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Molecular Oncology > Day 7: Cancer stem cells, intratumor heterogeneity, clonal growth, growth dynamics, population screening > Flashcards

Day 7: Cancer stem cells, intratumor heterogeneity, clonal growth, growth dynamics, population screening Flashcards

HC 17, 18, 19

1
Q

HC17: The intestinal stem cell niche: cells and signals

A

In base of the crypt
> Niche cells: Paneth cells and mesenchymal cells like fibroblasts
> Niche signals: Wnt, Notch, EGF, and ECM proteins (morphogens)
> Notch and EGF from Paneth cells
> Wnt from Paneth cells and mesenchymal cells (different types of Wnt ligands)

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2
Q

Classic development CRC

A

APC loss, hyperproliferation to adenoma, than KRAS, Smad4 and TP53 to CRC

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3
Q

Intratumor heterogeneity

A

All the normal cell types from the niche found back in tumor
> also cancer stem cells
> multilineage differentiation
> non-malignant cells in micro-environment
> stem cell like cells: cancer stem cells

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4
Q

Distinction cancer stem cells in breast cancer based on CD44 and CD24

A

High CD44 and low CD24

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5
Q

Cancer stem cells character

A

Can form new tumor after injection in NOD/SCID mice
> CSCs found in multiple tissues

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6
Q

CD133 expression in CSCs

A

High expression
> has better outgrowth
> can differentiate into other tumor cell types

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7
Q

CD133+ cancer cells in xenograft

A

CSCs: outgrowth of new tumor

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8
Q

Spherical cancer cells in vitro resemble

A

CSCs > retain stemness

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9
Q

CK20 marker and character of these cells

A

For differentiated cell: can be increased when differentiation factors added to CSC culture medium
> these cannot form new tumors because differentiated

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10
Q

Wnt activity as CSC marker

A

High Wnt activity for CSCs
> Wnt reporter contruct: TOP-GFP with TCF promotor: Wnt reactive
> green cells in xenograft

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11
Q

Tumor induction capacity is .. for many malignancies

A

Low, only few cells can develop outgrowth: CSCs

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12
Q

Single CSCs display:

A
  • Self-renewal
  • Multilineage differentiation potential
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13
Q

CSCs in established cancer: CD133 and prognosis

A

High CD133+ cells: bad prognosis and low survival rate

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14
Q

Higher expression Lgr5 in TOP-GFP+ cells > ?? (Lgr5 promotor sensitive for b-catenin–TCF)

A

CSCs

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15
Q

CSCs and chemotherapies

A

Better resistance against it
> 5-FU and oxaliplatin (FOLFOX)
> CD133+ cells better resistant

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16
Q

Why are CSCs therapy resistant

A
  • Express high levels of anti-apoptotic proteins
  • Primed to respond to DNA damage
  • Protected by the niche
  • Express higher levels of drug efflux transporters
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17
Q

CSCs and immune escape

A
  • Express molecules that repress T-cells
  • Promote pro-tumor phenotype of macrophages
  • Adenosine production to inhibit T-cells and NK-cells
  • Promote M2 proliferation (tumorigenic macrophage)
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18
Q

CSC therapy response in conventional therapy

A

Differentiated cells die and CSCs survive: outgrowth and differentiation (repopulation)

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19
Q

CSC specific therapy

A

Target CSC to remain with differentiated cells which can be eliminated later

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20
Q

Are there successful cancer stem cell depletion therapies?

A

No, not yet because
> Tumor initiation or established tumor?
> Tumor heterogeneity: hard to specifically target
> Cancer stem cell plasticity: role micro-environment

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21
Q

… cells are required for metastasis

A

CSCs

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22
Q

CSC plasticity in established cancers: Lgr5 expression coupled to DTR test (toxin receptor) and with metastasis

A

> Give mice DT (toxin, only cells with DTR die)
Deplete CSCs
However, when treatment stopped: tumor grows back with the same rate as control: plasticity (can shift cell types)
CSCs are essential for metastasis: liver metastases: after CSC depletion and DT test and stopped treatment, metastatic tumor did not come back

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23
Q

iCaspase-9 gene coupled to Lgr5 promotor (Wnt target): apoptosis inducer. Treat mice with cancer with dimerizer so that iCasp9 dimerizes and induces apoptosis: effect?

