1. Cancer stem cells in solid tumors Laurie E Ailles and Irving L Weissman Stanford University School of Medicine Current opinion in biotechnology 2007, 18:
R4. Jae Joon Han, M.D.

2. The cancer stem cell model
Cells with stem cell quantities have been identified
in malignancies of hematopoietic origin and in some solid tumors.

3. Identification of markers for the prospective isolation of cancer stem cells
AACR(American association for cancer research) workshop
‘cancer stem cells can be defined experimentally by their ability to
recapitulate the generation of a continuously growing tumor’
The most widely accepted assay to validate a candidate CSC
Tumor initiation in immunocompromised mice
Serial transplantation in immunocompromised mice

4. The identification of AML stem cells
The AML stem cells with the phenotype CD34+CD38- and CD90-
Represented 0.1-1% of the total cells
Were the only cells able to initiate AML that resembled the human disease
in NOD/SCID mice
Adding in vitro assays
The phenotype of the AML stem cell indicates that they probably originate in the
hematopoietic stem/progenitor cell compartment, possibly from the multipotent progenitor
In CML that has progressed to blast crisis
The progression step is linked to the activation of a self renewal program
in the granulocyte-macrophage progenitor population
thus conferring cells that normally cannot self renew with CSC properties

5. Transformation
While CSCs may derive from normal stem cells, this may not be the case in all tumors.
More differentiated cells could acquire self renewal capacity
through multiple mutagenic events,
leading them to become the CSC that is driving tumor growth

6. In order to identify CSCs
Patient-derived tumor cells are stained with labeled antibodies to various cell surface markers, either singly or in combination
Techniques such as magnetic columns or fluorescence-activated cell sorting (FACS) are used to separate labeled versus unlabeled populations of cells.
These populations are then assayed for their ability to initiate tumor growth in immunocompromised mice. (NOD/SCID mice) 
Immunostaining and flow cytometric analysis of the engrafted tumor
Must then show recapitulation of all of the original populations that were in the primary tumor
Upon resorting of the CSC phenotype and retransplantation, a fully heterogeneous tumor must again be initiated.
Ultimately, clonal marking, for example by retroviral insertion sites, and the demonstration of multiple secondary tumors containing the same clone will be necessary to truly demonstrate self renewal.

7. In the case of solid tumors the challenge of identifying and characterizing CSCs is significant
The normal tissue developmental hierarchy has not been identified or characterized
This makes the section of candidate markers more difficult, and provides no basis for comparison between normal and malignant cells, or means to identify the cell of origin.
In vivo models of human solid tumors can also be challenging to achieve
In some cases orthotopic injection is not feasible or is technically challenging
e.g. lung, colon, and bladder cancers
In these cases, subcutaneous injection or implantation under the kidney capsule
can be successful, but it is essential to confirm that these xenografts recapitulate the
histology of the patient tumors.

8. The first to identify and prospectively isolate a minority subpopulation of cells from a human solid tumor
Lineage-CD44+CD24-/low : initiate tumors in immunocompromised mice with as few as 200 cells, while as many as 500,000 or more of the remaining cells in the tumor did not initiate new tumors in mice.

9. Cell surface phenotype of CSCs identified in human cancers
Tumor type
CSC phenotype
Breast
CD44+ CD24-/low
CNS
CD133+
Multiple myeloma
CD138-
Melanoma
CD20+
Prostate
CD44+ α2β1+ CD133+
HNSCC
CD44+
Colon
CD44+ EpCam+ CD166+
Pancreatic
CD44+ EpCam+ CD24+

10. CSCs identified based on their ability to form colonies in vitro
Sphere-forming ability as a measure of stem cells was first developed
for central nervous system (CNS) cells
Human CNS tumors can form spheres when cultured under the appropriate conditions.
The neurosphere-forming cells could be prospectively isolated from fresh tissue
using the cell surface marker CD133, and that the CD133+ cells did indeed initiate brain
tumors in vivo, without any in vitro manipulation, indicating that they do in fact represent CSCs.

