**Executive Summary of the Tumor Stem Cell & Self-Renewal Genes Think
Tank**

The Tumor Stem Cell and Self-Renewal Genes Think tank was convened to
identify the major scientific issues in the field and provide
recommendations to the NCI to help advance research in this area. The
participants came to the following scientific conclusions:

-   Efforts are required prospectively to isolate and purify the tumor
    initiating cells (T-IC) from a larger variety of hematological and
    solid tumors. Since many tissues do not have as rich a source of
    cell surface markers as the blood system, effort will need to be
    expended to develop the means for T-IC purification.

-   The normal tissue stem cell is a likely target for the initial
    carcinogenic insult, because it has the long life necessary for
    accumulating the multiple genetic or epigenetic changes required for
    malignant transformation.

-   Although the events required for malignant transformation may all
    accumulate in the normal tissue stem cell, this cell does not
    necessarily become the tumor stem cell.

-   It appears that the genetic and biochemical pathways regulating the
    "stem" phenotype in normal stem cells are subverted later to
    maintain the tumor stem cell phenotype.

-   There is evidence to suggest that epigenetic mechanisms can produce
    an inheritable tumor stem cell phenotype. Epigenetic control also
    underlies a great amount of stem cell regulation, as exemplified by
    the polycomb gene Bmi-1. Thus, there is a link between normal stem
    cell regulation and the control of cancer stem cells.

Finally, it was recommended that the National Cancer Institute:

-   Continue support for opportunistic basic research into tumor stem
    cell biology.

-   Develop a Research Consortium to encourage transdisciplinary
    approaches and to provide specialized research reagents.

    **Introduction**

The clonal nature of most malignant tumors is well established.
Experiments spanning several decades have shown, however, that as many
as 106 murine or human tumor cells are required to transplant a new
tumor from an existing one. Two theories have been developed to account
for the observation that apparently not every tumor cell is a tumor
initiating cell (T-IC). The stochastic theory predicts that every tumor
cell can form an entirely new tumor; however, entry into the cell cycle
is a stochastic event with low probability. Alternatively, tumor cells
may exist in a hierarchical state in which only a small number of cells
possess tumor initiating potential. If the stochastic model is correct
then tumor cells are biologically homogeneous and genetic or epigenetic
programs that allow for tumorigenesis are operative in the majority of
cells that comprise a tumor. The hierarchical model, however, predicts
the tumor cells possess a functional heterogeneity and that
quantitatively the cells capable of tumorigenesis are a relatively minor
population among the bulk of tumor cells.

Recent data from both hematologic malignancies and solid tumors have
suggested that there are only minor populations of cells in each
malignancy that are capable of tumor initiation. These tumor initiating
cells have the functional properties of a tumor stem cell. They appear
to be capable of asymmetric division and self renewal, and are only a
minor faction among the bulk of more differentiated cells in the tumor.
These observations have profound implications for tumor biology research
as well as successful tumor therapy. The Tumor Stem cell Think Tank
addressed several of the outstanding scientific questions that will
define research in this area, as well as the needs of the research
community to promote progress in this research.

\(1\) [What is the current evidence for tumor initiating stem cells
among tumors arising from a variety of tissues?]{.underline}

Currently, tumor stem cells have been isolated and characterized in
several hematologic malignancies and two solid tumors. The critical
experimental design that underlies all these studies is the development
and use of a functional assay for tumor establishment and the
prospective isolation of the T-IC. Often normal tissue stem cell markers
are used to identify these populations, but a functional assay such as
transplantation of human leukemic stem cells into immunodeficient murine
models such as the NOD/SCID mouse is most important in identifying tumor
initiating cells. One of the first tumors in which a stem cell was
identified was acute myeloid leukemia (AML). In this disease, the
frequency of the leukemic stem cell (LSC) was approximately 1 per
million AML blasts, establishing that not every AML cell had LSC
capacity. A CD34+, CD38- cell fraction representing 0.1-1% of the tumor
cells possessed all the leukemia initiating activity in the NOD/SCID
model. By contrast, the CD34+, CD38+ cells and the CD34- cells, which
comprise most of the cells in the tumor, could not initiate leukemia. A
multiple myeloma stem cell has also been characterized. Multiple myeloma
cell lines and primary patient derived cells express the cell surface
marker syndecan -1 (CD138). Expression appears during the course of
B-cell differentiation. A population of cells representing \<5% of the
cells in the bulk population of multiple myeloma cells were found to be
CD138- and possessed in vitro clonogenic potential. These cells also
engrafted successfully into NOD/SCID mice, whereas CD138+ cells did not
engraft. CD138- cells were also CD19+ and CD20+, and they expressed
higher levels of KI67 (a cell proliferation antigen) than CD138+ cells.
Recently a mammary carcinoma stem cell has been isolated primarily using
three cell surface markers (CD44, CD24, and epithelial specific
antigen). The tumor initiating capacity of the cells was verified in a
NOD/SCID engraftment assay, and the T-ICs represented only 2% of the
unfractionated cells.

