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"Cancer is a disease of stem cells”

Discuss in the context of your knowledge of normal tissue homeostasis.

Context

Stem cells are defined by their capacity to self-renew, while also producing progeny

with more restricted properties. Xenotransplantation assays suggest only a select

subpopulation of tumour cells possess the tumourigenic and metastatic phenotype (Reya et

al., 2001). These so-called cancer stem cells (CSC) share the defining properties of adult

stem cells and are cited to follow an organisational and functional hierarchy as seen in the

haematopoietic stem cell (HSC) paradigm (Sreepadmanabh & Toley, 2018). Indeed, an

acute myeloid leukaemia (AML) model provided the first evidence of a hierarchy of

leukaemic clones (Bonnett & Dick, 1997). The aesthetic appeal of a hierarchy, clinical

promise and potential for a grand unifying stem cell theory of cancer, initiated an expansive

search for professional stem cells fuelling cancer; CSCs were next identified in breast and

brain cancer. The CSC model offered novel therapeutic approaches and a mechanism

explaining the intra-tumour heterogeneity conferred to many cancers beyond that explained

by the stochasticity of the clonal evolution model and extrinsic differences (Shackleton et

al., 2009; Magee, Piskounova & Morrison, 2012).

However, I contest the relevance of the CSC model in lieu of CSC plasticity and the

growing relevance of tumour microenvironment (TME). The unidirectional CSC model

tacitly assumes rarity, quiescence, asymmetric division and non-plasticity to be hallmarks

of CSCs based on a HSC-centric approach (Batlle & Clevers, 2017). Elucidating stem cell
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paradigms in the intestine, among others, has shown the HSC system to be an outlier and

unidirectionality to be an outdated concept. The model has suffered over-interpretation

without due caution (Clevers, 2011). Findings largely rely on inadequate assays of

tumorigenesis in immunodeficient mice and stem cell markers, while extrapolating causality

from associations alone. The clonal evolution model, stands in contrast to the CSC model,

but the dichotomy is largely artificial (Wang et al., 2014). A bidirectional hierarchy

appreciating the intrinsic roles of (epi)genetic modifications and extrinsic role of the TME

in ascribing stemness establishes a more relevant picture of cancer evolution. Stem cells are

highly relevant to cancer, and clearly offer important insights. But, the evidence for a

unifying mechanism of cancer evolution, based on CSCs as cells of origin is weak. I reframe

the statement from “cancer is a disease of stem cells” to acquisition of stemness is required

for tumourigenesis. I explore techniques that have heretofore been relied on, the relevant

intrinsic and extrinsic factors directing cancer evolution and metastasis, to present a model

unifying the CSC- and stochastic model of cancer.

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Fig 1 Clonal Evolution versus CSC models.

Stochastic genetic and epigenetic changes confer heritable heterogeneity in tumours (Nowell;
Baylin & Jones, as cited in Magee, Piskounova & Morrison, 2012). The unidirectional CSC
model shows a phenotypically distinct subpopulation of cancer cells that are tumourigenic which
give rise to non-tumourigenic and tumourigenic progeny (Reya et al., 2001). The CSC plasticity
model showcases an element of bidirectionality which ascribed stemness to non-CSCs. The
modified CSC plasticity model showcases the influence of the tumour microenvironment (TME)
on stem-cell properties.

Adapted & modified from Marjanovic, Weinberg & Chaffer (2013) with permission from the American
Association for Clinical Chemistry
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Changing identities
The CSC model arose from an understanding of HSCs as a stereotypic template of

adult stem cells. The classical paradigm showed the transplantable long-term HSC, to

maintain and expand the haematopoietic compartment (Carroll & Clair, 2018). This

hardwired HSC produces progeny of increasingly restricted potential and fate, a feature

supposedly mirrored in cancers. Cells capable of initiating AML in NOD-SCID (non-obese

diabetic/severe combined immunodeficiency) mice have a CD34+CD38- phenotype akin to

normal HSCs (Bonnett & Dick, 1997). Normal HSCs are therefore a target for leukaemic

transformation. CSCs fuel a hierarchy of tumourigenic and non-tumourigenic (NT) cells.

Cancer therapeutics focused heretofore on targeting the most proliferative cells i.e. non-CSC

tumour bulk, if the CSC model is valid, chemo- and radiotherapy resistance and relapse

could be explained (Batlle & Clevers, 2017).

