Cancer Stem Cells

CAMB 512
February 21, 2008
What makes a stem cell a stem cell?
1. Self-renewal - when a stem cell divides,
each daughter cell can either retain its
stem cell identity, or can become a
progenitor. Stem cells appear to retain
capacity for limitless division.



2. Multipotency - progenitors (also called
transit amplifying cells) have limited
proliferative capacity and are committed
to terminally differentiate into multiple
lineages (multipotent). Often have high
proliferative rates in the short term,
whereas most stem cells divide rarely.
Progenitor cell
Stem cell
A B C D
Different cell types
Embryonic stem cells derived from blastocysts - E3.5 mouse embryos
Pluripotent (able to contribute to all embryonic tissues in vivo)

Can be primed to differentiate into an increasing number of cell types in vitro

Human ES cell linestechnical, ethical, oversight issues abound
Paradigm - hematopoietic stem cells (HSCs) - found in
bone marrow in adults (yolk sac blood islands, AGM, and
fetal liver in developing embryos).

Self-renew and are multipotent - but, very few in number, hard
to identify. How can we study in the laboratory?
Bone marrow
biopsy
What are the criteria by which stem cells can be identified?
Adoptive transferthe idea behind Bone Marrow Transplant in
patients
One can also separate marrow cells by virtue
of the cell-surface proteins they express by
fluorescence activated cell sorting (FACS)

By testing these separated cells, identified
some that produce long-term (ie, permanent)
reconstitution of all blood cell lineages in
recipient mice - at least some of these are
stem cells. Short-term reconstitution
characteristic of progenitor cells.

LTR cells express specific markers, pump out
rhodamine or Hoescht dyes through ABC
transporters (side-population cells).

Very few cells (~1/10,000) in bone marrow
have LTR stem cell properties
Current view of hematopoietic differentiation from HSC - gradual process of
restricting fate. CSFs and lineage-specific transcription factors, drive
expression of critical genes
Criteria for identifying adult stem cells - need for caution
Self-renewal and
pluripotency - require
relevant assays!!

Cell purification -
ideally, single cell
resolution (markers).

Single LacZ
+
Lin
-
CD29
hi
CD24
+
cell
repopulates a
mammary fat pad.
Shackleton et. al, (2006) Nature 439: 84-88
Huntly and Gilliland (2005) NRC 5: 311-321
Recognized fairly recently
that a subset of cancer
cells seem capable of self-
renewing and producing
tumors either in vitro or in
vivo.

1994 - first real identification
of a true cancer stem cell
(AML), which can
repeatedly confer disease in
recipient mice.

How do stem cells relate to
cancer?
John Dick:
Leukemia Initiating cell: CD34
+
CD38
-

1/250,000 AML cells

NOT: CD34
+
CD38
+

CD34
-
CD38
-


Lapidot et al. (1994) Nature 367:645-
648.
Cells initiating acute myeloid leukemia
after transplantation into SCID mice
Human AML originates from primitive HSCs?
(but frequency varies)
Bonet and Dick (1997) Nat. Med. 3:730-737
Model for normal and AML hematopoietic system
Cells from 9 breast cancer patients
(pleural effusion) either purified directly
(UP) or passaged once in mice (P)

Based on previous work looking at cell
surface markers on breast ca cells,
authors purified a subpopulation of tumor
cells that express high levels of CD44
expression, low levels of CD24, etc.

ONLY these CD44
+
, CD24
-/low
cells
(~15% of total) reproducibly formed
tumors when injected into the mammary
pads of immunodeficient mice. Other
cells did NOT form new tumors, even
when 10 X more cells were injected
(exception - T7).

Consistent with idea of cancer stem
cells
Non-hematopoietic cancer stem cells
Al-Hajj et. al (2003) PNAS 100: 3983-3988
Would you have any concerns about this approach?
Histology from the injection site
Dick (2003) PNAS 100:3547-3549
Where would a cancer stem cell
come from? Transformation of
normal SC?

