistry of biopsy samples to measure cancer stem cell content, or will cell sorting be needed, and if so, how difficult will it be to do these assays on small tissue samples? Alternatively, perhaps the measurement of circulating cancer stem cells in patients can be used as a readout of treatment effect. Several recent reports using microfluidics-based technology and spectral imaging suggest that this may be possible (20 – 22), avoiding the potential need to biopsy the pancreas to assess treatment response. It may be that combination therapies that target both the pancreatic cancer stem cell population and the differentiated, nontumorigenic bulk population of pancreatic cancer cells will be most efficacious in treating patient symptoms associated with tumor mass and resulting in longterm cure. Upcoming clinical trials will help us determine if this indeed is the case. Disclosure of Potential Conflicts of Interest D.M. Simeone receives research funding from Oncomed pharmaceuticals. References 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57: 43 ^ 66. 2. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730 ^ 7. 3. Al-Hajj M,Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983 ^ 8. 4. O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445:106 ^ 10. 5. Collins AT, Berry PA, Hyde C, et al. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 2005;65:10946 ^ 51. 6. Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 2007;104:973 ^ 8. 7. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature 2004;432: 396 ^ 401. 8. Yang ZF, Ho DW, Ng MN, et al. Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008;13:153 ^ 66. 9. Fang D, Nguyen TK, Leishear K, et al. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 2005;65:9328 ^ 37. 10. Matsui W, Huff CA, Wang Q, et al. Characterization of clonagenic multiple myeloma cells. Blood 2004; 103:2332 ^ 6. 11. Li C, Heidt DG, Dalerba P, et al. Identification of pancreatic cancer stem cells. Cancer Res 2007;67: 1030 ^ 7. 12. Hermann PC, Huber SL, HerrlerT, et al. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 2007;1:313 ^ 23. 13. Michor F, HughesTP, IwasaY, et al. Dynamics of chronic myeloid leukaemia. Nature 2005;435:1267 ^ 70. 14. Bao S,Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444: 756 ^ 60. 15. Todaro M, Alea MP, Di Stefano AB, et al. Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 2007;1:389 ^ 402. 16. Ishibashi S, Sakaguchi M, Kuroiwa T, et al. Human neural stem/progenitor cells, expanded in long-term Clin Cancer Res 2008;14(18) September 15, 2008 5648 neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils. J Neurosci Res 2004;78:215 ^ 23. 17. Lee J, Kotlairova S, KotlairovY, et al.Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 2006;9:391 ^ 403. 18. Hwang RF, Moore T, Arumugam T, et al. Cancerassociated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 2008;68:918 ^ 26. 19. Garber K. Arrested development? Notch emerges as new cancer drug target. J Natl Cancer Inst 2007;99: 1284 ^ 5. 20. Balic M, Lin H, Young L, et al. Most disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 2006;12:5615 ^ 21. 21. Zheng S, Lin H, Liu J-Q, et al. Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells. J Chromatogr A 2007;1162:154 ^ 61. 22. Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature 2007;450:1235 ^ 9. www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 2008 American Association for Cancer Research. Pancreatic Cancer Stem Cells: Implications for the Treatment of Pancreatic Cancer Diane M. Simeone Clin Cancer Res 2008;14:5646-5648. Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/14/18/5646 This article cites 22 articles, 9 of which you can access for free at: http://clincancerres.aacrjournals.org/content/14/18/5646.full.html#ref-list-1 This article has been cited by 10 HighWire-hosted articles. Access the articles at: /content/14/18/5646.full.html#related-urls Sign up to receive free email-alerts related to this article or journal. 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