Download Pancreatic Cancer Stem Cells: Implications for theTreatment of

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.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from clincancerres.aacrjournals.org on June 18, 2017. © 2008 American Association for Cancer
Research.