Stem cell dynamics in homeostasis and cancer of the intestine.

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Nature Reviews Cancer | AOP, published online 12 June 2014; doi:10.1038/nrc3744

Stem cell dynamics in homeostasis and cancer of the intestine Louis Vermeulen1,2 and Hugo J. Snippert3

Abstract | Intestinal stem cells (ISCs) and colorectal cancer (CRC) biology are tightly linked in many aspects. It is generally thought that ISCs are the cells of origin for a large proportion of CRCs and crucial ISC-associated signalling pathways are often affected in CRCs. Moreover, CRCs are thought to retain a cellular hierarchy that is reminiscent of the intestinal epithelium. Recent studies offer quantitative insights into the dynamics of ISC behaviour that govern homeostasis and thereby provide the necessary baseline parameters to begin to apply these analyses during the various stages of tumour development. Self-renewal The ability of a cell to maintain itself while producing enough offspring to repopulate the tissue. As a result of intestinal stem cell dynamics, self-renewal is achieved by the population, rather than on a single cell level.

Laboratory for Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. 2 Cancer Research UK — Cambridge Institute, University of Cambridge, Robinson Way, CB2 0RE, Cambridge, UK. 3 Molecular Cancer Research and Cancer Genomics Netherlands, Center for Molecular Medicine, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. e-mails: [email protected]; [email protected] doi:10.1038/nrc3744 Published online 12 June 2014 1

The inner lining of the intestine is one of the most rapidly renewing tissues in the human body: every ~5 days, the entire epithelial surface is replaced. This process is sustained by intestinal stem cells (ISCs) that reside at the bottom of crypt-like invaginations and generate precursor cells that subsequently give rise to the specialized differentiated cells that fulfil the physiological functions of the intestine: mainly, the uptake of nutrients by enterocytes, as well as the transport and removal of faeces that is facilitated by mucin-producing goblet cells1 (FIG. 1). Additional abundant differentiated cell types in the intestine include hormone-producing enteroendocrine cells and Paneth cells. Paneth cells reside at the bottom of the crypt, where they form an important part of the ISC niche and also function in intestinal innate immunity 1,2 (FIG. 1). Probably as a result of its high turnover rate, the intestinal epithelium is frequently subject to malignant transformation, and colorectal cancers (CRCs) are one of the top three cancer-related causes of death3. Intriguingly, the biology of ISCs and CRCs is highly interconnected4. Signalling pathways that govern ISC function, such as WNT signalling, are often aberrantly activated in CRCs5, and it is generally thought that most intestinal cancers originate from initial transforming events in the ISC compartment 6. In addition, cells that share properties of ISCs, including multilineage potential and self-renewal, are observed in CRCs and are referred to as colon cancer stem cells (CSCs)7,8. However, the importance of these cells to the biology of CRC remains unclear. In recent years, it has become apparent that ISCs are not static entities that solely generate more differentiated progenitor cells. Rather, they are involved in many dynamical processes. For example, ISCs can transition from one ISC subset to another: rapidly dividing

leucine-rich repeat-containing G protein-coupled receptor 5‑positive (LGR5+) crypt base columnar (CBC) ISCs can generate relatively more quiescent BMI1+ ISCs and vice versa9,10. Moreover, committed progenitor cells can acquire ISC properties by dedifferentiation11,12 and ISCs are continuously lost and replaced by neighbouring stem cells in a stochastic manner 13,14, which has important implications for the initiation of cancers, as it prevents the accumulation of mutated lineages in the intestine15,16. Excitingly, stem cell behaviour can now be fairly accurately described in quantitative terms, thereby providing tremendous insights into ISC dynamics that govern homeostasis and tumour initiation. Furthermore, similar quantitative models can be applied to describe the behaviour of the stem cell-like compartment of adenomas and CRCs. These recent advances are the main topic of this Review, and we highlight how these studies affect our understanding of CRC and its future clinical management.

ISCs It has been known for decades that multipotent adult stem cells fuel the proliferative capacity of the intestinal epithelium, although the exact identity of these cells remained uncertain17. Biotechnological innovation and the recent discovery of cellular markers for multiple candidate stem cell populations provided new insights into the functioning of the ISC compartment. Unfortunately, the long-lasting debate about the identity of the ISC has not ended and has instead become confused, owing in part to the use of many different terms for the same cell or cellular processes. We think, however, that a coherent theory of the biology of the ISC is emerging, which we cover below in a series of subsections — ISC phenotype, ISC activity, ISC potential and

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REVIEWS Key points • Intestinal stem cells (ISCs) are not static entities but are instead involved in many dynamical processes. • ISCs are equipotent and continuously replace each other in neutral events. • The ISC phenotype is the sum of all markers and features that are commonly associated with stem cells in the intestine. Therefore, the ISC phenotype is continuously changing as new markers and features are being identified. • ISC activity is the ability of cells to initiate clonal long-term, multipotent lineages and is typically assessed by lineage tracing experiments. • ISC potential refers to the display of ISC activity solely in a specific context but not during homeostasis; for example, during regeneration after tissue injury. Examples of intestinal cells with ISC potential are label-retaining Paneth cell precursors and Delta-like 1‑positive (DLL1+) secretory precursors. • The functional ISC compartment is the number of cells with ISC activity corrected for their relative contribution to the total output of the stem cell compartment. • Mutations that are commonly found in colorectal cancer (CRC), such as adenomatous polyposis coli (APC) inactivation and KRAS activation, act on ISC dynamics and give a competitive advantage to the cell in which they occur. • The benefit of mutated ISCs over wild-type ISCs is not absolute, and mutated ISCs are frequently outcompeted by wild-type ISCs. • CRCs contain cells with stem cell-like activity; however, the frequency of these cells remains unknown, as does the importance of these cells for the biology of CRCs. • Differentiated cancer cells and cancer stem cells are in constant flux, which is influenced by signals that emanate from the tumour stroma.

Cancer stem cells (CSCs). Stem cell-like cells within an adenoma or cancer that fuel tumour expansion and progression. Like normal intestinal stem cells, they are multipotent and display the ability to self-renew.

Adenomas Benign intestinal tumours that strongly resemble the tissue architecture of the healthy tissue.

ISC activity An intrinsic intestinal stem cell (ISC) property that is defined by multipotency and self-renewal. It can be shown using lineage tracing.