Finally, a putative brain tumor stem cell has also been isolated. These
cells appear to be between 0.3 - 25 % of the cells in the brain tumors
examined. They are positive for the neural stem cell marker CD133 and
have a marked capacity for self renewal and differentiation.
Transplantation of these putative neural tumor stem cells into the
forebrains of NOD/SCID mice yields tumors phenotypically resembling the
tumors from which the stem cells were isolated.

The think tank participants arrived at a consensus that:

A.  Isolation of stem cells from more hematological malignancies and
    solid tumors is required to validate the general nature of the
    presence of stem cells in tumors.

B.  Functional assays, such as NOD/SCID mouse engraftment and single
    cell tumor initiating capacity assays, are necessary to establish
    the true stem nature of isolated cells. Cell surface morphological
    markers alone are insufficient to accurately characterize stem
    cells.

C.  Efforts are needed to isolate prospectively and purify the T-IC.
    Since many tissues do not have as rich a source of cell surface
    markers as the blood system, effort should be expended to develop
    the means for T-IC purification.

    \(2\) [Is the tumor stem cell a derivative of a normal tumor stem
    cell or a later more differentiated progenitor cell?]{.underline}

It is unclear whether tumor stem cells arise exclusively from normal
tissue stem cells, or from progenitors that have differentiated from the
stem cell itself. From a theoretical standpoint, it has been well
established that neoplasia arises as a consequence of the acquistion of
multiple oncogenic events. Thus, the initial events must occur in cells
that persist. Hierarchical differentiation of blood cells from a stem
and progenitor population has been well studied and characterized in the
hematopoietic system. It is thus not surprising that a great deal of
evidence on the nature of the tumor stem cells is available from studies
of hematopoietic malignancies. In AML, the stem cell population appears
to share many of the markers of the normal hematopoietic cell (e.g.
CD34+, CD38-, HLA-DR-); however the leukemic stem cell appears to
overexpress the IL-3 Rα subunit (CD123) relative to normal hemapoietic
stem cells (HSC). The similar cell surface properties, self-renewal
properties, and complexity of stem cell hierarchies, as determined by
clonal stem cell tracking of both LSC and normal stem cells, suggest
that they are closely related and have been interpreted to suggest that
LSC are derived from HSC. In the blood system, only the stem cells have
the long-life required to accumulate all the initiating mutations and
these could be passed to the progeny that are continuously produced from
such stem cells.

In experimental murine systems, it has been found that the more
committed myeloid progenitor cells, especially those employing the MLL
oncogene, are also capable of becoming tumor stem cells. This gene codes
for a transcription factor that regulates Hox gene expression. The Hox
genes have been implicated in stem/progenitor cell expansion and self
renewal. MLL fusion proteins created by chromosomal translocations are
frequently associated with acute lymphoid and myeloid leukemias.
Transduction of an MLL fusion protein into purified populations of
either hematopoietic stem cells or more committed granulocyte macrophage
progenitors gives rise to cells that produce a rapid AML. Although the
disease is similar with both populations, the stem cell fraction is much
more potent. Thus, a more differentiated cell can also act as a tumor
initiating stem cell if the genetic alteration endows this cell with
self-renewal capacity. In chronic myelogenous leukemia (CML), the
t(9;22) translocation joining the BCR and ABL genes is found in
hematopoeitic stem cells, but the mRNA and protein for the fusion gene
are found only in later progenitrors. In AML patients carrying the AML-1
Eto translocation in hematopoietic stem cells, the stem cell fraction is
still capable of normal differentiation, but the more commited cells
possess clonogenic leukemia potential only, suggesting that the AML/ETO
translocation created a pre-leukemic stem cell. Additional alterations
occurred in the more committed cells to create a frank leukemia. Thus,
the tumor stem cell can arise in hematologic malignancies from multiple
alterations occurring in normal stem cells, or the initial alteration
could occur in the stem cell, with additional hits occurring in a more
differentiated progenitor cell. Finally, it is also possible that under
some rare circumstances, the initiating event could occur in the
committed progenitor to convert it into a self renewing stem cell. It
will be important to determine if the leukemogenic pathways obtained in
experimental murine systems are recapitulated in the human disease.