A

In treated cells: proliferation cells takes place in non-stem cells (this is normally not the case
> first decrease in size but then growth again
> High level plasticity: differentiated cells can return to undifferentiated state
» towards Lgr5+ CSCs again
> Reversion from differentiated population to CSC population

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24
Q

Cell plasticity in tumors is strongly dependent on …

A

the micro-environment
> differentiated cells become stem cell like again: reversion

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25
Q

CSC niche shown with TOP-GFP marker

A

a-SMA for myofibroblasts
> TOP-GFP cells close to a-SMA cells: niche!
> Differentiated cancer cells without myofibroblasts injected: no regerneration CSCs
> Injected with myofibroblasts: regeneration CSCs! (nude mice)
> Factors from micro-environment induce stemness in cancer cells
> CSC is not an intrinsic propery
> Myofibroblasts induce CSCs in vivo

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26
Q

CSCs are tumor initiating cells. What is their role in established cancers based on their location?

A

In vivo clonogenicity located at surface of the tumor > stem cell functionality
> interactions with micro-environment on surface of tumor

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27
Q

Correlation stem cell marker expression and stem cell functionality (in cancer)

A

No correlation
> homogeneous expression across tumor (centre and surface tumor cells)

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28
Q

Presence fibroblasts and stem cell functionality correlation (in cancer)

A

Significant
> On outiside tumor; a-SMA+ cells (fibroblasts) > induce stem cell functionality on surface of tumor

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29
Q

Xenograft with only the centre tumor cells of primary tumor taken

A

Were not clonogenic in primary tumor but still outgrowth because signals micro-environment > expression stem cells markers also in centre of the tumor
> low TOP-GFP in primary tumor, decreased outgrowth potential

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30
Q

CSC functionality in initiation tumor and established tumor

A

CSC marker expression: tumor initiation
Presence of niche: clonogenicity in established tumor

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31
Q

Functional stem cell properties are … dependent in established cancer tissue

A

location

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32
Q

Prevent plasticity in tumors

A

Therapy: target CSCs and tumor micro-environment (niche cells)
> prevent tumor relapse

33
Q

Cancer stem cells have been identified in multiple cancer types using tumor initiation assays in immunodeficient mice. Do the identified cancer cell sub-populations reflect the cancer cells with stem cell functionality within established tumor tissue?

A

No, cancer stem cell functionality in established cancers is dependent on the niche signals from the micro-environment

34
Q

HC18: Lineage tracing in intestinal lining

A

Stem cells fuel clonal growth
> mark the stem cells (stem cell markers like LGR5)

35
Q

Confetti staining lineage tracing of crypt base stem cells

A

Cells per crypt are renewed by stem cells
> crypts turn into a single color each
> one stem cells will imprint the entire crypt
> villi are fueled by multiple crypts, multiple colors

36
Q

Why do crypts turn monoclonal? Randomness:

A

In crypt base, limited space for set amount of stem cells (with Paneth cells)
> some stem cells get pushed out upon division
> until one color from the confetti staining remains (one clone from set starting point will make up for all stem cells in crypt, the rest pushed out)
> one color lost per division: random which one remains as the last one (50% chance between 2)
» no preference for a certain cell

37
Q

Stem cell competition results in …

A

clonal fixation (monoclonal crypt in stem cells)

38
Q

Neutral drift dynamics

A
  • Division of one stem cell pushes out another for differentiation
  • No bias in this: neutral drift (random 50/50)
  • Stain one stem cell in all crypts red, mostly disappears but some crypts develop entirely red
  • Clone size increases over time when becoming fixated
39
Q

Clone size and clonal fixation

A

More fixation, larger size

40
Q

Clonal competition in cancer when first hit: loss of APC (conventional development pathway)

