What is Cancer? How do we get it? How does it kill us?
Two distinct cancer research themes:
1. tumor onset (how does it all start? Is it really just a question of bad luck?)
2. metastasis and phenotypic plasticity (what has personalized medicine to do with that?)
Mythbusting Cancer Research
`For every complicated problem there is a solution that is simple, direct, understandable, and wrong.
H.L. Mencken
Colorectal Cancer: the second deadliest
sequential clonal expansions (Darwin docet!)
Cancer: a multistep genetic process
2nd hit (gene B mutation)
1st hit (gene A mutation)
3rd hit (gene C mutation)
Cancer: a multistep genetic process
intestinal
epithelial cell ACF adenoma
p53
chr. 17p LOH
carcinoma
The Adenoma-Carcinoma Sequence in Colorectal Cancer: a genetic paradigm for tumor initiation, progression, and metastasis
APC
K-RAS SMAD2/4
chr. 18q LOH
What about the rate-limiting, initiating (APC) mutation event? Is it just a ‘random’ event (bad luck, shit happens), or
do extrinsic (environmental) factors favor its occurrence?
Cancer: the role of “bad luck” vs. environmental risk factors
Cancer: the role of “bad luck” vs. environmental risk factors
Colorectal Cancer: the second deadliest, highest stem cell division rate (1012 over 60 yrs), and yet largely due to environmental factors.
Cancer: the role of “bad luck” vs. environmental risk factors
Why do we get colon cancer? 4 main etiological factors:
1. Age
2. Diet (life style)
3. Inflammation
4. Genetics
It’s all in the stem cell niche!
intestinal
epithelial cell ACF adenoma
p53
chr. 17p LOH
carcinoma
APC K-RAS SMAD2/4
chr. 18q LOH
stem cell niche alterations (i.e. expansion of cell targets for tumor
initiation and progression)
metaplasia (stem cell reprogramming)
Western-style diet Aging
IBD Predisposition
Why do we get colon cancer? 4 main etiological factors:
1. Age
2. Diet (life style)
3. Inflammation
4. Genetics
It’s all in the stem cell niche!
How to model Western-Style diet-induced colon cancer in the mouse?
How to model Western-Style diet-induced colon cancer in the mouse?
NWD1: - 25% of C57BL/6J mice develop colon tumors (n= 1-2); - Tumors arise at 1.5-2 years of age; - Ratio carcinoma-to-adenoma of ~10%; → First and only model of dietary induced sporadic CRC NWD2: Increased Vit D3 and Ca2+ rescue the tumorigenic effect
0%
100%
200%
300%
400%
500%
Fat Vit D3 Ca2+ Fiber Methyldonors
% o
f co
ntr
ol d
iet
AIN76A
NWD1
NWD2
Inflammatory Bowel Diseases: Crohn’s and Ulcerative Colitis
1 2 3 4 5 6 7 8 9 10
3% DSS H20
SACRIFICE
Lgr5-EGFP
1 2 3 4 5 6 7 8 9 10
H20
SACRIFICE
Lgr5-EGFP
Modelling inflammatory bowel disease by Dextran Sodium Sulfate (DSS)
The intestinal stem cell niche
cKit+ Paneth-like cells (in colon)
LyscreERT2
(J. Van Es, H. Clevers)
cKitcreERT2
(D. Saur)
Lgr5EGFP-IRES-creERT2
(N. Barker, H. Clevers)
The intestinal stem cell niche
GFP
Co
ntr
ol
3%
DSS
GFP GFP/RFP
GFP/RFP GFP/YFP
GFP/YFP GFP
Co
ntr
ol d
iet
NW
D1
die
t
GFP GFP
Ki67
GFP
Ki67
→ fewer Lgr5+ stem cells
→ Lgr5+ stem cell lineage tracing is inhibited
Both Western-style diet and inflammation suppress Lgr5+ stem cell function
→ Lgr5+ stem cells cease to proliferate
NWD1 inhibits proliferation, lineage tracing, and tumor-initiating capacity of Lgr5+ stem cells through the vitamin D receptor.
Western-style dietary factors and in particular vitamin D depletion inhibit Lgr5 stem cell function
The western-style NWD1 diet (low in vitamin D) represses the stem cell function of Lgr5+ cells and their capacity to underlie cell turnover in the small an large intestine.
Are there other stem cells involved? Or are the dietary effects mediated by the niche?
‘Mini-Gut’ Organoid Assay
small intestine or colon
crypts 500 crypts/ well
Matrigel + growth
factors
0h 5h 1day 5 days
AIN
76
A
NW
D1
N
WD
2
5x 20x 10x
0
20
40
60
80
100
120
140
160
180
200
AIN76A NWD1 NWD2
nu
mb
er
of
org
ano
ids
→ notwithstanding the fact that the Western-style diet suppresses stemness of Lgr5+ cells, mini-gut organoid forming capacity is increased.
Hence, a distinct stem cell type is activated upon Western-style diet.
Intestinal stemness is increased by Western-style diet
5 day old organoids
The intestinal stem cell niche
1000 Lgr5+ CBCs from mouse A
1000 Paneth cells from mouse B
Plate out in matrigel for ‘mini-gut’ assay
30’ co-incubation at 37°C
optional: genetic (e.g. siRNA) or biochemical modification of sorted lineages
exp. mouse A (e.g. control diet)
exp. mouse B (e.g. NWD1 diet)
Single cell digest of intestinal mucosa
FACSort stem (Lgr5+) and niche (Paneth) cells
separately
Functional analysis of stem vs. niche effects: the organoid reconstitution assay
Lgr5 cells Paneth cells
Western style diet
Lgr5+ stem cells inactivation
Homeostasis Paneth cells provide niche
support to Lgr5+ stem cells.