Mammary tumor stem cells are CD44+, CD24- , and epithelial stem antigen
positive, and this phenotype overlaps with that of epithelial stem
cells. Definitive evidence, however, that these cells arise from the
normal mammary tumor cells is thus far lacking.

The tumor stem cell isolated from a variety of brain tumors, including
slowly proliferating astrocytomas and highly malignant medulloblastomas
and glioblastomas, contain tumor stem cells that express the CD133
antigen and nestin found on normal neural tumor stem cells. The neural
tumor stem cell does not contain any of the markers characteristic of
more differentiated neural cells. Again, these data suggest that the
normal neural stem cell is the precursor of the brain tumor stem cell.
This conclusion about neural tumors is supported by molecular genetic
studies performed in neural cells. Simultaneous knockout of p53 and NF-1
in neuronal precursor cells results in the development of brain tumors.
Imaging studies demonstrate that the tumors arise in two areas: the
subventricular zone of the lateral ventricle and the hippocampus. These
areas of the brain have been previously shown to be reservoirs of normal
neural stem cells. Studies of the generation of neurofibromas from
Schwann cells have also shown that primitive neural crest cells can
become tumor stem cells. The transcription factor Krox20 participates in
the differentiation of these Schwann cells, but has recently also been
shown to become expressed in the primitive neural crest. Homozygous
deletion of the Krox 20 gene using a Krox 20 -- Cre system causes some
of the deleted cells to become neurofibromas. It is postulated that the
cells must accumulate other stochastic events (e.g. loss of NF-1) to
become fibromas, because all the cells do not become transformed.

The participants concluded the following:

A.  Among the solid tumor stem cells identified there is an overlap
    between the phenotype of the normal tissue stem cell and the tumor
    stem cell, but definitive evidence as to whether tumor stem cells
    are actually transformed normal stem cells is not yet available

B.  In the hematologic and neural malignancies there is evidence that
    tumor stem cells may be heterogeneous. Some may arise from the
    normal stem cells population, but others may arise from more
    differentiated progeny cells that have acquired self renewal
    capacity

C.  The normal tissue stem cell is a likely target for the initial
    carcinogenic insult, because it has the long life required to
    accumulate the multiple genetic or epigenetic changes required for
    malignant transformation.

D.  Although the events required for malignant transformation may all
    accumulate in the normal tissue stem cell, this cell does not
    necessarily become the tumor stem cell.

    \(3\) [What genetic pathways may be important in maintaining the
    tumor stem cell state?]{.underline}

The proteins involved in self renewal in normal tissue stem cells appear
to be subverted in tumorigenesis to allow the tumor initiating cells to
maintain self renewal capacity. Two families of proteins related to self
renewal were considered in detail: the poycomb gene Bmi-1 and the Wnt
signaling pathway proteins. The polycomb genes have an essential role in
embryogenesis, regulation of the cell cycle and lymphopoieisis. These
genes are essential for the silencing of other families of genes. It has
been shown by RT-PCR analysis that knockout of the polycomb gene Bmi-1
in mice results in a progressive loss of all hematopoietic lineages.
This loss results from the inability of the Bmi-1 (-/-) stem cells to
self renew. Bmi-1 (-/-) cells displayed altered expression of the cell
cycle inhibitor genes p16 INK4a and p19ARF, and down regulation of a
gene coding for an inhibitor of apoptosis. The p16 and p19 proteins
interact with the p53/Rb regulated cell cycle pathways. Introducing
genes known to produce acute myeloid leukemia (AML) into Bmi-1(-/-)
hematopoeitic stem cells (fetal liver cells) induced AML with normal
kinetics. However, the Bmi-1(-/-) leukemic stem cells from primary
recipients were unable to produce AML in secondary recipients. These
results demonstrate that Bmi-1 is also required for self renewal of
leukemic stem cells in AML.

Another group of genes involved in self renewal are those involved in
the Wnt signal transduction cascade. The Wnt protein binds to a receptor
called Frizzled and activates cell fate decisions during tissue
development. It has been shown that deletion of the TCF-4 gene, a
transcription factors at the end of the Wnt signal transduction cascade,
causes early neonatal death in mice. The mice lacking the gene have a
single histological defect - the intestinal stem cell lining is absent.
It has also been shown that inhibitors of Wnt signaling leads to
inhibition of hematopoietic stem cell growth [in]{.underline}
[vitro]{.underline} and reduced hematopoietic reconstitution
[in]{.underline} [vivo]{.underline} . Activation of Wnt signaling in
hematopoeitic stem cells leads to increased expression of Hox B4 and
Notch-1 genes previously replicated in self renewal of hematopoietic
stem cells. The Wnt signaling pathways has been shown to be involved in
both hematopoietic malignancy and colon carcinoma.