11. CSCs identified based on their ability to form colonies in vitro
CD44+ α2β1+ CD133+ possess a significant capacity for self renewal, differentiation,
and invasiveness in vitro.

12. Understanding the biological properties of CSC
The characterization of their molecular and biological properties, in hopes of identifying ways to specifically target and eradicate these cells in cancer patients.
Global gene expression profiling
Identification of self renewal signaling pathways in CSCs
Identification of a CSC niche
Are CSCs selectively resistant to conventional therapies?

13. Global gene expression profiling
The gene expression profile of CD44+CD24-/low tumorigenic breast cancer cell
vs. normal breast epithelium.
186 gene invasiveness gene signature is strongly associate with
metastasis-free survival and overall survival in high-risk early breast cancer patients.

14. Global gene expression profiling
CSC phenotype expressed stem/progenitor-associated gene
Non-CSC expressed differentiation associated genes.
Correlation of their gene expression signature with patient clinical outcome
Identified a TGF-β signaling pathway specifically active in the CSC population
Inhibition of TGF-β signaling pathway in vitro led to differentiation

15. Identification of self renewal signaling pathways
BMP-1
Self renewal of hematopoietic and neural stem cells
Self renewal of leukemic stem cells
Overexpressed in human AML compared to normal bone marrow
The Hedgehog pathway
Recently related specifically to human CSCs for multiple myeloma
Hh pathway can be inhibited by cyclopamine treatment
BMPs and their antagonists
Regulating homeostasis of various organs and tissues via the control of differentiation, proliferation, and apoptosis

16. Identification of self renewal signaling pathways

17. Identification of a CSC niche
Normal stem cell and their microenvironment or niche
Maintain their quiescent and undifferentiated state
Maintain their proliferation and differentiation potentials
Nonepithelial stromal cells
Inflammatory cells
Vascular endothelial cells
Fibroblasts

18. BMPs can induce differentiation and block expansion of normal stem and progenitor cells.
BMP antagonists can reverse this effect and favor expansion.
Signals that are critical for stem cell self renewal, such as BMP antagonists,
derive from the stem cell niche. BMPs may often come from epithelial or stem cells.
In tumor biology, BMPs can also inhibit expansion of tumor cells.
BMP antagonists secreted by the tumor cell niche may reverse this inhibition, allowing tumor cell expansion to continue.

19. Conventional model of tumor-cell drug resistance
Rare cells with genetic alterations that confer multidrug resistance (MDR) form a drugresistant clone.
Following chemotherapy, these cells survive and proliferate, forming a recurrent tumour that is composed of offspring of the drug-resistant clone.

20. Are CSCs selectively resistant to conventional therapies?
Normal stem cells ≈ tumor stem cell
relatively quiescent
expression of drug efflux pumps
active DNA-repair capacity
resistance to apoptosis
Tumours contain a small population of tumour stem cells (red) and their differentiated offspring, which are committed to a particular lineage (blue). Following chemotherapy, the committed cells are killed, but the stem cells, which express drug transporters, survive. These cells repopulate the tumour, resulting in a
heterogeneous tumour composed of stem cells and committed but variably differentiated offspring.

21. Are CSCs selectively resistant to conventional therapies?
AML stem cells
Selectively resistant to daunorubicin and Ara-C
Largely quiescent
Express drug efflux molecules : ABCG2 (ATP-binding cassette drug transporter)
CD133+ brain tumor stem cells
Selectively resistant to radiation
Activation of DNA damage checkpoint response
Increase in DNA repair capacity

22. Conclusion
CSCs are associated with the stromal components of the tumor, including fibroblasts and/or blood vessels, which make up the CSC ‘niche’. The niche cells secrete factors that support CSC self renewal.
CSCs retain differentiation potential, giving rise to non-self renewing tumor cells that make up the bulk of the tumor.
CSCs may be drug and/or radiation resistant, and may express CSC-specific antigens.