A

Hyperproliferation > bias towards the clonal survival of the APC mutated stem cell and the loss of other wild-type stem cells
> APC mutant wins most of the times: but not always

41
Q

Chances of growth dynamic results when one APC mutant in the stem cell crypt

A
  • Mutant cells can be extinct and pushed out: 45%
  • Mutant outcompetes it and fixation of crypt: only APC mutants: 55%
    > Limited ability of fixative effect
42
Q

Normal competition between two WT stem cells in clonal dynamics to win and push other out, and with APC+/- and APC-/-

A

WT/WT > 50%
APC+/- /WT > 62%
APC-/- /WT > 79%
APC-/- /APC+/- > 69%

43
Q

APC-/- stem cells: why benefit over WT stem cells?

A

They actively disadvantage WT cells

44
Q

Probability to reach fixation in 5 stem cell situation crypt with:
- KRAS G12D
- APC-/-
- APC+/-
-WT

A

KRAS G12D (70%) > APC -/- (55%) > APC +/- (40%) > WT (20%)
- Important in prevention tumor formation: especially in inherited deficiencies
- Early oncogenic mutations provide limited clonal advantage over other stem cells in niche (crypt)

45
Q

APC mutants in WT organoid stem cell niches in co-culture

A

Outcompeted by APC mutants
> WT organoids are disappearing

46
Q

APC mutant stem cells disadvantage WT stem cells, how?

A

Secrete Wnt antagonists

47
Q

Wnt antagonists made in vitro APC-/- organoids and in vivo in adenoma

A

Notum, Wif1, Dkk2

48
Q

Function Wnt antagonists

A

Inhibit Wnt or Wnt receptor more upstream (extracellular)

49
Q

LiCl as treatment

A

Lithium Chloride inhibits Gsk3b (part of b-catenin destruction complex)
> inhibited destruction complex
> stimulated Wnt pathway for WT stem cells
> outcompete APC mutant
> disadvantage of WT stem cells by APC mutants overcome
> rescue loss of stemness in vivo

50
Q

Lgr5 expression in Notum+ stem cells and Notum+ stem cells treated with LiCl

A

Notum+: low
Notum+ LiCl+: recued expression, no different to control

51
Q

LiCl effect on biased drift

A

Reduce advantage of APC mutants
> lower clone size (lower clone fixation)
> reduced polyp burden, less polyps formed in mice when APC deficient (FAP)

52
Q

Neutral drift to biased drift to corrected drift to treated

A

Neutral drift
> APC mutation
Biased drift
> LiCl treatment
Corrected drift
> Removal of mutant ISCs (intestinal stem cells)
(prevent ougrowth adenomas in patients with inherited risk: FAP)

53
Q

Intestinal stem cells compete for …. in crypt

A

the space in the niche (to not get pushed out)

54
Q

Some mutations cause super-competition in ISC niche. What is super-competition.

A

Active inhibition of neighbouring cells (with Wnt antagonists) which can lead to complete disappearance of WT stem cells: fixation of mutant stem cell clone (but this potential is limited!, 55% in APC -/-)

55
Q

APC-/- Culture Medium (CM) induces … in WT stem cells

A

Differentiation

56
Q

Chemo-preventive strategies like LiCl can …. the advantage of mutant stem cells

A

Reverse to normal (random)

57
Q

HC19: Mortality rate and incidence CRC in Netherlands is …

A

High

58
Q

Development CRC in time

A

Slow progress over years

59
Q

Polypectomy in adenomatous polyp stage leads to…

A

No development of cancer

60
Q

When patients are symptomatic of CRC, then:

A

Already in advanced CRC stage

61
Q

5-years survival rates for stage 1 and stage 4 CRC

A

1: >90%
4: <10%
> survival rate depends on stage

62
Q

Strategies to reduce CRC-related mortality

A
  • Prevention: reduce risk factors, no alcohol, smoking, reduce processed foods
  • Screening: testing asymptomatic persons for disease or risk factors
63
Q

