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PATH30001 - Mechanisms of Disease > Lecture 33 - Cancer Stem Cells > Flashcards

Lecture 33 - Cancer Stem Cells Flashcards

1
Q

When were cancer stem cells first characterised?

A

1995/7
i.e. only 20 years ago

This is quite a new field

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2
Q

In general, what does tumour initiation stem from?

A

Spontaneous/stochastic
or
Environmentally induced

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3
Q

Is initial genetic damage leading to tumour growth lethal or non-lethal?

A

Must be non-lethal (i.e. bypass cell checkpoints

If the cell dies there is no propagation of the tumour

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4
Q

What must be the proliferative capacity of cells that give rise to tumours?

A

Must be able to proliferate

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5
Q

Describe the role of driver genes in tumour growth

A 
Driver genes:
 • Oncogenes
 • TSGs
 • Genes involved in apoptosis
 • Genes involved in DNA repair

Tumour development frequently requires alteration in at least two of these driver genes

Alterations usually need to affect both alleles of a driver gene for maximal impact

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6
Q

What are passenger mutations?

A

Do not have an impact on or drive the tumour

They arise because the cells are becoming more and more genetically unstable

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7
Q

Describe heterogeneity between cancer types

A 
Heterogeneity between:
1. Cancer types
 • Hepatocellular carcinoma
 • Breast cancer
 • Adenocarcinoma etc.
2. Tumours of the same organ in different patients
 • Different genetic alterations
 • Different cell-of-origins
 • Different micro-environment context
e.g. 
 • Melanoma
 • Basal cell carcinoma
  1. Primary tumour and metastases within the same patient
  2. Phenotypic heterogeneity within a single tumour
    • Presence of different cell types in different proportions
    • Genetic or epigenetic level
    • Environment / context-driven heterogeneity
    • Dominance of different clones in different areas of the tumour
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8
Q

Describe the two models for cancer heterogeneity

A
  1. Stochastic model / clonal evolution
    • Self renewal and differentiation are random
    • Extrinsic factors (environmental) that determine the differences between the cells
    • Any cell type can be taken from the tumour, and a new tumour can be generated from it (i.e. all cells have equal but low probability of initiating the tumour growth)
    • Strong influence of the microenvironment
  2. Cancer stem cell model
    • Intrinsic factors are most important (as opposed to extrinsic microenvironment)
    • No matter what the external environment, most cells are unable to generate a tumour
    • Distinct classes of cells within a tumour (CSCs, and the rest)
    • Only a small definable subset has intrinsic ability to initiate tumour growth (CSCs)
    • These cells will again generate a tumour that is heterogeneous (not unlike tumour of origin)
    • Heirarchical organisation with cancer stem cells (CSCs) as the source of other cells
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9
Q

Describe detection of cancer stem cells in leukaemias

A

Suspicion that some cells were different from the others and had special markers that differentiated them

FACS: (flow activated cell sorting aka flow cytometry)
• Separates cells based on surface markers
• Surface markers detected with Abs specific for them
• FACS plot generated

In leukaemia:
• Two sub populations of cells observed:

  1. CD34+
    • CSC, stem cell potential
    • These cells were put into irradiated mice (no BM)
    • Bone marrow reconstituted from the CSCs
    • Mice will have leukaemia
    • Whole haematopoietic system regenerated from these cells, thus, they must have some stem cell characteristics
  2. CD34-
    • When injected into irradiated mice, the cells were unable to reconstitute the haematopoietic system
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10
Q

Describe detection of cancer stem cells in solid tumours

A
  1. Derivation of cells from solid tumour to create a cell suspension
  2. FACS
  3. Two sub-population of cells identified:
    a. CD24+CD44+
    • No tumour in mouse

b. CD24-CD44+
• Tumour generated in mouse

Thus, CD24-CD44+ cells in solid tumours are the stem cells, capable of self-renewal and tumourigenesis

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11
Q

Describe the central characteristics of cancer stem cells

A

Cancer stem cell capable of:

  1. Self-renewal (cell line never dies)
    •Can form a human tumour when given to an immunodeficient mouse
  2. Differentiation into progeny that cannot self-renew
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12
Q

What are the similarities of CSCs with normal SCs?