ISC potential The ability of non-stem cells to re‑obtain intestinal stem cell (ISC) activity. The degree of ISC potential probably correlates with the level of differentiation.

Multipotency The ability of a cell to differentiate into any cell type of the tissue of residence.

ISC functionality (FIG. 2a,b). We think that these terms best show that ISC properties are context-dependent and ISC fate is not an intrinsic property. Instead, it is a cellular state that can be lost but also regained by progenitor cells via dedifferentiation. Moreover, we argue that important stem cell characteristics, such as longterm self-renewal, are in fact features that apply to the ISC compartment as a whole, rather than necessarily to individual stem cells. ISC phenotype. Until recently, research into ISCs was principally descriptive, as these cells could only be identified on the basis of their phenotype, including morphology and marker expression, or by simple assays to assess their cell cycle rate, which was erroneously assumed to be slow (quiescent) during normal tissue homeostasis. Importantly, the definition of an ISC phenotype is continuously changing as new markers to identify ISCs, or subsets of them, accumulate in the literature (for an excellent review, see REF. 18) (FIG. 2a). ISC activity. The minimal definition for stem cell activity is the combined characteristics of multipotency and self-renewal1. In homeostasis, the current ‘gold standard’ to assess stem cell activity is clonal lineage tracing 19. In this technique, a permanent genetic mark, such as the expression of a fluorescent protein, is induced in a (candidate) stem cell, and this mark is inherited by all of the descendants of that cell. The presence of multiple cell types in a single traced clone that remains present for the entire lifetime of the organism reveals multipotency and self-renewal capacity of the original labelled cell. Moreover, it reveals stem cell activity within endogenous environments in the absence of stress or injury responses. A clear downside of lineage tracing

techniques is the required knowledge of marker gene expression patterns to identify single markers that are unique for ISCs. In addition, the fate of cells without known marker expression usually remains untested. After initial lineage tracing experiments with the CBC cell marker Lgr5 (REFS 9,20), various knock‑in alleles of ISC markers — including the additional CBC marker SPARC related modular calcium binding 2 (Smoc2)21, the ‘+4’ markers Bmi1 (REF. 22), HOP homeobox (Hopx)10 and telomerase reverse transcriptase (Tert)23, and the more ubiquitously expressed SRY-box 9 (Sox9)24 and prominin 1 (Prom1)25,26 — revealed more information about ISC activity, mainly in the small intestine. These findings included that the expression patterns of these markers were partially overlapping 21,27 and that CBC and +4 ISCs can interconvert 10, indicating that a distinction between these ISC populations is ambiguous. It is now commonly thought that most CBC cells (with the exception of label-retaining cells (LRCs)) have stem cell activity. ISC potential. Cells that only show stem cell activity in specific contexts, such as during in vitro clonogenicity assays or in a regenerative response, are said to have ISC potential. Intestinal LRCs are non-dividing secretory precursors that express Lgr5 as well as +4 ISC markers 11. Lineage tracing from these cells in the homeostatic small intestine revealed strict terminal differentiation towards the Paneth cell lineage. However, upon tissue damage, these LRCs can revert their fate into cycling cells with ISC activity to develop long-term multipotent lineages11 (FIG. 2c). Intriguingly, ablation of the entire LGR5+ cell population in the mouse intestine did not give rise to a phenotype9,28. Therefore, ISC potential is present beyond the LGR5+ compartment. Indeed, although lineage tracing of LGR5‑negative secretory progenitors expressing the Notch ligand Delta-like 1 (DLL1) typically marked a few goblet or Paneth cells in homeostasis, there was a significant induction of stable clones following radiation-induced damage12. Thus, the DLL1+ precursors can also regain ISC activity during tissue regeneration (FIG. 2c). The plasticity of intestinal progenitors is probably due to the fact that, following damage, these cells regain physical contact with Paneth cells. These cells provide dominant stem cell niche signals, of which WNT and Notch ligands seem to be the most important 12,14. This concept is also in agreement with the recent observation that ablation of the entire LGR5+ compartment or severely damaging the crypt progenitors by radiation are mutually dispensable, but damaging both compartments simultaneously completely disrupts the crypt–villus architecture28. The plasticity of intestinal progenitor cells might be partly due to similar epigenetic signatures between different progenitor cell types29,30. Intestinal lineage commitment does not seem to occur via hardwired epigenetic determinants, as is observed in the haemato poietic system31. Rather, epigenetic similarity between crypt progenitor and stem cells allows cell states to be reverted with relative ease towards uncommitted ISCs,

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REVIEWS b Stem cell differentiation in the normal intestine

a The small intestine and colon

Enterocytes

Goblet cells

Enteroendocrine cells

Small intestine Colon

DLL1+ ‘+5 cell’ secretory progenitor Notch

Delta

LRC Paneth cells ISCs Enterocytes Goblet cells

Enteroendocrine cells ‘+5’ secretory progenitor

‘+5’ enterocyte progenitor LRC — Paneth cell precursor

Deep-secretory cells (colon) Paneth cells (small intestine)

CBC ISCs Mesenchymal cells

Figure 1 | Intestinal epithelium. a | The intestinal epithelium contains crypts and finger-like protrusions (villi). The crypt Nature Reviews | Cancer bottoms contain rapidly cycling intestinal stem cells (ISCs) and transit-amplifying progenitors110. Major differentiated cell types are enterocytes (which are involved in the uptake of nutrients), goblet cells (which produce mucus), enteroendocrine cells (which produce hormones) and Paneth cells. Except for Paneth cells, all cell types migrate as clonal lineages to the tip of the villi within 4–5 days, where they are shed into the lumen. Post-mitotic Paneth cells are relatively long-lived (5–6 weeks)111 and intermingle with ISCs at crypt bottoms to secrete ISC niche factors and function in innate immunity. The colon has a flat-surface epithelium that lacks Paneth cells; instead, deep secretory cells are thought to be responsible for the ISC niche function112. b | Schematic representation of the cellular hierarchy of the intestine. Actively dividing ISCs compete for niche residency. Once contact with Paneth cells is lost (the ‘+5’ position), cells make a binary fate decision that is mediated by Notch. Active Notch signalling allows multiple cell cycles while cells become fated towards enterocytes113. Delta signalling forces cell fate towards the secretory lineages114. Presumably, the lack of additional cell divisions of secretory progenitors accounts for different absolute cell numbers. CBC, crypt base columnar; DLL1, Delta-like 1; LRC, label-retaining cell.

