http://www.mayfieldclinic.com/pe-braintumor.

htm

GS6882A Biology of Disease

Brain tumors & Cancer Stem Cells

Derrick Ong, Ph.D.

Department of Physiology, NUS
phsostd@nus.edu.sg
Office: 65163232

1
 Learning outcome
At the end of the lesson, students are expected to be
able to:
1) Explain how cancer stem cells (CSCs) are clinically
relevant to brain tumors.
2) Describe various approaches to model brain CSCs in
vitro and in vivo, including their advantages and
disadvantages.
3) Discuss emerging concepts in GSC biology.

https://www.futurelearn.com/courses/educational-design/0/steps/26427 2
 What is a brain tumour?

 A mass of abnormal tissue growth in the brain.

 Non-cancerous (Benign) - grows slowly, has distinct boundaries,

and rarely spreads.

 Cancerous (Malignant) - grows quickly, has irregular boundaries,

and spreads to nearby brain areas.

o Primary: originate in the brain.

o Secondary or metastatic: come from another part of the

body (e.g. breast and lung).

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 Brain Cancer Statistics
USA
In 2015, about 166,039
people living with brain
and other nervous
system cancer in USA.
https://seer.cancer.gov/statfacts/html/brain.html

UK

http://www.cancerresearchuk.org/health-professional/cancer-
statistics/statistics-by-cancer-type/brain-other-cns-and-intracranial-tumours

Uncommon cancer in Singapore. Between 1968 and 2007, there were

about 1,903 cases reported. Most were astrocytomas and glioblastoma.
https://www.nccs.com.sg/PatientCare/WhatisCancer/TypesofCancer/Pages/brain-cancer.aspx?p=/PatientCare
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 Brain Tumour Grading Scale
2016 WHO classification of gliomas
 Brain tumour grading is a
category system that
describes the brain tumour
cells and indicates how likely
the tumour is to grow and
spread.

http://www.mayfieldclinic.com/pe-braintumor.htm
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Reifenberger et al, Nat Rev Clin Oncol, 2017.
 Types of brain tumours (WHO classification)

Normal brain
https://medicine.stonybrook
medicine.edu/pathology/ne
uropathology/chapter1

6
Huse & Holland, Nat Rev Cancer, 2010, 10, 319.
Diagnostic approach for integrated histological and molecular
classification of diffuse gliomas according to the 2016 WHO
Classification of Tumours of the Central Nervous System

Reifenberger et al, Nat Rev Clin Oncol, 2017. 7

 Isocitrate dehydroengase (IDH) mutations are associated with
better prognosis of glioma patients.

Mutations at R132 in IDH1 and R172 in IDH2 in gliomas.

• Widespread epigenomic dysregulation

through generation of the glioma-CpG island
methylator phenotype (G-CIMP)
hypermethylator phenotype.
• Contribute to transcriptional remodeling &
deposition of histone modifications at
specific genomic loci → emergence of a
CD24+ stem-like population & increased
genomic instability.

Yan et al, New Eng J Med, 2009, 360, 765; Gimple et al, Genes Dev, 2019, 591; Turcan et al, Nature, 2012, 483: 479-483;
Turcan et al, Nat Genet, 2018, 50: 62-72. 8
Molecular subgroups of glioblastoma, as defined by distinct
genetic and epigenetic profiles

Reifenberger et al, Nat Rev Clin Oncol, 2017. 9

Promoter methylation of MGMT is a prognostic biomarker in
glioblastoma patients treated with temozolomide.

(O6-methylguanine-DNA
methyltransferase)

Wick et al, Nat Rev Neurol, 2014, 10, 372-385;

Hegi et al, New Eng J Med, 2005, 352: 997-1003. 10
Possible cell-of-origin of various brain tumours

11
Huse & Holland, Nat Rev Cancer, 2010, 10, 319.
Timeline of the important discoveries of cancer stem cells

A subset of cancer cells are

tumorigenic and pluripotent.
1988 Pierce: Early
definition of CSC concept
Lapidot et al, Nature,
1994; 367:645-648.
Hematopoietic stem cell
research flourished.
Bone marrow
transplantation was
introduced in the clinic.
Development of FACS and
mouse xenograft assays
for human hematopoietic
stem cells.

University of Toronto

Al-Hajj et al, PNAS,

2003; 100(7):3983-8.