Screening criteria CRC

A
  • Important health problem
  • Patient should directly benefit upon identification: follow-up treatment should be available
    > Suitable test available
    > Accepted treatment for patients with recognized disease
    > Test should be acceptable to population
  • Identification early stage
  • Condition with important health problem: high incidence and mortality for CRC
64
Q

Population-based screening for CRC

A

Search for asymptomatic individuals in population with disease markers
> detect CRC in earlier stage
> detect and remove advanced precursors (big polyps, to reduce mortality)

65
Q

Screening types CRC

A
  • FIT (stool test)
  • Sigmoidoscopy
  • Colonoscopy
  • Molecular markers: not very promising
  • (also colonography or colonocapsule)
66
Q

Stool test CRC

A
  • Polyps shed blood
  • Blood markers in stool test: cancer marker
67
Q

Fecal immunochemical test (FIT) (stool)

A
  • Antibodies to detect human Hb only
  • Non-invasive, 1 stool sample
  • Done at home and sent to lab
  • Reduced CRC related mortality with 22%!
68
Q

Sigmoidoscopy

A
  • Inspection left side of colon
    > if positive > colonoscopy
    > Every 3-5 years
    > reduce CRC-related mortality: 31%
  • Annama to get feces out the colon
  • Complication risk of 0-0.003%
  • Low participation rate: 32%, makes it less effective
69
Q

Colonoscopy

A

Reference standard: through entire colon
> detection and removal in one procedure
> effect on CRC incidence: 18% reduction, not significant mortality reduction in NL, but with 50% when everyone participates (prediction)!
> higher participation grade in US
> Participation 22% in NL
> complication risk 0.1-0.3%: perforation and bleeding: is severe: instant surgery needed
> invasive procedure

70
Q

Factors in choice of screening program

A
  • Features
  • Participation rate
  • Yield of test
  • Risk and burden
  • Capacity endoscopy
  • Costs
    » choice differs for each country
71
Q

National Bowel Cancer Program Netherlands

A

All Dutch citizens 55-75 years
> FIT every 2 years
> Participation rate lowered when coronavirus outbreak: lowered governmental trust

72
Q

Participation rate by age and gender in CRC screening programs

A

-Men are less willing worldwide
> higher risk men in CRC though (counterinuitive)
- Participation rate trend is negative overall over years
- Blood in stool test > 5% > ask for follow-up with colonoscopy
- More in increased age

73
Q

FIT positivity rate by age and gender

A

FIT positivity increases with age and more with men

74
Q

Possible findings when follow-up colonoscopy after positive FIT

A
  • Nothing
  • Serrated and non-advanced adenomas
  • Advanced adenomas
  • Colorectal cancer
75
Q

What happens when polyps found (adenomas, serrated or advanced) when colonoscopy?

A

Easy removal with resection
> small chance of developing CRC

76
Q

Stage discovery distribution of screened and unscreened CRC

A

Before screening program
> a lot stage 4 and little stage 1
Screening
> a lot stage 1 and low stage 4
» mortality rates are dropping, slow downwards trend CRC incidence and mortality

77
Q

Possible pitt falls in CRC screening

A
  • Sensitivity for adenomas and sessile serrated lesions
  • Type of screening test and willingness to participate
  • Inducing more inequity in health care: persons with highest social economic status and more healthy lifestyle are the ones participating in screening, but they have approx. better outcomes as risk probability
    > extra attention needed for other groups
  • FIT screening is based on detection of blood in stool sample: not all lesions bleed
78
Q

Cancers can be missed in FIT”FIT interval CRC

A

CRC between 2 screening rounds
> 0.10% of iCRC after negative FIT
> sensitivity of the test is 75%

79
Q

Future perspective CRC screening

A
  • Find protein biomarkers in feces for stool test
  • Fecal DNA marker test: used in US
    > downside: lower-cost effectiveness and more false positives
  • Risk stratification: select patients better based on risk factors
  • Barriers in participation researched and decision-making

Molecular Oncology (7 decks)

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