A
  1. Expression of specific markers that enrich cells with tumourigenic potential
  2. Self-renewal
    • Allows indefinite tumour growth
  3. Potential for differentiation into phenotypically diverse mature cell types
    • Gives rise to a heterogeneous population of cells that composes the tumour
    • NB lack the ability for unlimited proliferation
  4. Regulated by similar signalling pathways
    • Pathways in normal stem cells are generally dysregulated in CSCs
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13
Q

Describe how the following are determined experimentally:
• Tumourigenicity
• Self-renewal
• Differentiation potential

A

– Tumourigenicity –

  1. FACS separation of cells into groups depending on surface markers
    • CD133+
    • CD133-
  2. Comparison of the two sub-populations
    • Cell culture of the two sub-populations in vitro in suspension (cells cannot attach to plate)
    (Clonogenic sphere assay)
    a. CD133+
    • Stem cells start dividing and forming spheres

• i.e. a tumour forms

b. CD133-
• No formation of spheres

– Self-renewal –

  1. Seropassaging of spheres:
    Dissociation of cells into single cell suspension
  2. Continuous rounds of clonogenic sphere assays
  3. Only stem cells will be able to continue generating spheres time after time etc
    NB Some progenitors may be able to regenerate for a short time, then conk out

– Differentiation potential –

  1. Look for differentiated cells in the tumours
  2. Differentiation assay:
    • Growth of cells in particular conditions that force differentiation

CD133+ populations:
• Evidence of differentiation

CD133- populations:
• No evidence of differentiation

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14
Q

Compare surface markers for healthy tissue cells and tissue cancer cells, using the example of the gut

A

Certain markers that are shared between healthy intestinal epithelium and colon cancer cells:
• LGR5
• ALDH1

It is hard to find one marker the completely differentiates these two stem cell populations

Thus, the more markers investigated at once, the better the ability to differentiate tissue stem cells and the cancer stem cells

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15
Q 
What are some markers for CSCs in the following cancers:
 • Breast cancer
 • Medulloblastoma
 • Colon cancer
 • Leukaemia
 • Melanoma

Describe the implication of this

A
  1. Breast cancer
    • CD44+CD24(low): CSC
    • CD44+CD24+: not CSC
  2. Medulloblastoma
    • CD133+: CSC
    • CD133-: not CSC
  3. Colon cancer
    • CD24+: CSC
    • CD24-: non-CSC
  4. Leukaemia
    • CD34+: CSC
    • CD34-: non CSC
  5. Melanoma
    • There are no marker that enriches tumorigenic potential
    • No matter which marker was used, they couldn’t separate out the CSC
    • Thus, melanoma does not conform to the cancer stem cell model
    • Tumorigenicity is random
    • Any cell has the potential to generate a new tumour

Implication:
Some cancers conform to the CSC model, and some (like melanoma) do not

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16
Q

Describe the illustration of differentiation potential of CSCs

A

Colon cancer cells

  1. FACS isolation of CSCs based on CD133+ and CD24+
  2. Cell cultured in differentiation conditions
  3. Over time:
    • Acquisition of differentiated cell markers (CK20)
    • Disappearance of stem cell markers

Implications:
• CSCs have the capacity to differentiate

17
Q

Describe the pathways involved in CSCs:
• What is the normal function of these pathways?
• List which cancers arise from dysregulation of them respectively

A 
These pathways are involved in self renewal in adult stem cells:
 • Haematopoietic system
 • Epidermis
 • Gut epithelium
 • Germ cells
 • Neural stem cells

In cancer:
Become dysregulated

It makes sense that pathways that are normally involved in maintenance of stem cells can become dysregulated to drive cancer

  1. Wnt pathway

Normal role:
• Haematopoietic SCs
• Epidermal SCs
• Intestinal SCs

Observed in:
• Colon carcinoma
• Epidermal tumours

  1. Hedgehog pathway

Normal role:
• Haematopoietic SCs
• Neural SCs
• Germ line SCs

Involved in:
• Neural cancers (Medulloblastoma)
• Basal cell carcinoma

  1. Notch pathway

Normal role:
• Haeatopoietic SCs
• Neural SCs
• Germ line SCs

Cancers:
• Leukaemia
• Mammory tumours
• Colon cancers

18
Q

What do people with cancer die from in most cancers?