Stem cell niche A microenvironment that imposes intestinal stem cell (ISC) activity on adjacent proliferative cells via a diverse set of stimuli. The ISC niche consists of, among others, Paneth cells and mesenchymal cells along the crypt base.

Functional ISC compartment The average number of intestinal stem cells (ISCs) per crypt that contribute to long-term homeostasis. Individual ISCs cannot be assigned as ‘functional’ with 100% certainty, only by probability.

when exposed to niche stimuli. The experimental observations mentioned above indicate that the existence of a dedicated reserve stem cell pool is not a necessity and the profound regenerative capacity of the intestine is probably provided by the ISC potential that resides in progenitor cells. ISC functionality. ISC activity and ISC potential are intrinsic cellular characteristics. By contrast, ISC functionality refers to a property of the total ISC population within each crypt. As a result of the spatial organization of intestinal crypts, not all cells with ISC activity equally contribute to long-term homeostasis32. Recent data indicate that the probability of these ISCs generating long-lived lineages increases once their positioning is closer to the bottom of the crypt32 (FIG. 2b). Owing to the probabilistic nature, it is impossible to tell with certainty to what extent an individual ISC contributes to the functional ISC compartment. However, the number of functional stem cells can be defined by the total

number of cells with ISC activity, corrected for their average contribution to long-term homeostasis. This concept is discussed below.

Stem cell population dynamics in homeostasis Analysis of chimeric mice from strains with various polymorphic markers has revealed that nascent intestinal crypts are polyclonal and gradually become clonal in the weeks after birth33,34. As a result of this process, a clonally related lineage of cells populates each adult murine crypt, and similar observations have been made in humans35–38. Importantly, although crypts are phenotypically clonal, this does not imply that they are sustained by one master stem cell. Instead, each crypt contains multiple stem cells that constantly replace each other in a dynamic process: stem cells are randomly lost from the ISC population and become primed for differentiation, and vacant niches are repopulated by the offspring of neighbouring stem cells13,14 (FIG. 3a). Ultimately, because of the stochastic nature of this process, all crypt



REVIEWS a ISC phenotype: commonly associated markers CBC ISCs

• LGR5 • ASCL2 • OLFM4 • TERT • BMI1

‘+4’ position

• SMOC2 • TNFRSF19 • RNF43 • LRIG1

• HOPX • TERT • BMI1 • LRIG1

b Stem cell dynamics in the normal intestine ISC activity

ISC potential

Gradient

• EPHB2 • PROM1 • SOX9 • LIMK2 • LRIG1

LRC — Paneth cell precursor

• LGR5 • KIT • LRIG1 • CHGA • PROM1 • MMP7

Probability for functional ISCs

c Fate plasticity during regeneration ‘+5 cell’ enterocyte progenitor

DLL1+ ‘+5 cell’ secretory progenitor LRC

ISCs reappear within a few days

Time

Figure 2 | Intestinal stem cell (ISC) phenotype, activity, potential and functionality. a | The ISC phenotype is commonly associated with various markers. In general, crypt base Nature Reviews columnar (CBC) ISCs are associated with the expression of the following markers:| Cancer 20 leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) , achaete–scute homolog 2 (ASCL2)24, olfactomedin 4 (OLFM4)115, SPARC related modular calcium-binding protein 2 (SMOC2)21, tumour necrosis factor receptor superfamily member 19 (TNFRSF19)116 and RNF43 (REF. 117). ISCs at the ‘+4’ position are associated with BMI1 (REF. 22), HOP homeobox (HOPX)10, telomerase reverse transcriptase (TERT)23 and leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1)118. However, other evidence21,119 suggests broader expression patterns of markers (arrows), such as ephrin B2 (EPHB2)120, SRY-box 9 (SOX9)121, prominin 1 (PROM1)25,26 and LIM domain kinase 2 (LIMK2)122. Label-retaining cells (LRCs), which are Paneth cell precursors, express ISC markers and markers of secretory cells11. b | Most CBC and +4 ISCs are a homogeneous population that all display ISC activity — that is, they have the ability to self-renew and are multipotent. Progenitor cells outside of the ISC niche have lost ISC activity but have the potential to re‑obtain it. Their degree of fate plasticity determines their ISC potential. Owing to favourable positioning within the ISC niche, ISCs that are located at the very bottom of the crypt are endowed with a bias for survival and are the most likely to participate as functional ISCs. c | In the case of a response to injury, cells that contain ISC potential can re‑obtain ISC activity on re‑association with Paneth cells. However, to date, it has not been formally shown that enterocyte precursors can regenerate the ISC population. CHGA, chromogranin A; MMP7, matrix metalloproteinase 7.

stem cells will become descendants of the same ISC. This course of action resembles the spread of genetic variants without selective advantage (neutral) in populations of individuals, and the term neutral drift was therefore borrowed from population genetics to describe it. One-dimensional neutral drift. The geometry of the stem cells within crypts can be approximated by a ring structure (FIG. 3a) that accommodates the dynamical properties of intra-crypt neutral drift using a relatively simple one-dimensional stochastic model. In essence, the behaviour of the stem cell pool can be adequately defined by only two parameters: the number of functional ISCs per crypt (N) and the rate at which these are lost and replaced by a neighbour (λ)13,14,39 (BOX 1). Initially, data to support this model were derived from pulse–chase experiments that consisted of clone induction and the subsequent analysis of clone size distributions at various time points13,14. As predicted, owing to the stochastic expansion, retraction and loss of clones, on average, clones became larger and less frequent with time. At later time points (>3 weeks), a substantial proportion of the clones occupied a whole crypt; that is, clones reached fixation (FIG. 3b). It is predicted that 1/N of labelled stem cells will ultimately reach fixation, as all stem cells at a given time have an equal fixation probability (Pfix) (BOX 1). These studies provided compelling evidence for the neutrality of the process and robust measurements of the rate of the neutral drift, which is proportional to the derived parameter λ/N2. In mice, it was shown that colonic crypts drift faster to clonality than crypts in the small intestine. Using a lineage tracing strategy that was independent of cellular markers and that instead relied on the continuous, stochastic activation of a reporter gene, the individual numbers for N and λ could be estimated with a high accuracy 39. As expected, as clone occurrence and neutral drift remain constant throughout life, numbers of fixed clones linearly increased with the age of the animal and clones partially occupying crypts remained present at a constant frequency (FIG. 3c). Spatial regulation of ISC fate. Intriguingly, the numbers of functional stem cells per crypt (N = 5–7) that were estimated using the mouse model described above39 were much lower than the amount of LGR5+ CBC cells detected in each crypt (n~14)14. This disparity has been largely resolved in a recent study employing in vivo live imaging of LGR5+ cells32. CBC cells in the upper part of the crypt niche can be displaced from Paneth cell contact through the divisions of neighbouring stem cells, which suggests that ISC activity is imposed by the niche and is not directly associated with stem cell division. Importantly, the success rate for individual LGR5+ CBC cells to generate long-term clonal lineages (functionality) depends on their relative positioning within the niche, with stem cells that are remote from the niche boundary being three times more likely to successfully colonize a crypt (fixation) (FIG. 3d). Thus, although most of the LGR5+ CBC cells have the intrinsic ability to generate self-renewing lineages, the position within the crypt renders the central LGR5+ cells mostly responsible for the long-term functionality of