+4
Lgr5+
Lyz+Paneth
Inflammation
Depletion of Lgr5+ stem cells
Regeneration
Paneth cells acquire stem cell properties and repopulate the intestinal epithelium.
Lin
eag
e t
racin
g
Lin
eag
e t
racin
g
Regeneration
Niche reinforcement by Paneth cells, quiescent stem cell (+4) activation.
+4
Lyz+Paneth
Overall, colon cancer risk factors such as diet and inflammation profoundly affect
both niche and stem cells resulting in improved regeneration upon tissue injury.
The recruitment of new stem cell types
and the overall increased proliferation rate ultimately results in an increased mutation
rate and cancer risk.
For every complicated problem there is a solution that is simple, direct, understandable, and wrong.
H.L. Mencken
Two distinct cancer research themes:
1. tumor onset (how does it all start? Is it really just a question of bad luck?)
2. metastasis and phenotypic plasticity (what has personalized medicine to do with that?)
20 30 40 50 60 70
Age (years)
(ep
i)ge
net
ic m
uta
tio
n lo
ad Tumor Evolution at the Primary Site
(Darwin docet: sequential clonal expansion)
2nd hit 1st hit 3rd hit
2nd hit
x
3rd hit
x
EMT
surgery
Dissemination (CTCs & DTCs)
. .
.
. .
Tumorigenesis and Metastasis: Discontinous Evolution
.
. x genetic hit
(mutation)
epigenetic hit (red: EMT; yellow: MET)
Distant Site Metastases
x x
.
.
1st hit
x
Phenotypic Plasticity
The current ‘hype’ on personalized cancer therapy is misplaced and unjustified
‘a large majority of driver gene mutations are common to all metastases’
‘the driver gene mutations that were not shared by all metastases are unlikely to
have functional consequences’
2002
2018
Tumor cells are capable of contributing to normal development and generate phenotypically normal chimeric organism with tumor cells present in most adult tissues (mouse teratocarcinoma cells: Illmensee & Mintz, 1976; chicken embryonic cells infected with Rous sarcoma virus: Rous, 1979; Dolberg & Bissell, 1984; Stoker, Hatier, & Bissell, 1990).
Some early (70’s and 80’s) experimental evidence that things are not as simple as that:
Mina Bissel, PhD:
phenotype is dominant over
genotype!!
Notably, when cultured in vitro, these embryonic cells rapidly reveal pronounced tumorigenic features and undergo neoplastic transformation.
Hence, the embryonic environment seems to attenuate oncogenesis possibly by inducing differentiation and suppressing cancer stemness.
Type, density and location of tumor-infiltrating immune cells in large cohorts of human colorectal cancers represent the strongest prognostic factors in terms of freedom from disease and overall survival at all stages of clinical disease (Galon et al., Science 2006).
Jerome Galone, PhD
The Overhyping of Precision Medicine Science has a history of inflated promises when it comes to disease treatment. by Nathaniel Comfort, Dec. 16, 2016 www.theatlantic.com/health/archive/2016/12/the-peril-of-overhyping-precision-medicine/510326
Phenotypic Plasticity
The invasion-metastasis cascade: two main theories
….
“seed and soil” (single CTCs) vs. “collective cell migration”
Epithelial markers: • E-cadherin • Cytokeratin • Desmoplakin • Laminin
Mesenchymal markers: • Vimentin • Fibronectin • N-cadherin • 𝛂-SMA
Epithelial-to-mesenchymal transition: not a B&W process
EMT transcription factors: ZEB1, ZEB2, Snail, Slug, Twist, FoxC2
Functional features: • Tight cell-cell adhesion • Non-motile • Non-invasive
Functional features: • No cell-cell adhesion • Motile • Invasive
Functional features: • Weak cell-cell
adhesion • Metastable
E/M markers:
??
Phenotypic Plasticity and Metastasis: conventional immortalized cell lines as experimental models
Common colon cancer cell lines encompass a CD44highEpCAMlow subpopulation with mesenchymal and highly motile and invasive features
Andrea Sacchetti
CD44highEpCAMlow colon cancer cells express EMT markers and transcription factors (ZEB1)
CD44hiEpCAMlow =19%
CD44hiEpCAMlow = 84%
EpCAM-FITC
CD
44
-AP
C
HCT116 - Sorted EpCAM low + 34 days
CD44highEpCAMlow cells have stem-like characteristics
CD44highEpCAMlow cells have high-metastatic capacity in vivo
HCT116 - Spleen injection
…and are resistant to chemotherapy
CD44hiEpCAMhigh CD44hiEpCAMlow
CD44hiEpCAMhigh CD44hiEpCAMlow
HCT116 – Top differentially expressed genes
EpCAM
E-Cad
ZEB1 VIM1
RSPO1
RNAseq analysis: functional and omics characterization
HCT116 and SW480: combined pathway analysis
HCT116 and SW480: MDS analysis
CD44highEpCAMlow cells correlate with EMT signatures and the CMS4 subtype of colon cancer
HCT116 – chemotherapy
Post-treatment
Pre-treatment
Recurrence Post-treatment
Pre-treatment
Recurrence
SW480 – chemotherapy
CD44highEpCAMlow cells correlate with signatures of chemoresistance and have a worse clinical outcome
Diagnostic and prognostic CTC markers
Targeted CTC therapy
Future Plans: detection and targeting of single CTCs and CTC cluster in blood
Single CTCs vs clusters
Epithelial vs hybrid E/M vs mesenchymal
http://labriccardofodde.nl