Stanford University
Clevers, Nat Med, 2011, 17(3): 313-319. 12
Parallels between normal and cancer stem cells (CSC)

 Populations of cells that

derive from stem cells are
organized in a hierarchical
fashion, with the stem cell
residing at the apex of the
Self-renewal developmental pathway.

 CSC cannot modulate and

balance differentiation and
Differentiate self-renewal according to
environmental stimuli and
genetic constraints.

Pardal et al, Nat Rev Cancer, 2003, 3: 895-902; 13

Dalerba et al, Ann Rev Med, 2007, 58: 267-84.
 Why is the “Cancer Stem Cell (CSC) Hypothesis”
attractive?
Intertumour heterogeneity Intratumour heterogeneity

Burrell et al, Nature, 2013.

• Not all cells in tumours are created equal (tumour heterogeneity):

rapidly proliferating cells, as well as postmitotic, differentiated cells.
• Which subclones to target for effective anti-cancer treatment?
• CSC hypothesis promises the development of more effective
treatments, aimed not at reducing tumour bulk, but rather at
targeting the ‘beating heart’ of the tumour.
Clevers, Nat Med, 2011, 17(3): 313-319. 14
 Why study brain cancer stem cells?

GLIOBLASTOMA Median survival

CLINICAL RELEVANCE:
RT + TMZ:
• Intratumor heterogeneity
14.6 months • Therapeutic resistance
RT only: • Tumor recurrence
12.1 months • Invasive
• Angiogenic

https://emedicine.medscape.co
m/article/340870-overview
Stupp et al, NEJM, 2005.

TUMOR REMISSION RECURRENCE

RT +
Chemo

Therapy
resistance

Chen et al, Nature, 2012.

Glioblastoma stem cells (GSC) Patel et al, Science, 2014.

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Publications on cancer stem cells in glioblastoma

Glioma stem cells Glioblastoma stem cells

(Result count: 4,744) (Result count: 3,300)

Brain tumor initiating cells

(Result count: 752)  “Glioma stem cells” =
“Glioblastoma stem cells” =
“Brain tumor initiating cells”

 Intensive cancer stem cell

research in brain tumors since
2003.

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 Evidence of brain cancer stem cells
Tumorspheres

Multipotency

Galli et al, Cancer Res, 2004, 17(3): 313-319. 17

 Evidence of brain cancer stem cells

CD133- tumour cells

CD133+ tumour cells initiate tumours upon intracranial transplantation into the
adult NOD-SCID mouse forebrain.

Singh et al, Nature, 2004, 432: 396. 18

 Evidence of brain cancer stem cells

Intratumoral glioblastoma heterogeneity

quantified by single-cell RNA-seq.

Patel et al, Science, 2014, 344: 1396-1401. 19

 Evidence of brain cancer stem cells

A relatively small proportion of cells associated with

cell cycle meta-signature from each tumor,
consistent with Ki67 staining of tumors.

Stemness gradient is modestly

anticorrelated to the cell cycle meta-
signature, consistent with stemlike
cells dividing at lower overall rates.
Patel et al, Science, 2014, 344: 1396-1401. 20
 How do we model glioblastoma?

https://pngtree.com/freepng/patient-
infusion_3179553.html

http://clipart-library.com/mouse-
cartoon-images.html
https://png.pngtree.com/png_detail/20181019/
cancer-cell-vector-png-clipart_3500523.png

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 Modeling glioblastoma #1
Extensive overlap between GSC markers and markers of neural stem cells Eg. SOX2, OLIG2,
MYC, and NESTIN (intracellular) and CD133, L1CAM, CD44, and A2B5 (cell surface markers).
Enriched GSC
Cell sorting

Vescovi et al, Nat Rev Cancer, 2006, 6: 425. 22

 Modeling glioblastoma #1

Sorting using cellular markers (CD133, CD15, and others)

• Advantages: Prospective identification of putative stem populations is
possible.
• Disadvantages: Individual stem markers are frequently disputed; no
functional criteria are used; may deplete GSC heterogeneity; integrity of
surface markers may be affected during single-cell dissociation.