Alternative - dedifferentiation of a
committed progenitor through
which it gains ability to self-renew.
Recent evidence strongly supports
this idea

In either case, multiple additional
mutations are almost certainly
needed to create full-blown cancer
stem cell

Progeny of CSC arent normal, but
have limited proliferative potential
(similar to normal progenitors).
Pardal, Clarke, Morrison NRC 2003
August 2006 - report in Cell that fibroblasts can be converted into embryonic
stem-like cells when engineered to express only four specific genes!! (not
really true ES cellsbut pretty close!)
So, aspects of stem-ness can be conferred on differentiated cells with
minimal genetic changes! Implications for cancer stem cells are huge...
Takahashi and Yamanaka 2006 Cell 126: 1-14
Fibroblasts engineered to express only four specific genes - Oct-4, c-Myc,
Sox2 and KLF4, can be converted into cells indistinguishable from bona fide
ES cells. These iPS (induced pluripotent cells) can generate live-born mice!
Demonstrates that true stem-ness can be conferred on non-stem cells
From Wernig et. al (2007) Nature 448: 318-325
See also Okita et. al, (2007) Nature 448: 313-17
Maherali et. al (2007) Cell Stem Cell 1: 55-70
Also 2006, Scott Armstrongs group* (HMS) showed that if they introduced a
specific leukemic translocation (MLL-AF9) into committed progenitor cells
(GMPs), and then injected these transformed cells into mice - they get
cancer (AML)! Called cells that cause this leukemia L-GMPs.

But - the disease is transferable to new mice - hence, gained self-renewal
1/6 cells by limiting dilution. Culturing cells in differentiation medium
reduces the number of L-GMPs - so they can also differentiate into other
cells...

*Krivtsov et. al (2006) Nature 442:818-822
Self-renewal correlates to changes in
gene expression - 363 genes altered
in the self-renewing L-GMPs
compared to initial GMPs. 91 of those
363 genes have been linked to self-
renewal in other stem cells.

Implication - one transforming event
(in this case, the MLL-AF9 transgene)
may produce conditions that allow for
selection of a self-renewal program...

Details will differ for different tissues,
but - it may not require a large
number of genetic changes to make
a tumor initiating cell

Time will tell -
AML (acute myeloid leukemia)
medulloblastomas
glioblastomas
colon cancer
ALL (acute lymphoblastic leukemia)
Cancer Stem Cells Identified in Human Cancers:
Identification of human CD133
+
brain tumor
initiating cells
Singh et al. (2004) Nature 432:396-401
100 CD133
+
cells sufficient to
induce medulloblastoma in mice;
10
5
CD133
-

insufficient
OBrien et al. (2007) Nature 445:106-110
Human colon cancer cells initiating tumors in SCID mice
CC-ICs are CD133
+

Limiting Dilution Analysis
Ricci-Vitiani et al. (2007) 445:11-115
Rare CD133
+
cells in colon cancer
The big question - whats different about cancer SCs (or tumor-initiating
cells - TICs) from normal SCs?

Traditional therapies may be best at debulking majority of cancer cells in
tumors, but may be less effective at eradicating cancer SCsalso, SCs
are particularly resistant to many chemotherapeutics (ABC transporters,
etc.) - side population cells particularly good at pumping out drugs,
making them resistantobvious problem!

Multi-drug resistance (MDR)

Are we targeting the right cancer cells.?!?

If we could target cancer SCs, what about your skin, blood, gut
lining, etc.? How selective could we be?
Huntly and Gilliland (2005) NRC 5:311
Rich (2007) Cancer Res. 67:8980-8984
QuickTime and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Wang (2007) Cell Stem Cell 1:497-501
Are Cancer Stem Cells Necessarily Rare?
Somervaille and Cleary (2006) Cancer Cell 10:257-268
LSCs are frequent in mice with MLL-AF9 leukemia
Kelly et al. (2007) Science 317:337
Tumor growth need not be driven by rare cancer stem cells
Bottom line
Cancers may contain cells with some capacity to self-renew and produce
multiple transformed cell types (TICs or CSCs). Not necessarily transformed
stem cells These may represent important targets for future therapies

Genetic instability inherent to cancer cells generates mutations that can be
selected during treatment, resulting in more resistant form of the disease

Successful treatment of cancer - transforming it from a fatal to a chronic
disease - will require a combination of different treatments tailored to each
cancer as it develops. Some will target common features, some individual
ones


The challenge - how do we identify the critical Achilles heel for a given tumor
(given the large number of mutations, some of which are important, some
perhaps not), and how do we treat it without killing normal cells?