The validity of the HSC, professional stem cell model is disputed in the

haematopoietic system. Some AMLs do not follow that CSC model as leukemogenic activity

is not restricted to the CD34+CD38- fraction (Magee, Piskounova & Morrison, 2012). But

even if true, the liquid organ is an outlier. Other systems resembling this paradigm include

the mammary stem cells; whether multiple independent stem cells or a rare professional

multipotent stem cell produces this function is unclear (Stingl et al., as cited in Clevers &

Watt, 2018). The skin shows the existence of slow-cycling stem cells but in normal

homeostasis the transit-amplifying cells predominate in maintaining the epidermis (Mascré

et al., 2012). But stemness may be attributed to a far larger population of undifferentiated

cells neutrally competing for niche space. The neutral competition model states that stem

cell daughters are not intrinsically divergent, but division can result in zero, one or two new

stem cells depending on niche space which contradicts the classical view of an intrinsic,

asymmetric, invariably mitotic process. Stem cells may be: facultative, as potentially in the
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pancreas; or dedifferentiate from terminal tissues; and in some instances, systems can allow

differentiated cells to directly enter the cell cycle, as in the liver (Clevers & Watt, 2018).

Closely following this more complex model of stemness in adult tissue, cancers may

arise in a hierarchical fashion. But cancer stem cells of origin may be progenitors or

differentiated cells, too. Crypt stem cells are the cells-of-origin of intestinal cancer (2009),

but the Gretan group show that elevated NF-β signalling enhances Wnt activation and

induces dedifferentiation of nonstem cells that go onto acquire tumour-initiating capacity in

a colorectal cancer model (Schwitalla et al., 2013). To suggest only a small subpopulation

of CSCs initiate tumours is an oversimplification, given that even in hierarchical systems,

the cell of origin may be a progenitor or differentiated cell.

Markers and Assays

Considerable attention has been paid to CSC surface markers (CSC-sm). The brain

CSC was exclusively isolated in a cell fraction expression neural stem cell surface marker

CD133, and this formed the basis of identifying CSCs in other systems (Singh et al., 2003).

Of the >40 CSC-sm known, 73% are present in embryonic and adult stem cells. The

remaining are significantly expressed in even normal tissue cells despite extensive validation

as CSC-sm (Kim & Ryu, 2017). Difficulty reproducing CSC markers is a common problem;

in the brain, subsequent studies find both CD133+ and CD133- fractions to be tumourigenic

(Wang et al., 2008). Elucidating changing glycan patterns in malignant cells offers a

promising approach to differentiating CSCs from normal stem cells (Karsten & Goletz,

2013). However even with such approaches, the central problem of how analysis of a small

number of surface markers can account for intra-tumour genetic heterogeneity is unclear.

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Cell purification (CSC-sm) and xenografting into immunodeficient mice, to test

tumourigenic potential, has been the gold standard in identifying CSCs (Prasetyanti &

Medema, 2017). If the tumour can be serially transplanted and act as a phenocopy of the

patient’s original tumour, it is thought to be representative (Singh et al., 2004). But several

caveats exist. There is no direct evidence that cellular behaviour after xenotransplantation

reflects the hierarchical position within the original tumour. The cancer cell ends up in a

context vastly different to the original tumour niche. In the 2004 experiments outlining

human brain tumour initiating cells, human cancer cells were transplanted into mice. This

inevitably impedes crucial niche functions. Even if this cross-species niche alteration can be

discounted, there is strong evidence that the act of transplantation itself profoundly affects

stem cell behaviour. For instance, quiescent hair bulge stem cells will generate only hair.

Upon transplantation however, the same cells readily generate epidermis and sebaceous

gland in addition (Blanpain, Horsley & Fuchs, 2007). Our reliance on immunodeficient

mouse models also omits the importance of the immune system, which is highly relevant in

immune-mediated tumour growth control as has been shown in sarcoma (Koebel et al.,

2007). Much of our understanding of CSCs arises from severely limited experimental

platforms.

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Fig 2 Comparison of transplantation assays and lineage tracing.
Recent adoption of Cre-mediated genetic lineage tracing has less biased approach and has
revealed Lgr5+ stem cell activity in mouse intestinal adenomas (Schepers et al., 2012). One
important advantage of this method, is the ability to resolve individual cell fate. Lineage tracing
studies have identified cancer cells of origin in multiple other systems, including the prostate
(Choi et al., 2012). Xenograft evidence corroborated by lineage tracing holds critical value. The
inability to lineage trace in humans however, is a shortfall. For example, mice prostate anatomy
is significantly different and the organ does not express Prostate Specific Antigen. Additionally
in mouse models, the promotor is turned on instantly. This stands in stark contrast to the
sequential acquisition of mutations in human cancer (Rycaj & Tang, 2015).

Reproduced from Rycaj & Tang (2015)


The Niche with permission from the American Association for Cancer Research

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The Niche

In adults, stem cells reside in a specialised microenvironment, or niche (Morrison &

Spradling, 2008). Stem cell function hinges on its niche, and similarly CSCs occupy TMEs

– which govern self-renewal, differentiation, metastatic potential and cellular plasticity in

CSCs (Plaks, Kong & Werb, 2015). Humanising immunodeficient recipient mice has helped

to ameliorate the major limitation of unrepresentative TME, thereby improving assay utility

(Rongvaux et al., 2013).