In vitro neurosphere culture

• Advantages: Uses a functional assay for self-renewal capacity; high
throughput.
• Disadvantages: Inability to determine tumor formation capacity, identify
quiescent stem populations, and model diverse cellular interactions;
reliance on artificial and unphysiological medium conditions with loss of
tumor heterogeneity; contain populations of stem and differentiated cells;
not ideal for assays in which homogeneity is required; percentage of
enriched GSCs will vary with every sphere depending on size, passage, and
technique used in propagation and culture medium.

23
Gimple et al, Genes Dev, 2019, 591.
 Modeling glioblastoma #1

Two-dimensional adherent culture on poly-L-lysine/laminin plates

• Advantages: May reduce differentiation compared with neurosphere
culture; ideal for assays in which homogeneity is required; high throughput.
• Disadvantages: Inability to determine tumor formation capacity, identify
quiescent stem populations, and model diverse cellular interactions;
reliance on artificial and unphysiological medium conditions with loss of
tumor heterogeneity.

24
Gimple et al, Genes Dev, 2019, 591.
 Modeling glioblastoma #2

3D Organoid
Culture System

Hubert et al, Cancer Res, 2016, 76: 2465-77. 25

Modeling glioblastoma #2

Organoid-derived
glioblastoma
xenografts recapitulate
the diffusive
phenotype of the
original patient tumor.

Hubert et al, Cancer Res, 2016, 76: 2465-77. 26

 Modeling glioblastoma #2

• Organoids maintain functionally divergent tumor cell populations from distinct

tumor regions.
• Showed marked heterogeneity and reproduced the indistinct margins and
single-cell infiltration present in the original patient sample.

Hubert et al, Cancer Res, 2016, 76: 2465-77. 27

 Modeling glioblastoma #2

Three-dimensional GSC organoid culture systems and biomaterial

scaffolds
• Advantages: Recapitulate in vivo environment with higher fidelity than in
vitro systems; model cellular and stromal interactions; model hypoxic and
other biologic gradients; model multicellular and microenvironmental
interactions
• Disadvantages: Decreased throughput compared with two-dimensional
methods; complex procedure for initiation and maintenance; lack certain
cellular interactions, including with vasculature, microglia, and others

28
Gimple et al, Genes Dev, 2019, 591.
 Modeling glioblastoma #3
A PDGFRα-driven mouse model of glioblastoma:
• Overexpression and chronic activation of non-
mutant, WT PDGFRα is the second most common
genomic aberration of a receptor tyrosine kinase;
• 48% of these PDGFRα-positive GBMs are associated
with loss-of-function mutations within the tumor
suppressor gene TP53.

poorly
differentiated
giant
cells
multinucleate
d cells

areas of
pseudo-
palisading
necrosis highly infiltrative
tumor cells

Variable GFAP No NeuN

expression expression

29
Jun et al, Nat Comms, 2018, 9: 3116.
 Modeling glioblastoma #3
Nes-CreERT2;
QkL/L; PtenL/L;
Trp53L/L (QPP)
mouse model of
glioblastoma

pseudopalisading microvascular
necrosis proliferation

Intratumor
heterogeneity

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Shingu et al, Nat Genet, 2017, 49: 75-86.
Glioblastoma stem cells (GSCs) promote radioresistance.

Enhancement of intracranial tumour

formation by increased CD1331 fraction.

Enrichment of CD133+ tumour

subpopulations after irradiation.

CD133+ tumour cells showed characteristics

consistent with cancer stem cells.

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Bao et al, Nature, 2006, 444: 756-60.
Glioblastoma stem cells (GSCs) promote radioresistance.

CD133+ tumour cells show

radioresistance and lower sensitivity
to radiation-induced apoptosis than
CD133- tumour cells.

CD133+ glioma cells

preferentially activate the
DNA damage checkpoint…

and repair IR-induced DNA

damage more efficiently than
CD133- cells.

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Bao et al, Nature, 2006, 444: 756-60.
PAF promotes stemness and radioresistance of GSC.

Ong et al, PNAS, 2017. 33

 PAF promotes stemness and radioresistance of GSC.

Replicative Impaired DNA replication

polymerase 5’

x
3’ 5’ and cell cycle
5’ DNA damage progression
3’ 5’
5’
PAF 3’ 5’ Impaired TLS

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Ong et al, PNAS, 2017.
GSCs promote tumor angiogenesis.

GSCs form more vascular

and necrotic tumors
when grown in the brains
of immunocompromised
mice.