REVIEWS a

c Continuous clonal labelling *

Expansion

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R26R-Confetti tracing — 8 weeks post-induction

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Clone number (per 105 crypts)

V 1,000

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0

200

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Cpart 600

Border CBC cells

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Central CBC cells

Neutral drift Continuous, on‑going competition between equipotent, active dividing intestinal stem cells for positioning within the niche. Passenger mutations do not affect competitive behaviour.

Fixation The point at which the descendants of one cell (the most recent common ancestor) have colonized a whole crypt and cannot be outcompeted anymore. Neutral drift towards clone fixation continues between relatives.

Fixation probability (Pfix) The probability of an individual intestinal stem cell (ISC) reaching fixation.

Figure 3 | Intestinal homeostasis results from neutral competition between intestinal stem cells (ISCs). a | Schematic Nature Reviews | Cancer representation of neutral competition between functional ISCs that are hypothetically labelled in different colours. Owing to restricted niche space, mitotic events lead to displacement of neighbouring ISCs from the niche. As a result, clonal expansion (arrows) is compensated by retraction of other clones. Moreover, the loss of the last cell of a clone leads to extinction (asterisks). Ultimately, it is inevitable that one clone (blue) will stochastically replace the other cells (fixation). b | Mouse small intestine in which R26R‑Confetti (a multicolour Cre-reporter) visually reveals neutral competition between ISCs (each endowed with one of four fluorescent proteins). Longitudinal sections (left-hand panels): few clones survive at the expense of multiple small clones that became extinct. Sagittal sections (right-hand panels): cross-sections though the crypts, showing that most crypts became clonal over time (single coloured). c | A mutation-prone [CA]30-repeat sequence allows continuous clonal labelling and a precise estimation of the size of the functional ISC population. When mice age, the amount of fixed crypts linearly increases over time, with the rate ΔCfix, while the amount of partially populated crypts (PPCs; on‑going competition) remains constant (Cpart). See BOX 1 for more details. A whole populated crypt (WPC) and a PPC are shown, with associated crypt (C) and villus (V) regions. Scale bars represent 25 μm. d | In vivo live imaging showed that relative location within the niche correlated with the probability of a cell to be functional. The central ISCs are biased for survival and contribute most extensively to the long-term ISC functionality. CBC, crypt base columnar. Top left-hand and bottom right-hand images in part b are adapted with permission from REF. 14, Elsevier. The bottom left-hand image in part b is reprinted from Cell, 145, Simons, B. D. and Clevers, H., Strategies for homeostatic stem cell self-renewal in adult tissues, 851–862, Copyright (2011), with permission from Elsevier65. The images in part c are reprinted from Cell Stem Cell, 13, Kozar, S. et al. Continuous clonal labelling reveals small numbers of functional stem cells in intestinal crypts and adenomas, 626–633, Copyright (2013), with permission from Elsevier39. Data in the graph in part c are from REF. 39.

the stem cell compartment. In 1979, a similar model was suggested by Chris Potten and colleagues40, who proposed a ‘focal point’ (the crypt base) surrounded by ‘shells’ of cells with decreasing stem cell potential. The highly consistent dynamics that have been observed throughout all of these studies — encompassing pulse–chase experiments using various promoters14,15, low- and high-dose tamoxifen14,15,28 and continuous stochastic labelling 39 — attest to the robustness of the ISC

lineage tracing system, and it is unlikely that, for example, tamoxifen administration substantially affects the properties of the functional ISC population as has recently been suggested41. Drosophila melanogaster midgut stem cells follow similar stochastic dynamics, which indicates the generality of the principles discussed above42 (see Supplementary information S1 (box)). Unfortunately, no detailed study of neutral drift dynamics has been described in humans.



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Cell of origin The cell that acquired the initial mutation that initiated tumour development.

Biased drift Unequal competition between wild-type intestinal stem cells (ISCs) and mutant ISCs for positioning within the niche. Driver mutations confer a selective advantage to a clone.

Tumour initiation CRC development has been seen as a paradigm of stepwise tumorigenesis because each of the subsequent histopathological stages that precedes invasive neoplastic growth is associated with specific and an increasing number of genetic aberrations5,43 (FIG. 4a). This conceptual framework, which is often described as the ‘adenoma–carcinoma sequence’, has had a marked impact on our understanding of cancer biology and is also central to the idea that cancer is a disease in which pre-malignant or malignant cells have an evolutionary competitive advantage over neighbouring cells and, as such, cause disruptive tissue overgrowth. Interestingly, the stochastic models used to study stem cell dynamics in homeostasis can be adapted to cover both tumour initiation and stem cell properties within intestinal adenomas. Cell of origin of colorectal cancer. For many malignancies, it is assumed that stem cells are the ‘cell of origin’; that is, a stem cell acquires the initial mutations that are necessary for malignant conversion44. Direct evidence for the preferential transformation of stem cells has