Although the Wnt ligands themselves are only rarely involved in
tumorigenesis, mutations mimicking Wnt receptor (Frizzled) activation
induce a set of genes associated with repression of differentiation and
potentiation of self renewal. In general, these mutations involve Wnt
signal transduction proteins: activation of ß- catenin and inactivation
of the (APC) adenomatosis polyposis coli protein. In myeloid leukemia,
non-phosphorylated ß-catenin accumulates in granulocyte macrophage
progenitors as they progress toward leukemia. These normally more
committed progenitors can thus acquire self renewal properties. A
similar accumulation of non-phosphorylated ß-catenin has also been
observed in multiple myeloma cells. In colon cancer, the APC gene is
mutated early in the development of 90% of colon carcinomas. Similarity
in gene expression patterns between populations of colon cancer cells
and colon epithelial stem cells has also been observed by DNA microarray
analysis. It is possible that mutations in the Wnt signaling pathway
maintain the program of stem cell genes in the "on" position.

Two other proteins that may play a role in tumor stem cell biology are
nucleostemin and the tumor suppressor PTEN. Nucleostemin is abundant in
self renewing cells such as mouse embryonic and neural stem cells as
well as several human cancer cells. Although the exact function of
nucleostemin is not yet known, it behaves like a molecular switch to
control cell division, perhaps through binding to p53. Knockout of the
PTEN phosphatase in prostate cancer cells allows the expression of genes
associated with metastasis. As metastatic cells are likely prostate
tumor stem cells, it is likely this gene may regulate expression of stem
cell related genes.

In conclusion, it appears that the genetic and biochemical pathways
regulating the "stem" phenotype in normal stem cells are subverted to
maintain the tumor stem cell phenotype.

\(4\) [Can the tumor stem cell phenotype be epigenetically programmed or
reprogrammed?]{.underline}

Human tumor cells often demonstrate abnormal patterns of DNA
methylation. DNA methylation provides an epigenetic mechanism for
altering gene expression by silencing genes. Hypermethylation frequently
underlies the silencing of tumor suppressor genes. The opposite
condition, in which DNA is hypomethylated, often in concert with
regional hypermethylation, has been observed in a spectrum of human
tumors. That such hypomethylation can have a fundamental effect on tumor
cell development has recently become clear. Transgenic mice that are
heterozygously deleted for a DNA methyltransferase show a substantial
decrease in total genomic methylation in all tissues. At 4 to 8 months
of age, these mice develop an aggressive T-cell lymphoma with a high
frequency of trisomy in Chromosome 15. There appears to be a link
between DNA hypomethylation and chromosomal stability. Chromosome 15 is
frequently duplicated in T-cell lymphomas and the c-myc oncogene is
located on this chromosome. c-Myc is overexpressed in many of the
hypomethylated tumors and in T-cell lymphomas as well.

Further evidence that hypomethylation may have a causal role in
carcinogenesis was obtained by crossing the DNA methylase heterozygote
mice with mice prone to develop soft tissue sarcoma with simultaneous
loss of heterozygosity (LOH) of the NF-1 and p53 genes. The resulting
transgenic mice develop sarcomas at an earlier age and demonstrate an
increase in LOH in the hypomethylated versus the normally methylated
cells. The increase in the rate of LOH is the result of a specific
effect of hypomethylation on the stability of pericentric and
centromeric chromosomal regions.

Other experiments with nuclear cloning provide evidence that the
tumorigenic phenotype of tumor stem cells can be reprogrammed
epigenetically. Introducing the nucleus of a murine melanoma cell into
an enucleated murine ovum with subsequent transfer into a surrogate
female resulted in a normal offspring. The neural crest derived cells
including melanocytes were all normal in the resulting mouse. Thus, the
micro-environment induced a genetic reprogramming. However, the genetic
mechanisms that existed in the tumor nuclei were maintained as these
mice had a high incidence of neoplasia. Thus, both genetic and
epigenetic mechanisms may be active in neoplastic development. This
result with melanoma was a rare experimental success among a number of
tumor nuclei tested, and the reasons for the lack of repeated success
with nuclei from other tumors is under investigation.

That the microenvironmental niche that a cell resides in can have
profound effects on the cellular phenotype is also demonstrated by
studies on the fate of embryonic stem cells derived from the blastocyst
inner cell mass. When they are transplanted back into another embryo,
they maintain their normal phenotype. If, however, they are transplanted
into a differentiated microenviroment such as kidney or liver, the cells
form a teratoma.