GSCs express elevated level

of VEGF.

35
Bao et al, Cancer Res, 2006, 66: 7843-48.
GSCs promote tumor angiogenesis.

VEGF neutralizing
antibody blocks GSC-
induced endothelial cell
migration and tube
formation.

VEGF neutralizing antibody

bevacizumab specifically
suppresses GSC tumor
growth and hemorrhage in
vivo.

36
Bao et al, Cancer Res, 2006, 66: 7843-48.
GSCs contribute to chemoresistance and recurrence.

Nes-TK-GFP transgene:
• Marks adult NSCs (and endogenous
glioma cells).
• Harbours a cassette containing a
modified version of the herpes simplex
virus (HSV) thymidine kinase (DTK),
allowing for temporally regulated
ablation of dividing neural progenitors by
systemic ganciclovir (GCV)
administration.
• IRES-GFP cassette to mark Nes-TK-
expressing cells in the absence of GCV.

Rostral migratory stream (RMS), formed by neural progenitor cells migrating from
the SVZ to the olfactory bulb, was severely diminished in Nes-DTK-GFP mice after
GCV treatment.

37
Chen et al, Nature, 2012, 488: 522-26.
 GSCs contribute to chemoresistance and recurrence.
Bromodeoxyuridine (BrdU) labeling
• Label cells born at the time of BrdU administration, specifically in the S phase
of the cell cycle when cells replicate DNA.

BrdU
https://prezi.com/suew-
pjyeyva/how-does-brdu-work/

• Short term labeling: study proliferating cells.

• BrdU label retaining assay: study slowly dividing cells that retain BrdU.
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GSCs contribute to chemoresistance and recurrence.

(2 hours)

Temozolomide targets proliferating derivatives but

not the GFP+ quiescent cell population.

Large majority of
(1d later) CldU+ and IdU+ cells
(3d later) contained GFP →
renewed tumour cell
proliferation was
hierarchical and
derived from the
GFP+ cells.

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Chen et al, Nature, 2012, 488: 522-26.
GSCs contribute to chemoresistance and recurrence.

40
Chen et al, Nature, 2012, 488: 522-26.
Epigenetic heterogeneity in glioblastoma

• Enhancing region (ER): defined by

disruption of the blood–brain barrier at
areas of angiogenesis.
• Necrotic region (NR): hypoxic.
• Enhanced margin (EM): intermediate
tumor region.

• ER- high expression of proneural genes.

• NR- high expression of mesenchymal
genes.
• EM- mixed transcriptional signature
comprising classical and proneural gene
expression signatures.

• GSCs in the ER were

exclusively SOX2+ and
OLIG2+.
• NR GSCs were exclusively
CD44+ and YKL40+.
• Segregation of proneural and
mesenchymal GSC markers.
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Jin et al, Nat Med, 2017, 23: 1352-61.
 Epigenetic heterogeneity in glioblastoma

• Dichotomous distribution of
cells positive for H2AK119Ub
and H3K27me3 in hypoxic
(necrotic) and vascular
(enhancing) regions,
respectively.

PRC2 binds to chromatin and its catalytic subunit, EZH2, trimethylates histone H3 at residue K27
(H3K27me3). H3K27me3 is then recognized by PRC1, which contains BMI1, then monoubiquitination of
histone 2A on K119 (H2AK119Ub) causes chromatin compaction and pausing of RNA polymerase II.

• Tumors with proneural

signatures showed enrichment
of an EZH2 activation
signature, whereas tumors
with mesenchymal
transcriptional profiles showed
enrichment of a BMI1
activation signature.

42
Jin et al, Nat Med, 2017, 23: 1352-61.
Epigenetic heterogeneity in glioblastoma

• High EZH2 protein levels, high

expression of OLIG2, a proneural
marker.
• High BMI1 protein levels, high
expression of CD44, a
mesenchymal marker.

• EZH2 and BMI1 expression or

activation signatures were
associated with poor patient
prognosis, but the worst prognosis
was associated with high
expression of both proteins or
signatures.

43
Jin et al, Nat Med, 2017, 23: 1352-61.
Epigenetic heterogeneity in glioblastoma

• PTC596: BMI1 inhibitor.