Box 1 | Analytics of stem cell dynamics • N is the number of functional stem cells per crypt. This is the total number of cells with stem cell activity corrected for the average contribution of each of these cells to the clonal output of the stem cell population. • λ is the replacement rate of stem cells expressed as replacements per stem cell per day. λ = 0.2 indicates that, on average, each stem cell will be replaced by a neighbour every 5 days. • PR is the probability of replacement, where 1 is 100%. In the case of neutral drift during homeostasis, PRWT→WT = 0.5. This indicates that during a replacement event involving two neighbouring wild-type (WT) intestinal stem cells (ISCs), both cells have an equal probability of replacing each other. During biased drift — for example, in the case of a replacement event at the border of a KrasG12D mutant and a WT clone — PRKras→WT ≈ 0.8. This means that the Kras-mutant ISC has a probability of ~0.8 of replacing its WT neighbour. Conversely, the WT ISC has a probability of ~0.2 of replacing the Kras-mutant ISC15,16. The bias (δ) can also be directly expressed as an imbalance in the involvement in replacement events, assuming that a mutant ISC replaces a WT ISC at a rate of λ(1+δ), whereas the reverse process occurs at a rate of λ(1 – δ)16. Evidently, both biased drift parameters, PR and δ, are directly related. Of note, replacement probabilities of ISCs with the same mutation are predicted to be neutral; for example, PRKras→Kras = 0.5. • ΔCfix indicates the rate at which fixed clones accumulate in a tissue using a continuous, neutral labelling model that is reliant on stochastic and replicationdependent activation. An example of such a model is the [CA]30 mouse model, in which instability of a CA repeat sequence results in activation of a reporter gene39 (FIG. 3c). This parameter is solely dependent on the rate of stem cell replacement and the rate of label activation (α): that is, ΔCfix = αλ (REF. 39). • Cpart reflects the number of partial clones (not fixed), observed in a tissue using a continuous stochastic labelling model as described above39. This parameter is only dependent on N and α: that is, Cpart = 0.5αN(N – 1). When the rate of neutral drift dynamics between ISCs and the rate of labelling are constant over time, Cpart is a stable value (FIG. 3c). • Pfix is the probability of a single stem cell reaching fixation within the crypt (FIG. 4b). In the case of neutral drift, all stem cells are equipotent and have the same Pfix value. Therefore, when PR = 0.5, Pfix = 1/N. In the case of biased drift (PR ≠ 0.5), the probability of fixation can be calculated as Pfix = [1 – (1 – PR)/PR]/ [1 – (1 – PR)/PR]N (REFS 15,54). Of note, 1 – Pfix represents the proportion of clones that will ultimately become extinct owing to replacement by non-labelled or non-mutant stem cells.

been obtained in several tissues using mouse models that allow for the induction of transformation events specifically in either stem cells or more differentiated cells44. In the small intestine, a rapid onset of adenomatous growth is indeed seen upon aberrant activation of the WNT pathway in the bottom of the crypt, either in LGR5+, PROM1+ or BMI1+ cells6,22,25. By contrast, inactivation of the negative WNT regulator adenomatous polyposis coli (Apc) in more differentiated cells only sporadically results in robust adenoma formation. Instead, small indolent cyst-like structures emerge that persist for the lifetime of the animal, which leaves open the possibility that subsequent hits could enable these lesions to develop into tumours6. It has more recently been reported that hyper-proliferation mediated by loss of Apc can also be induced in the more differentiated cell compartments in the intestine. Following aberrant activation of the nuclear factor-κB (NF-κB) pathway in villus cells (for example, as a result of intestinal inflammation or by Kras mutations), these cells become susceptible to transformation45. This indicates that solitary activation of the WNT pathway in differentiated villus cells is insufficient to initiate an adenomatous expansion. For tumour initiation, additional signals are required, which are inherently present in the ISC niche and can be substituted by NF-κB pathway activity. The presence of such signals expands the range of cells that are susceptible to malignant conversion, and it is speculated that the increased risk of developing CRC that is seen in the hamartomatous polyposis syndromes 46 (such as Peutz–Jeghers) or severe colitis is related to this mechanism. Intriguingly, distinct cells of origin might contribute to the widespread inter-patient heterogeneity of CRCs, as a result of either cell intrinsic differences or different local morphogenic gradients47 (BOX 2). Quantifying the clonal advantage of mutations. Despite our tremendous qualitative insight into the cellular consequences of frequently occurring mutations48,49, there are only limited quantitative data on the population effects of these mutations. This is especially remarkable because, in essence, cancer is a disease of unwanted expansion of cell populations that are predicted to follow fundamental laws of population dynamics50. Insights into these dynamics might provide important knowledge through which therapy and preventive strategies could be improved. We have (independently of one another) recently adapted the neutral drift framework to allow the incorporation of non-neutral effects to quantitatively express the competitive advantage of oncogenic events within the intestinal crypt 15,16. On sporadic induction of oncogenic KrasG12D in cells for which the resulting lineages could be tracked using the expression of a fluorescent reporter, we found that these clones are, on average, larger and drift more rapidly towards crypt fixation than wild-type (WT) clones. The variability in clone sizes of KrasG12D lineages follows a biased drift model and, assuming that N and λ remain constant, the competitive advantage of mutant stem cells can be expressed as an



REVIEWS a Competitive behaviour of cancer mutations Normal intestine Adenoma

Carcinoma Stem cell-like cells • KRAS • TP53 • SMAD4

APC

* b

Oncogenic mutation

*

1/2 1/2

* 30–40%

1/2 1/2 1/2

1/4 1/4

Time

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3/4 Biased competition between WT and mutant ISCs

* 1/2 1/2 1/2

c Apchet versus WT

0.38 0.38 0.62

0.69

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d Clonal expansion by crypt fission

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Neutral competition between WT ISCs

0.48 0.42

0.58

0.42 0.58

Figure 4 | Competitive behaviour of cancer mutations. a | Schematic representation of the adenoma–carcinoma sequence. In principle, aberrant activation of the WNT pathway, typically via inactivation of the tumour suppressor adenomatous polyposis coli (APC), underlies adenoma formation. Additional mutations, such as those in KRAS, TP53 and Nature Reviews | Cancer SMAD4, are associated with progression towards carcinoma in situ. b | Oncogenic mutations alter the competitive fitness of intestinal stem cells (ISCs) with respect to their wild-type (WT) neighbours. As a result, the chance that a mutant ISC will displace a WT-neighbouring ISC (clonal expansion) is higher than the mutant ISC itself being displaced (retraction). Therefore, oncogenic mutations frequently colonize the entire niche. Importantly, however, the advantage is a bias and not deterministic; that is, there is still a chance that ISCs with an advantageous mutation will be displaced from the niche and become extinct. Between ISCs containing the same mutation, replacement events are neutral again. c | The relative competitive advantage for different cancer mutations. Note that the parameters are context-dependent, such as in the case of Trp53 mutations in homeostasis versus colitis. BOX 1 highlights how the proportion of fixation can be calculated using these values. d | The mutant clone can clonally expand through the epithelium via enhanced rates of crypt fission. In the case of field cancerization, such patches of mutant epithelium, although histologically often appearing normal, can predispose to the development of cancer. The dashed arrow represents a bifurcation furrow and crossed arrows represent the direction of probable clonal expansion. DN, dominant negative; het, heterozygous; hom, homozygous.



