Tumor heterogeneity and cancer cell plasticity

Introduction

The cancer stem cell model provides one explanation for the phenotypic and functional heterogeneity among cancer cells in some tumors1–5. The model posits that some cancers are hierarchically organized into subpopulations of tumorigenic cancer stem cells and their non-tumorigenic progeny V体育平台登录. In these cases, cancer stem cells are thought to drive tumor growth and disease progression, perhaps including therapy resistance6–8 and metastasis9,10. However, difficulty replicating solid cancer stem cell markers, variability from patient to patient, and variation in results from different xenograft models have meant that it remains unclear what fraction of cancers follow this model – most, or only a minority11.

Even in cancers that clearly contain a hierarchy of tumorigenic and non-tumorigenic cells, this hierarchy must co-exist with other sources of heterogeneity including clonal evolution12, heterogeneity in the microenvironment 13,14, and reversible changes in cancer cell properties that can occur independently of hierarchical organization15–18. Under these circumstances it is not necessarily clear which phenotypic and functional differences among cells arise from which sources of heterogeneity VSports注册入口. To what extent do metastasis, therapy resistance, and disease progression reflect intrinsic properties of cancer stem cells as opposed to genetic evolution or other sources of heterogeneity. Integration of results from multiple experimental approaches will be necessary to distinguish the relative contributions of these sources of heterogeneity to disease progression.

New experimental approaches have provided perspective and insight into these questions. Genetic approaches to fate-map the contributions of cancer cells to tumor growth in mice have provided evidence in support of the cancer stem cell model in some contexts and evidence against the model in other contexts19–23. Since transplantation assays evaluate the potential of cancer cells to form tumors, rather than their actual fate in the native tumor, fate-mapping complements what we have learned from transplantation assays (Figure 1) V体育官网入口. High-coverage sequencing of human tumors has also provided new insights into genetic heterogeneity within tumors and the cells responsible for relapse after therapy24–28. In this review, we will evaluate the implications of these new data for the cancer stem cell model and the extent to which this model accounts for clinically important forms of heterogeneity in cancer.

Testing tumorigenic potential

The central idea in the cancer stem cell model is that tumor growth and disease progression are driven by minority populations of tumorigenic cells, and that many other cancer cells have little or no capacity to contribute to tumor growth. This means that therapeutic strategies should particularly focus on killing the tumorigenic cells. Experimentally, the cancer stem cell model has primarily been tested using transplantation assays, which test the potential of a cancer cell to form a tumor. These assays have demonstrated the existence of phenotypically distinct subpopulations of tumorigenic/leukemogenic and non-tumorigenic/non-leukemogenic cells in a number of human cancers including acute myeloid leukemia (AML)29,30, chronic myeloid leukemia (CML)31, breast cancer32, glioblastoma 6,33, colorectal cancer 34–36, pancreatic cancer 37, and ovarian cancer 38–40 V体育2025版. Operationally, the cells that formed tumors in these studies were rare. Nonetheless, in principle a cancer could have common tumorigenic cells and still be hierarchically organized consistent with the cancer stem cell model41.

Tumorigenic potential can only be tested in a permissive environment. A persistent concern is that there may be cancer cells that have the potential to contribute to tumor growth and disease progression in patients but that do not have the opportunity to exhibit this potential after transplantation VSports. There are many reasons why a transplantation assay might underestimate the frequency of cancer cells with tumorigenic potential. Human cells must be transplanted into highly immunocompromised mice to escape the powerful xenogeneic immune response that kills human cells in mice. Although a succession of increasingly immunocompromised mice has been used to assay the tumorigenic potential of human cancer cells, from nude, to severe combined immunodeficient (SCID), to non-obese diabetic SCID (NOD/SCID), to NOD/SCID IL2Rγnull mice (NSG), all of these mice retain some xenogeneic immune barrier 42. There is no opportunity to test whether human cells have tumorigenic potential if they are killed by a xenogeneic immune response. The frequencies of human AML cells30,43, acute lymphoblastic leukemia (ALL) cells44,45, melanoma cells46, and lung cancer cells47 with leukemogenic or tumorigenic activity are much higher in more highly immunocompromised mice.

Other mechanisms also contribute to an underestimation of tumorigenic potential. Many mouse malignant peripheral nerve sheath tumor (MPNST) cells have tumorigenic potential, but cells of one genotype depend upon exogenous laminin to form tumors while cells of another genotype express laminin cell-autonomously⁴⁸. This raises the arresting possibility that tumors from different patients may require different assay conditions to read out their full spectrum of tumorigenic cells. There are other cases in which key adhesion molecules or growth factors are required for cells to exhibit clonogenic activity but are not available to human cells in mouse tissues because of the inability of mouse ligands to bind human receptors⁴⁹. Xenotransplanted tumors can also lack the architecture and stroma of tumors growing in native sites⁵⁰. There may be many variables that influence the permissiveness of the environment for tumorigenesis, including uncharacterized factors that have not yet been taken into account in assays.