In conclusion, there is evidence to suggest that epigenetic mechanisms
can produce an inheritable tumor stem cell phenotype. Epigenetic control
also underlies a great amount of stem cell regulation as exemplified by
the polycomb gene Bmi-1, thus providing a link to the control of cancer
stem cells.

[Future Directions:]{.underline}

At the conclusion of the Think Tank, a number of important future basic
research questions were synthesized from the scientific discussion. They
are summarized below:

1)  What governs the rate of proliferation of stem cells and in
    particular tumor stem cells? A corollary question would be whether
    the size or quality of the stem cell microenvironment (niche) acts
    as a constraint on stem cell growth?

2)  What creates the stem cell niche for a tumor stem cell (i.e. do
    tumor stromal cells constitute the tumor stem cell niche?)

3)  Does the transition from semi-linear to exponential cell growth
    occur when the tumor cells become independent of the stromal niche?

4)  Can oncogenes and their associated mutations affect asymmetric
    versus symmetric divisions in stem cells?

5)  Is the object of Darwinian selection in the tumor the T-IC, rather
    than the more differentiated tumor cells? It is likely that alleles
    for malignancy spread in a tissue because they arise in stem cells
    and provide advantages to the tumor stem cells that carry them.

6)  Are the phenotypes of invasion and metastasis uniquely connected to
    the tumor stem phenotype?

7)  Stem cell quiescence versus growth and differentiation must
    ultimately be understood in terms of progression through the cell
    cycle. It will be important to determine whether the retinoblastoma
    (Rb) gene product is as critical in this process as it appears. Rb
    plays a key role in cell cycle progression and differentiation in a
    number of tissues. Hypophosphorylation of Rb forces cells to leave
    the cell cycle and enter Go, therefore regulation of this protein is
    likely to play a role in regulating true stem cell state. This is
    substantiated by studies on the Bmi-1 gene and the genes it regulate
    that interact with the Rb/p53 cell cycle pathway.

8)  Most human carcinogens are strong tumor promoters and weak
    initiators of carcinogenesis. If tumor promoters work by increasing
    the size of the target population, then they must work by increasing
    the population of already initiated cells. Thus, it is important to
    understand whether tumor promoters work on initiated epithelial stem
    cells or on stromal cells, as the stromal cells may control the size
    of the stem cell niche.

9)  Can the roles of mutation and epigenetic mechanisms be distinguished
    in the generation of the tumor stem cell phenotype?

    In addition a number of questions related to the future of cancer
    therapy were also considered.

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1)  Can the current benchmark for measuring the success of cancer
    therapy, tumor shrinkage, be changed to something more biologically
    relevant? The success of therapy can only really be measured by
    understanding the effect of the therapy on the tumor stem cell.
    Unfortunately this is difficult to obtain because of the lack of
    tumor stem cell markers.

2)  Can we develop xenograft models that recapitulate the stem cell
    transition to more differentiated progenitors in a tumor? Such
    models might be useful to begin understanding the effects of therapy
    on tumor stem cells.

3)  Are there methods for treating tumors that might cause a collapse of
    the stem cell niche? It is likely that most human tumors depend on
    stromal cells that define the niche and can control the size of the
    stem cell niche.

4)  Are the cells that form the tumor stem cell niche different from
    those forming the normal tissue stem cell niche? Might any
    differences present an opportunity for directing selective therapy
    to tumor stromal cells?

5)  Finally, might tumor stem cells be more or less sensitive to
    apoptosis inducing stimuli? Such information is critical in
    designing therapies that destroy the tumor stem cells responsible
    for continued expansion of the tumor and subsequent metastasis.

    **Specific Recommendations for the NCI:**

    [Technical Recommendations:]{.underline}

1\) Develop methods to encourage surgeons involved in clinical research
to collect tumor specimens directly into trypsin disaggregation
solutions and provide viable freezing of cells in large banks to allow
scientists to isolate unselected populations of tumor stem cells.

2\) Encourage the development of real time PCR technology to examine
gene expression in tumor stem cells. This technique appears to give more
valuable information than DNA microarray technology for analysis of stem
cells.

3\) Improve [in vivo]{.underline} and [in vitro]{.underline} functional
assays for tumor stem cells to allow for more accurate identification of
these cells in concert with cell surface phenotype identification.

4\) Improve [in vivo]{.underline} organotypic assays to understand
symmetric versus asymmetric cell division.

**[Administrative Recommendations:]{.underline}**

1\) Continue National Cancer Institute support for opportunistic basic
research into tumor stem cell biology.

2\) Develop a Research Consortium to facilitate transdisciplinary
approaches and to provide specialized research reagents that will
advance research in this area.