REVIEWS increased potency of these cells to occupy a neighbouring vacant niche15,16. Although neutral drift is characterised by an equal probability of two neighbouring stem cells replacing each other (PRWT→WT = 0.50), KrasG12Dmutant clones have an increased probability of replacing a WT stem cell (PRKras→WT = 0.78–0.89) (BOX 1; FIG. 4b,c). Crucially, the probability that the mutant stem cell replaces the neighbouring WT stem cell is not 100%, which implies that mutant stem cells are also frequently lost by replacement with WT stem cells. The competitive advantage of KrasG12D-mutant ISCs could mostly be explained by the increased cell cycle rate of these cells16. These findings are in line with those of a recent study that found that although NrasG12D and KrasG12D both increased haematopoietic stem cell (HSC) proliferation, only NrasG12D gave a lasting competitive advantage over WT HSCs51, owing to a preserved long-term selfrenewal capacity, whereas KrasG12D resulted in stem cell exhaustion. Additionally, similar to aberrant Kras activation, both heterozygous and homozygous inactivating mutations in the Apc gene of ISCs give a competitive advantage over WT ISCs15 (FIG. 4c). Apc+/− ISCs show a small but significant increased fitness over WT ISCs (PRApc+/−→WT = 0.62), which is in line with observations that heterozygous loss of Apc already gives a phenotype52,53. The advantage of Apc−/− ISCs over Apc +/− ISCs was estimated to be of a similar order (P RApc−/−→Apc+/− = 0.69). By contrast, Trp53 mutations only cause a competitive advantage in colons affected

by colitis, which shows that the evolutionary advantage of mutations is dependent on the context in which they arise15 (FIG. 4c). Fixation rate of mutations within the crypt. Clearly, ISCs that have mutations that commonly occur in CRC have an advantage over WT stem cells. However, the competitive benefit is not absolute, and mutant stem cells are often replaced by normal stem cells in stochastic events. The proportion of mutant stem cells that is terminally lost or that reaches fixation within a crypt can be calculated using the biased drift theory and the experimentally determined values for the competitive advantage of mutations15,54 (BOX 1). For example, the chance that KrasG12D clones reach fixation in the murine small intestine is 60–70% (as compared with 12.5–20% for WT clones)15,16, which indicates that 30–40% of these oncogenic lineages are lost, and this number is even higher for Apc+/− clones. Evidently, the unique tissue architecture of the intestine, paired with the continuous stochastic replacement of stem cells, minimizes the accumulation of mutated lineages. This contributes to the prevention of cancer development, as many mutant clones are terminally lost from the tissue and never get the chance to accumulate further genetic aberrations. Importantly, these studies quantified the competitive advantage of mutation-bearing clones after a proliferative lineage was established, thereby ignoring potential cellular defence mechanisms, such as senescence and apoptosis, which also protect against the accumulation of mutations.

Box 2 | Colorectal cancer heterogeneity Colorectal cancer (CRC) is a heterogeneous disease, as shown by its clinical presentation and response to therapy. Recent studies have sought to address this by generating unbiased classifications of CRCs using gene expression105–109. Importantly, in most cases, different CRC subtypes cannot be recognized on the basis of the mutation spectrum. This indicates that the developmental history of CRCs is likely to be pivotal in defining the characteristics of the resulting tumour. Indeed, tumours that develop via the serrated pathway display an importantly distinct phenotype and clinical characteristics106. Similarly, the cell of origin might contribute to CRC heterogeneity, as has been shown for other malignancies, particularly those of haematological origin44. It is perceivable (but, to date, it has not been shown) that transformation of, for example, secretory progenitor cells would yield a different tumour phenotype compared to transformation of crypt base columnar (CBC) cells (see the figure). These distinct initiating events might also have a marked impact on the functional properties of the stem cell compartment of the resulting tumours. Indeed, heterogeneity of hierarchical organization of CRCs has been detected93 and is likely to directly affect clinical presentation of the disease, including metastasis formation and therapy response97. Therefore, investigating the underlying mechanisms for the phenotypic variability in the biology of intestinal cancers, including the role of the cell of origin, is an important avenue of future study. CBC cell transformation

Normal crypt

Secretory progenitor transformation

Nature Reviews | Cancer 8 | ADVANCE ONLINE PUBLICATION


REVIEWS Pre-malignant clonal expansion In neonatal mammals, the intestinal epithelium expands through crypt fission55,56. Although the rate of crypt fission in homeostasis is low, the frequency of crypt fission can increase during regeneration or as a result of mutations16,57,58. Crypt fission is therefore a likely mechanism for mutant cells to expand beyond the borders of a crypt 59, thereby generating areas of genetically altered cells that predispose tissues for cancer development (field cancerization)59,60. Indeed, in the murine small intestine, KrasG12D-mutant lineages, after reaching fixation in an individual crypt, expand via an increased rate of crypt fission (>30‑fold)16. Similar fields of mutant tissue can be obtained via crypt fission of Apc-deficient crypts and might have an important role in adenoma formation and expansion61,62. Despite the pathophysio logical importance of crypt fission, surprisingly little is known about the process itself. In humans, fields of KRAS-mutant epithelium with a normal appearance have been detected surrounding CRCs63,64. This suggests that KRAS mutations could be initiating events in a proportion of CRCs — in line with our findings that KrasG12D-mutant cells have a potent competitive advantage over WT cells. More generally, these insights lend support to the notion that alternative routes to CRC, such as the serrated pathway, might be associated with an alternative order in which mutations are accumulated. Intriguingly, most tissues that require robust regenerative capacity, such as the intestine, stomach and skin, contain cycling stem cell populations that are compartmentalized in niches65. By stark contrast, the bone marrow harbours HSCs that are slow-cycling and positioned in one, virtually continuous niche66. Indeed, mutations that confer a growth advantage to HSCs, such as those seen in chronic myeloid leukaemia67 or in paroxysmal nocturnal haemoglobinuria68, can rapidly overtake the entire bone marrow of a patient. It is therefore likely that compartmentalization of stem cell populations in repetitive small niches gives an optimal balance between robust regenerative capacity by active cycling stem cells and restraining the spread of potential oncogenic mutations. Opportunities for CRC prevention Preventing the accumulation of mutant clones is an attractive preventive strategy. The process in which mutant ISCs are stochastically replaced by WT ISCs could potentially be exploited to achieve this. Targeting of the pathways that are derailed in mutant ISCs might substantially reduce the relative fitness of mutant versus WT ISCs, thereby increasing their replacement frequency. CRC-associated syndromes that might benefit from this approach include familial adenomatous polyposis (FAP; caused by a germline mutation in APC), Cowden syndrome (caused by a germline mutation in PTEN), juvenile polyposis (caused by a germline mutation in SMAD4) and Peutz–Jeghers syndrome (caused by germline mutations in LKB1)5. In all of these conditions, loss of the second WT allele commonly underlies the onset of CRC development. Reducing the