Given these concerns about xenotransplantation it is reassuring that syngeneic transplantation of mouse cancers has also supported the cancer stem cell model. Studies of cells from mouse germ lineage cancers ⁵¹, AML^52,53, chronic myelomonocytic leukemia (CMML)⁸, CML⁵⁴, breast cancer^55,56, and medulloblastoma^57,58 have all been consistent with the cancer stem cell model. Many of these studies demonstrated that some fractions of cancer cells are enriched for tumorigenic activity even when there is no xenogeneic barrier to engraftment. The cancer stem cell model cannot, therefore, be entirely an artifact of xenotransplantation.

Improved transplantation assay conditions have revealed that some cancers have common leukemogenic/tumorigenic cells (some AMLs⁵⁹, many melanomas^15,46,60, some ALLs^59,61,62, and mouse MPNSTs⁴⁸), while other cancers continue to have only rare leukemogenic/tumorigenic cells no matter what assay conditions are used^31,41,47. More work will be required to determine what fraction of cancers falls in each category.

For all of the reasons described above, it will be critical to continue to optimize transplantation assays to most accurately estimate the spectrum of cancer cells that retain the potential to contribute to tumor growth. This will require systematically testing variations in xenotransplantation assay conditions, including the addition of human cytokines ⁶³ or other variables that might influence human cell engraftment⁶⁴. Many human cancers continue to be transplanted into heterotopic sites in mice without a careful comparison of the consequences of heterotopic versus orthotopic engraftment. It will not be surprising if we are continuing to vastly underestimate the frequency of cells that can contribute to disease progression in some human cancers despite the advances made in xenotransplantation assays over the past several years.

Testing hierarchical organization

Beyond showing a cancer has tumorigenic and non-tumorigenic cells, the other criterion that must be satisfied according to the cancer stem cell model is that the tumorigenic cells give rise to the non-tumorigenic progeny. Without demonstrating a lineage relationship, the functional differences between cells may reflect genetic differences. Tumorigenic human cancer cells from AML ^29,30, CML ³¹, breast cancer ³², glioblastoma ^6,33, colorectal cancer ^34–36, pancreatic cancer ³⁷, and ovarian cancer ^38–40 formed more tumorigenic cells as well as phenotypically distinct non-tumorigenic cells in immunocompromised mice. For example, CD34⁺CD38⁻ AML cells from many patients are not only enriched for leukemogenic activity but also form CD34⁻ and CD38⁺ non-leukemogenic cells upon transplantation⁶⁵. Similar observations were made in mouse models of germ lineage cancers⁵¹, AML^52,53, CMML⁸, CML⁵⁴, breast cancer^55,56, and medulloblastoma⁵⁷. This is the basis for the idea that cancer stem cells form heterogeneous tumors by undergoing epigenetic changes akin to the differentiation of normal stem cells.

There are also cancers for which there is compelling evidence against the stem cell model. Tumorigenic cells are common and phenotypically diverse in stage III and IV human melanomas^15,46. Nonetheless, Boiko et al.⁶⁶ suggested that only CD271⁺ melanoma cells can form tumors in immunocompromised mice. Civenni et al.⁶⁷ reported that CD271 negative melanoma cells can form tumors in NSG mice but that they cannot form CD271⁺ progeny or tumors in NOD/SCID mice. In our hands, CD271 negative melanoma cells readily form tumors in both NOD/SCID and NSG mice and all of the tumors are heterogeneous for CD271 expression^15,46. In an attempt to resolve the inconsistencies we have compared the tumorigenic capacity of CD271⁺ and CD271 negative melanoma cells isolated from multiple patients using our dissociation protocol^15,46 as well as the dissociation protocols described by Civenni et al.⁶⁷ and Boiko et al.⁶⁶. Irrespective of which dissociation protocol we used, whether or not we injected the cells with Matrigel, or whether we transplanted into NOD/SCID or NSG mice, both CD271⁺ and CD271 negative melanoma cells readily formed tumors and all the tumors were heterogeneous for CD271 (unpublished data). We have now tested over 20 heterogeneously expressed markers in tumors obtained from many patients but have not been able to detect any fraction of melanoma cells that lacks the potential to form a tumor^15,46.

Difficulty reproducing cancer stem cell markers has been a common problem in solid cancer studies. For example, CD133 appeared to robustly distinguish tumorigenic from non-tumorigenic brain tumor cells in early studies^6,33 but a series of subsequent studies found tumorigenic cells in both CD133⁺ and CD133 negative fractions^68–71. Since the existence of markers that can distinguish tumorigenic from non-tumorigenic cancer cells is the experimental basis for the conclusion that these cancers follow the stem cell model, the inability to widely confirm markers undermines the evidence supporting the model.