• EPZ6438: EZH2 inhibitor.
• GW120918: dual ABCCB1
and ABCG2 inhibitor; to
enhance EPZ6438 uptake
into brain.

44
Jin et al, Nat Med, 2017, 23: 1352-61.
GSCs are defined by a series of functional criteria, including tumor-
initiating capacity following serial transplantation, self-renewal, and
the ability to recapitulate tumor heterogeneity

45
Gimple et al, Genes Dev, 2019, 591.
 Microenvironmental Tumor cell
interactions with other interactions

tumor cells, neurons,

macrophages, T cells, Macrophage
and the vasculature interactions

are key for supporting

GSCs.
Neuron
interactions

Extracellular
matrix

Vascular T-cell
interactions suppression

Gimple et al, Genes Dev, 2019, 591. 46

Osteopontin mediates glioblastoma-associated macrophage infiltration.

Increased expression of OPN in

GBM and its negative correlation
with patient survival.

OPN expression in tumor

cells and non-tumor cells
influences survival
equally in GL261 glioma–
bearing mice.

47
Wei et al, J Clin Invest, 2019, 129: 137-149.
Osteopontin mediates glioblastoma-associated macrophage infiltration.

OPN deficiency
enhances T cell
effector activity and
reduces glioma
macrophage
infiltration.

48
Wei et al, J Clin Invest, 2019, 129: 137-149.
Osteopontin mediates glioblastoma-associated macrophage infiltration.

Preincubation of
GSC-CM with an
anti-OPN
antibody
attenuated the
OPN-promoting
effect of
macrophage
Recombinant OPN and conditioned
migration.
medium (CM) from human GSCs
attracted M0 and M2 macrophages.

Blockade of αvβ5 on M0 and M2

macrophages completely abolished their
migration to GSC conditioned medium.
49
Wei et al, J Clin Invest, 2019, 129: 137-149.
From basic science to novel therapeutics

Targeting GSCs:

Numerous avenues
exist for targeting
GSCs, including
selectively poisoning
epigenetic,
metabolic,
microenvironmental,
posttranscriptional,
and immune
interactions.

Gimple et al, Genes Dev, 2019, 591. 50

 What have we learnt today?

 Malignant brain tumours remain intractable.

 Cancer stem cells contribute to clinical features of malignant

brain tumours, including tumour heterogeneity, therapy
resistance, tumour recurrence, angiogenesis and invasiveness.

 Multiple approaches to model glioblastoma, including GSCs

defined by specific markers, neurosphere cultures, 2D adherent
cultures, 3D organoid cultures, genetically engineered mouse
models and patient-derived xenograft models.

 GSCs are supported by microenvironmental interactions with

other tumor cells, neurons, macrophages, T cells and
vasculature.

 Basic science has identified numerous avenues to target GSCs.

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 Selected readings
• Bao et al, Nature, 2006, 444: 756-60.
• Bao et al, Cancer Res, 2006, 66: 7843-48.
• Brennan et al, Cell, 2013, 155: 462-477.
• Chen et al, Nature, 2012, 488: 522-26.
• Clevers, Nat Med, 2011, 17(3): 313-319.
• Galli et al, Cancer Res, 2004, 17(3): 313-319.
• Gimple et al, Genes Dev, 2019, 591-609.
• Hegi et al, New Eng J Med, 2005, 352: 997-1003.
• Hubert et al, Cancer Res, 2016, 76: 2465-77.
• Huse & Holland, Nat Rev Cancer, 2010, 10, 319-331.
• Jun et al, Nat Comms, 2018, 9: 3116.
• Ong et al, PNAS, 2017, 114(43): E9086-E9095.
• Reifenberger et al, Nat Rev Clin Oncol, 2017, 14: 434-452.
• Patel et al, Science, 2014, 344: 1396-1401.
• Shingu et al, Nat Genet, 2017, 49: 75-86.
• Singh et al, Nature, 2004, 432: 396-401.
• Stupp et al, New Eng J Med, 2005, 352: 987-996.
• Turcan et al, Nature, 2012, 483: 479-483.
• Turcan et al, Nat Genet, 2018, 50: 62-72.
• Wick et al, Nat Rev Neurol, 2014, 10, 372-385.
• Yan et al, New Eng J Med, 2009, 360, 765-773.

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T H A N K

Y O U

http://thepirlo.com/consulting/research/
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