competitive advantage of cells that have lost their second allele in favour of the heterozygous affected ISCs by mutation-specific chemopreventive compounds is an interesting future avenue of study. Indeed, the observed preventive properties of aspirin69,70 might be partially explained by the possibility that it could limit the accumulation of fixed APC-deficient clones in cases of sporadic CRC by reducing the competitive advantage of these cells: aspirin reportedly impairs WNT signalling activity 71. An additional preventive approach is to limit clonal expansion beyond the niche by inhibiting crypt fission rates; in this respect, Fischer et al.61 showed that the non-steroidal anti-inflammatory drug sulindac reduces the formation of Apc-deficient fields, thereby preventing adenoma formation.

Stem cells in adenomas and cancer Adenomas and CRCs show a clear resemblance to normal intestinal tissue, as glandular structures (albeit aberrantly organized) are retained in most of them72. Additionally, heterogeneously differentiated cell types are found in individual CRCs73–77, and this contributes to the idea that tumours are ‘caricatures’ of normal tissue. This notion was further enforced by the discovery of stem cell-like cancer cells that express ISC markers and display multipotency, as well as the ability to self-renew8,78–80. These so-called CSCs are thought to be the cells that drive tumour growth and progression, but there are still many uncertainties regarding their identification, functional properties and importance81 (FIG. 5a). Controversies over transplantation studies. Colon CSCs have been identified, in analogy to leukaemia stem cells, by transplantation studies78,79,82,83. In short, distinct cell populations are isolated on the basis of marker expression, prior to assessing their tumorigenic capacity in xenotransplantation assays in immuno compromised mice. Such studies have revealed that cells expressing CD133 (the human homolog of mouse PROM1)78,79,82 and cells expressing CD44 and CD166 (CD44+;CD166+ cells)83 are tumorigenic, in contrast to the more differentiated CD133− and CD44−;CD166− cells. Tumours that arose in these studies were reminiscent of the original human malignancy, including a stem cell-like subpopulation that could initiate new tumours on secondary transplantation. These observations were interpreted to mean that CD133+ and CD44+;CD166+ populations contain CSCs. Notably, this assay has been criticized, and it is true that many caveats are present. For example, it is unclear whether the ability to initiate a tumour in a foreign environment represents the activity of CSCs in their native microenvironment or whether it instead measures CSC potential. There are also conflicting data on the usefulness of specific CRC stem cell markers84,85. Furthermore, it was established that the level of immunodeficiency of the recipient mouse strain influences the proportion of tumour cells that is able to induce tumour growth86. Altogether, more definitive proof or refutation of the CSC concept needs to be obtained, preferably in unperturbed systems.



REVIEWS CSC phenotype Common associated markers: • WNT-high • CD133 • CD44/CD166 • LGR5 • ALDH1

a

b

Cancer cells

Differentiation rate

CSC activity Lineage tracing in mouse cancer models: • LGR5 • DCLK1

CSC potential Transplantation experiments with WNT-low cells plus myofibroblasts

Dedifferentiation rate

Tumour microenvironment Cancer stem cells

Functional CSC Continuous clonal labelling in ApcMin mice

Figure 5 | Adenoma and cancer stem cells (CSCs). a | Adenomas and carcinomas are likely to be sustained by Nature Reviews | Cancer CSC-like cells. However, there are many uncertainties about their true identity and biological properties, especially with respect to human cancers. In the meantime, many markers have been associated with CSCs (CSC phenotype) in colorectal cancers (CRCs). Cell populations that express these markers or that have aldehyde dehydrogenase 1 (ALDH1) activity123 have been evaluated in xenotransplantation assays. Importantly, it is unclear whether this assay tests CSC activity or potential, and how it relates to these properties in unperturbed tumours. In mouse models, actual adenoma stem cell activity has been documented for leucine-rich repeat-containing G protein-coupled receptor 5‑positive (LGR5+) and doublecortin-like kinase 1‑positive (DCLK1+) cells via lineage tracing (represented by the blue-membrane clone). Through continuous clonal labelling, fewer functional CSCs than cells with CSC activity have been documented in ApcMin tumours, which is likely to relate to the spatial organization of adenoma glands (the probability for functionality is represented with a green gradient (darker green corresponds to increased probability)). Many cancer cells are thought to contain CSC potential (yellow gradient, darker yellow corresponds to presumed increased CSC potential). b | Cells with CSC potential can be activated either stochastically or by signals emanating from the tumour microenvironment. The population size of cancer cells with stem cell potential, as well as the rate at which differentiated tumour cells acquire stem cell activity, are crucial determinants of the CSC model124. Similarly to normal tissue and adenoma, CRC stem cells are in competition with one another within individual glandular structures.