A related problem is that cancer stem cell markers originally characterized in a limited number of tumors have often been assumed to be generalizable. Such markers have often been used in other tumors, or even in cell lines, without independently confirming that the markers were informative in these other contexts. For example, it was clear from the initial study of breast cancer stem cells that the CD44⁺CD24⁻ surface marker phenotype enriched tumorigenic cells in some, but not all, breast cancers³². Yet many studies subsequently characterized CD44⁺CD24⁻ breast cancer stem cells in other tumors or cell lines without confirming that these markers distinguished tumorigenic from non-tumorigenic cells in those contexts. Consequently, many studies of cancer stem cell properties are based upon markers of uncertain validity.

"VSports app下载" Variability among patients

One of the reasons for variability among studies is that markers expressed by tumorigenic cells differ among patients. Early studies in AML indicated that leukemogenic cells were highly enriched among CD34⁺CD38⁻ cells but not CD34⁺CD38⁺ AML cells ^29,30. More recent studies with larger numbers of samples showed that while the CD34⁺CD38⁻ fraction consistently contains leukemogenic cells, leukemogenic activity is also commonly found in the CD34⁺CD38⁺ and CD34⁻ fractions^43,65,72. Approximately half of AMLs have most leukemogenic cells in the CD34⁺CD38⁻ fraction while the other half have most leukemogenic cells in the CD34⁺CD38⁺ fraction^43,65. Many human AMLs with nucleophosmin mutations have leukemogenic activity exclusively in the CD34⁻ fraction, while some have leukemogenic activity in both CD34⁻ and CD34⁺ fractions⁷³. This indicates that differences in mutations can cause differences in the phenotype of leukemogenic cells among patients. The same is true in solid cancers, as Sca1⁺ cells are enriched for tumorigenic activity in mouse lung adenocarcinomas with Kras and p53 mutations but not in tumors with only Kras mutations⁷⁴.

Differences among patients may also reflect differences in the cell-of-origin. For example, some medulloblastomas arise in the cerebellum from sonic hedgehog pathway activation in granule neuron precursors and frequently have a poor prognosis⁷⁵. Other medulloblastomas arise in the dorsal brainstem and are highly curable⁷⁵. Similarly, neural progenitors from different regions of the central nervous system form different subtypes of ependymomas with different properties⁷⁶. Both hematopoietic stem cells and restricted myeloid progenitors can serve as the cell-of-origin for AML ^53,77–79 but the leukemogenic cells have somewhat different properties in each case ⁸⁰. The distinct developmental origins of tumors, both with respect to regional identity and position in the normal tissue hierarchy, contribute to differences among patients in tumorigenic cell properties.

Tumorigenic cell phenotype can also change over time. In some ovarian cancers, only CD133⁺ cells contain tumorigenic activity whereas in other ovarian cancers tumorigenic cells are in the CD133⁺ and CD133 negative fractions ⁴⁰. Ovarian cancers with only CD133⁺ tumorigenic cells sometimes give rise to CD133 negative tumorigenic cells upon passaging⁴⁰. If tumorigenic cell phenotypes commonly change upon passaging of tumors, this could explain some of the inconsistencies observed among studies that use small numbers of tumors.

The frequency of tumorigenic cells in some cancers also varies widely among patients. Side-by-side studies of AMLs from different patients revealed frequencies of leukemogenic cells in the CD34⁺CD38⁻ cell fraction that varied 1000-fold ⁶⁵. B-ALLs from different patients had frequencies of leukemogenic cells that varied 100-fold ⁴⁴. Ovarian cancers from different patients had tumorigenic cell frequencies that varied almost 1000-fold ⁴⁰. It remains uncertain to what extent this reflects biological variability in the frequency of cells that can contribute to tumor growth within patients as opposed to variability in the extent to which transplantation assays are permissive for tumorigenesis by cells of different genotypes. The variability in the frequency and identity of tumorigenic cells between patients shows that markers identified in one tumor cannot be assumed to distinguish cancer stem cells in other tumors or in other contexts.

A key question raised by the differences among patients is whether tumors of the same type differ in the extent to which they are hierarchically organized. For example, one possibility is that all breast cancers follow a stem cell model even though existing markers do not distinguish tumorigenic from non-tumorigenic cells in some tumors. Another possibility is that only a subset of breast cancers follows the stem cell model. Or perhaps the hierarchy is steep in one subset of each cancer, with rare tumorigenic cells that give rise to abundant non-tumorigenic cells, and shallow in another subset of each cancer, with common tumorigenic cells that form small numbers of non-tumorigenic cells (Figure 2). Until enough tumors are carefully studied to observe consistent patterns among patients with particular subtypes of disease, tumors will have to be tested individually to determine whether tumorigenic cells are common or rare and whether markers can distinguish tumorigenic from non-tumorigenic cells.