It is unclear the extent to which CSCs rely on the niche, this likely varies between

pathologies. The requirement of angiogenesis in maintaining tumour growth has long been

appreciated. Evidence of CSCs arising due to a microenvironment with growth-promoting

signals, was first indicated by models of neuroblastomas showing necessity of coordinated

mutations in both Schwann cells and supportive cells (Zhu et al., 2002). TMEs possess

diverse ECM scaffold, growth factors, vasculature and immune cells which influence intra-

tumour heterogeneity. Inflammation and hypoxia have been shown to enhance

tumourigenesis and metastasis (Gilkes, Semenza & Wirtz, 2014). Subtle changes in matrix

composition affect the phenotype of breast cancer (Shin et al., 2012).

Recently, the YAP/TAZ, transcriptional co-activators of the Hippo pathway –

present in TME - have been shown to sustain CSC features in several cancers in addition to

installing self-renewal capacity in non-CSCs (Cordenonsi et al., 2011; Mo, Park & Guan,

2014). Several intrinsic and extrinsic cues appear to override the YAP-inhibiting niche of

normal stem cells (Zanconato, Cordenonsi & Piccolo, 2016). Exploring this pathway offers

an exciting gateway into understanding how non-tumourigenic cells acquire stemness.

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Emerging concepts

Fig 3 Epithelial-to-mesenchymal Transition (EMT).


TWIST and SNAIL master EMT-transcription factors (TF) suppress E-cadherin which causes
loosening of cell-cell contact; this forms the basis for its association with metastasis (Cano et al.,
2000; Yang et al., 2004). Importantly, EMT-TFs regulate normal stem cell and CSC properties
(Mani et al., 2008). Increased tumorigenic and metastatic potential has been demonstrated in
mouse and human tumour cells expressing EMT-TFs (Ye et al., 2015).

Reproduced from Morandi et al. (2017) under the Creative Commons Attribution License.

EMT allows epithelial cells to acquire a mesenchymal gene programme. Although

associations between EMT, metastasis and stem cell state have been made, these are highly

disputed. Epithelial-mesenchymal plasticity supports metastasis in some models but it does

not play a critical role in differential stemness capacities (Beerling et al., 2016). The CSC

model implies that the metastatic phenotype is restricted to a subpopulation of cancer cells

which may explain why some cancer cells migrate, but do not colonise secondary sites. It

remains unclear whether intrinsic differences, stochastic events or TME play the dominant

role in defining the metastatic phenotype.

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Reproduced from Choi et al., 2015 with permission from the Royal Society of Chemistry (GB).

Fig 4 Microfluidic devices for tumour-on-a-chip.


Shown is a depiction of ductal carcinoma in situ in a mammary duct, with basement membrane,
epithelium, stroma and capillary blood flow.

In vitro overexpression/downregulation experiments are of limited value and in vivo data is


rarely reproducible. 3D tumour models and microfluidic-based approaches such as the
nanopattern assay allow single-cell tumour migration imaging by isolating cancer cells with
heterogeneous phenotypes from primary patient tumours (Smith et al., 2016). This is of
diagnostic and prognostic value clinically but such approaches will help to establish the role of
stemness in the metastatic phenotype. Recently cell-culture microfluidic devices allow for
precise control of hypoxic conditions, a state cited as pro-tumourigenic (Germain et al., 2016).
Organoids and microspheres offer another interesting system to study the TME. Co-
encapsulation techniques of neuroblastoma cells with mesenchymal stromal cells in collagen
microspheres appear to partially recreate the TME (Yeung et al., 2015). More precise
mathematical modelling of the TME niche variables will allow a greater understanding of how
stemness is installed in stem cells to promote carcinogenesis.
Perspective

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Perspective

The CSC model is disputed even in more convincing models, largely due to

inadequate experimental approaches. With the evolution of lineage tracing, 3D tumour

modelling and quantitative methods of measuring microenvironmental heterogeneity, our

understanding of how stemness plays a role in cancer initiation and progress will grow. CSC

markers lack robustness. It is important to highlight cancers that do not follow the stem cell

model. And I predict others, contain tumours demonstrating both hierarchical CSC-like and

non-hierarchical organisations. Anti-CSC therapy may be of greatest value in metastatic

disease if stronger links are drawn between stemness and the metastatic phenotype. In

cancers following a CSC hierarchy, A unified model displaying bidirectionality,

appreciating the intrinsic role of (epi)genetics and extrinsic role of TME is required.

Cancer is not a disease of stem cells, but rather stemness.

Word Count: 1590

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Appendix 1 License to reproduce Fig 1

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Appendix 2 License to reproduce Fig 2

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Appendix 3 License to reproduce Fig 4

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