Lineage tracing in murine adenomas. Lineage tracing of LGR5+ adenoma cells through ‘re‑tracing’ was recently used to study these cells in their native environment 87. This revealed that LGR5 + adenomatous cells have the potential to rapidly colonize a sizeable portion of an adenoma gland and display multi lineage differentiation potential, which is indicative of adenoma stem cell activity. Similarly, lineage tracing from rare cells that expressed the tuft cell marker doublecortin-like kinase 1 (DCLK1) 88 in adenomas in the Apc Min model resulted in substantial, longlived lineages 89. Ablation of the DCLK1 + compartment resulted in reduced tumour volumes, giving evidence for the functional importance of these cells in adenoma growth89. Interestingly, although DCLK1+ cells partially overlap with the LGR5+ adenomatous population, selective killing of LGR5 + cells in Apcdeficient tissue did not affect tissue morphology or the hyperproliferative phenotype28, showing that the exact relationship between LGR5 + and DCLK1 + adenoma cells needs to be further defined. To characterize the functional stem cell compartment in adenomas, Kozar et al. 39 used a continuous clonal labelling strategy that is marker-independent. All cells in an adenomatous gland are Apc-deficient and

they are therefore equipotent. Thus, the dynamics can be considered to be neutral. Using the relationship between the fully and partially clonally labelled adenomatous crypt-like structures, it was inferred that adenomatous crypts contain approximately nine functional stem cells that replace each other at a much accelerated rate compared with the normal intestine (about a tenfold increase). Again, in adenomas, the number of functional stem cells is much lower than the number of LGR5+ cells, and it is likely that a spatially orchestrated heterogeneity of the crypt-like glandular structure underlies this discrepancy — reminiscent of the normal tissue. Unfortunately, there are no practical mouse models to study invasive intestinal cancers, and although mouse studies give valuable scientific insight into cancer biology, caution needs to be applied when extrapolating this to the human disease. Human tumour stem cells and molecular clocks. Evidently, genetic lineage-tracing studies in endogenous human tumours are unfeasible. Therefore, the inference of stem cell dynamics in unperturbed human adenomas and cancer relies on so‑called ‘molecular clocks’. In principle, molecular clocks can be any stable and detectable neutral mutation. Crucially, the rate at which these



REVIEWS Fate plasticity The capacity of cells to dedifferentiate and re‑obtain intestinal stem cell activity.

hits occur needs to be sufficiently high to make ana lysis feasible. Individual mutations in genomic DNA, for example, are too infrequent, even in cancer 90. Two examples of molecular clocks that have been successfully used in humans are mutations in mitochondrial DNA that impair cytochrome c oxidase (CCO) activity and alterations in metastable methylation patterns of CpG-rich regions91–93. Analysis of CCO activity patterns in human adenomas has revealed that adenomatous glands, like normal glands, are predominantly clonal92. Furthermore these clonal glandular structures contain both secretory and absorptive cells, which indicates that multipotent stem cells populate them. Although the results of additional analysis of intra-crypt methy lation patterns were consistent with crypts containing multiple competing stem cells, the exact number could not be determined. Similarly, determination of single gland-specific methylation patterns in combination with computational models of tumour growth revealed a functional CSC population that represented 0.5–4% of cells in established CRCs93, although conflicting results have been reported using this method 94. It is anticipated that in the next few years, these types of studies in human tissue will benefit from technological advances in DNA sequencing that allow for greater depth and wider genomic coverage at a reduced cost. CSC potential and plasticity. Dedifferentiation of committed progenitor cells into ISCs can occur during a regenerative response following injury 11,12. Similarly, more differentiated tumour cells can revert back to a stem-like phenotype. An elegant study using breast cancer cell cultures revealed that stochastic state transitions of individual cells — moving from stem cell to differentiated cell or the reverse — could result in a phenotypic equilibrium in a population95. In CRC, it is likely that a similar dynamic equilibrium sustains the CSC population. Importantly, although cellular plasticity could be fully governed by stochastic fate decisions in homogeneous cell culture conditions, in tumours, the microenvironment is a substantial participant that can locally affect transition rates (FIG. 5b). Indeed, factors that are secreted by stromal myofibroblasts can directly induce functional and phenotypical dedifferentiation96. It will be essential to determine the rate at which dedifferentiation occurs in unperturbed CRCs, as it is a pivotal parameter to define the value of the CSC model97. For example, if fate transitions from differentiated cells to CSCs are highly frequent and many

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cells with CSC potential are present, it is inappropriate to specifically target the CSC population, as these cells will rapidly recur when treatment ends98. Interestingly, chemotherapy activates cancer-associated fibroblasts to secrete interleukin‑17A (IL‑17A), which increases their potency in promoting CRC stem cell activity 99. Furthermore, therapy can also directly induce CSC activity in otherwise dormant CRC cells100.

Conclusions and outlook Despite the immense insight that we have obtained in the past few years into the complex processes that govern intestinal homeostasis and malignant conversion, much still needs to be uncovered. This relates most prominently to the role and properties of stem cell-like cells in human cancers. We think that for a comprehensive understanding of stem cell biology, it is important to acknowledge the distinctions between stem cell phenotype, activity, potential and functionality, especially in the context of cancer. Studying CSCs in human CRC is complicated by widespread heterogeneity within tumours and between tumours of different patients47,101. For example, it is possible that the CSC markers are differently expressed between various CRCs, in analogy to breast cancer 102 and mouse cancer models 103. Furthermore, not all CRCs may follow the same hierarchical organization, as the proportion of functional stem cells might vary. Similarly, the composition of CRC stroma might vary per individual, as do the spatial characteristics of individual CRC glands; both will substantially affect the functions of CSCs. In general, it is expected that the relatively high fate plasticity of the normal ISC compartments is conserved in CRCs and that the acquisition of CSC activity by more differentiated cancer cells is a relatively frequent event. The ISC dynamics in the intestine and CRCs, in contrast to Waddington’s ‘epigenetic mountainous landscape’, are thus more reminiscent of a river delta that seems to lack insurmountable lineage commitments. An additional complicating factor in uncovering CRC stem cell dynamics is the clonal heterogeneity within individual CRCs. Indeed, in haematological malignancies, it has already been reported that stem cell frequencies differ between clones of the same malignancy 104. To achieve a comprehensive understanding of CRC biology in the future and to improve the treatment of patients suffering from this disease, it will therefore be essential to integrate clonal evolution data with intra-clonal stem cell dynamics.

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Acknowledgements

L.V. and H.J.S. are both supported by a KWF Fellowship from the Dutch Cancer Society (grant numbers 2011–4969 and 2013–6070, respectively). The authors wish to thank D. Winton and the members of his laboratory, as well as E. Morrissey, L. van der Flier and M. van de Wetering, for illuminating discussions.

Competing interests statement

The authors declare no competing interests.

SUPPLEMENTARY INFORMATION See online article: S1 (box) ALL LINKS ARE ACTIVE IN THE ONLINE PDF


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