“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 lines…technical, 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 transfer…the 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-

CD29hiCD24+ 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 aren’t 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 cells…but 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: 313-17Maherali et. al (2007) Cell Stem Cell 1: 55-70

Also 2006, Scott Armstrong’s 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; 105 CD133- insufficient

O’Brien 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 - what’s 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 SCs…also, SCs are particularly resistant to many chemotherapeutics (ABC transporters, etc.) - “side population” cells particularly good at pumping out drugs, making them resistant…obvious 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 aTIFF (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…?