A

The metastasis (90%)

Far fewer deaths are due to the primary tumours

19
Q

Describe the role of metastasis in CSCs

Describe how this was determined experimentally

A

Not all CSCs have the ability to metastasise

Colorectal tumour sample

FACS: Cell type separation into three groups:

  1. CD133+CD44+CD26+
    • Only these cells were detected circulating in the blood of patients
    • Able to generate tumour in the liver
    • As well as generate tumours
  2. CD133+CD44+CD26-
    • Not observed circulating (i.e. not metastatic)
    • Did not cause tumour in liver
    • Able to generate tumours
  3. CD133-CD44-CD26-
    • Unable to generate tumours

Thus, there are different types of CSCs (+/- ability to metastasise)

20
Q

Describe the therapeutic resistance in CSCs

A

Colon cancer

  1. Two groups of primary tumours in mice:
    a. No chemotherapy
    • Normal growth of tumour

b. Treatment with chemotherapy (Irinotecan)
• Slowed proliferation of tumour

  1. Cells from primary tumour isolated and proportion of CSCs determined
    a. No chemotherapy
    • Decreased proportion of CSCs compared to chemotherapy group
    b. Chemotherapy
    • Increased proportion of CSCs
  2. Ability to generate spheres analysed
    a. No chemotherapy
    • Cannot cause spheres
    b. Chemotherapy
    • Can still cause spheres

Conclusion:
• CSCs are resistant to chemotherapy
• Only the other cells in the tumours are targeted (thus, tumour looks smaller)

21
Q

What are the mechanisms for CSC resistance to chemotherapy?

A
  1. CSCs are not rapidly proliferating
  2. Not sensitive to pro-apoptotic signals
    • Contain increased proportion of anti-apoptotic proteins
  3. High capacity to metabolise drugs
    • Lots of enzymes to degrade / export / convert into less toxic metabolite
  4. Less prone and less sensitive to DNA damage
22
Q

Compare tumour growth over time with conventional chemotherapy and drugs that kill CSCs

A

Chemotherapy:
• Kill tumour cells
• Does not kill CSCs
→ Regrowth of tumour

Drugs that kill CSCs:
• target CSCs
→ Tumour degenerates

23
Q

Describe the difference between CSCs and ‘cell of origin’

A

CSC:
• The cell that, when isolated, can generate a new tumour in an immunocompromised animal

Cell-of-origin:
• Cell that underwent the original genetic damage
• Not always known
• May not be a stem cell
• Initial mutation occurs many years before the tumour gets large
• It is possible to have a mutation in a progenitor cell (not a stem cell) that generates a CSC

24
Q

Describe the role of cell-of-origin in inter-tumour heterogeneity

A

Cell-of-origin depends on cancer types and subtypes and will thus play a role in inter-tumour heterogeneity

25
Q

Describe EMT

What is its role in normal processes and in cancer?

What drives it?

A

Cells lose epithelial characteristics to become more mesenchymal:
• Motility
• Invasiveness
• Increased resistance to apoptosis

Involved in normal developmental processes
 • Gastrulation
 • Placenta formation
 • Somites
 • Heart valves
 • Neural crest etc.

Plays an important role in many cancerous processes:
• Metastasis
• Dissemination
• Acquisition of therapeutic resistance

Driving EMT:
• Highly sensitive to signals that cells receive from their stromal microenvironment
Thus, certainly not purely intrinsic

26
Q

Is the driving of EMT intrinsic or extrinsic?

A

Predominantly extrinsic

For example
1. Treatment of cancer cells
• TGF-beta
• Transfection of vector that over expresses Snail

  1. Cellular changes
    Upregulation of TFs that play a key role in EMT:
    • Snail
    • Twist
  2. Observations:
    • Migration
    • Increased proportion of CSCs (detected with FACS)
27
Q

Describe the importance of phenotypical plasticity of cancer cells

A

Paradigm:
• Once the cell is differentiated, it cannot go back to a stem cell phenotype

Plasticity model:
• It is not true that differentiated cells in tumours cannot go back to form CSCs

  1. Differentiated cells cultured in very plentiful conditions (no pressure to differentiate)
  2. Differentiated cells regain stem cell markers

– Negation of hierarchical paradigm of cancer cells –

28
Q

What is the clinical implication of tumour cell plasticity?

A

CSC-targeted therapy may not be effective on its own, because the tumour cells that aren’t targeted may convert back into stem cells to propagate the tumour

Combination therapy required:
• Conventional + CSC-targeted therapy

29
Q

What are challenges to the cancer stem cell model?

A
  1. Melanoma
    • There are no stem cell markers that enrich tumorigenic potential
    • All cells have equal tumorigenic potential
  2. EMT
    • Induction of the CSC phenotype through environmental factors
  3. Phenotypic plasticity
    • Differentiated cells can acquire CSC features
    • Negates ‘hierarchy’ of model
30
Q

Heterogeneity of cancers, according to the CSC model, is almost uniquely driven by…

A

… intrinsic factors

PATH30001 - Mechanisms of Disease (34 decks)

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