Fate versus potential in tumors in vivo

Which cells actually contribute to the growth and progression of tumors in vivo? Most cancer stem cell studies are designed to assess the identity of cancer cells with the potential to contribute to tumor growth. A separate question relates to the identity of cells fated to contribute to the growth and progression of specific tumors (Figure 1). The question of fate addresses what cells actually do in a specific circumstance while the question of potential addresses what cells can do under permissive conditions.

Some cells that have the potential to drive tumor growth do not actually do so in the native tumor because they are not in a permissive environment, they are killed by immune cells, or by therapy. In the native tumor slowly proliferating clones may be at a competitive disadvantage to rapidly proliferating clones and therefore may not contribute much to tumor growth. However, these slowly proliferating clones may form tumors after transplantation. Environmental cues from stromal cells can restrict the growth of cancer cells in the native tumor environment ⁸¹, but the absence of these cues may permit the same cells to form tumors after transplantation. In some circumstances, a high percentage of cells with tumorigenic potential may contribute to tumor growth (Figure 1). In other circumstances, there may only be a small percentage of cells with tumorigenic potential that actually contribute to tumor growth.

One context in which the fate of tumorigenic cells can be tracked is after xenotransplantion of human cancer cells. Primary human colorectal cancer cells were marked by lentiviral infection and the relative abundance of distinct clones was tracked as tumor cells were serially transplanted ⁸². Individual clones differentially contributed to tumor growth over time: some were always abundant, some were abundant then became rare, and some were rare then became abundant. When human B-ALL cells from a single patient were transplanted at limiting and non-limiting cell doses, different dominant clones emerged in each recipient mouse⁴⁴. Leukemogenic clones thus do not contribute equally over time after transplantation.

Lineage tracing experiments have recently been performed in mouse models of benign tumors to test whether many, or few, cells contribute to their growth ^20,21. Mice bearing benign papillomas were treated with low doses of tamoxifen, allowing Keratin14-CreER to recombine a conditional reporter in a small percentage of papilloma cells²⁰. These rare marked clones were tracked over time to assess their contribution to tumor growth. The frequency of clones declined over time, with only 20% persisting after seven weeks. Non-persisting clones appeared to be lost through terminal differentiation. The average size of persisting clones increased over time: by seven weeks the clones ranged from hundreds to thousands of cells. These observations demonstrate that only a minor subpopulation of tumor cells drives papilloma growth, though the rate at which these cells divide is increased relative to what is observed in the normal epidermis ⁸³. Benign papillomas are therefore hierarchically organized, consistent with the stem cell model, though this is perhaps not surprising for a benign tumor.

The same study also fate mapped cells in tumors after they progressed to squamous cell carcinomas²⁰. Cells in these tumors were more highly proliferative, more undifferentiated, and formed larger clones as compared to cells in benign papillomas. A much greater percentage of cells sustainably contributed to the growth of squamous cell carcinomas. The squamous cell carcinomas in this study, therefore, have only a shallow hierarchy, with few non-tumorigenic cells (Figure 2). Additional studies are required to determine whether there is any hierarchical organization among the persisting clones - perhaps some have more proliferative potential than others. Overall, the data suggest that as benign adenomas progress to carcinomas, the hierarchy becomes more shallow and more cells can contribute to tumor growth.

Schepers et al. used a multicolor conditional reporter to track the contribution of Lgr5⁺ cells to the growth of pre-malignant intestinal adenomas²¹. Lgr5 expression marks normal stem cells in the intestinal crypt ⁸⁴. Administration of tamoxifen activated Lgr5-CreER recombinase. This deleted the APC tumor suppressor in Lgr5⁺ cells, forming adenomas as well as activating the expression of a multicolor reporter in Lgr5⁺ cells and their progeny ²¹. Administration of a second pulse of tamoxifen reactivated Cre recombinase, recombining the multicolor reporter again, leading to a color switch within some Lgr5⁺ cells in the adenomas. Lgr5⁺ cells in the normal epithelium are thus capable of giving rise to adenomas and the Lgr5⁺ cells within the adenomas can contribute extensively to tumor growth. Since most of the progeny were Lgr5 negative cells it was speculated that Lgr5⁺ adenoma stem cells give rise to Lgr5 negative cells with little proliferative capacity; however, the Lgr5 negative cells have not yet been fate mapped and it is unknown whether they have less ability to contribute to tumor growth.

A recent study shows that Lgr5 negative cells can also act as the cell-of-origin for intestinal adenomas in the context of Wnt pathway activation and inflammation. Adenomas that arise in this context do not, based on currently available data, appear to follow the cancer stem cell model²². The Lgr5 negative cells in these tumors give rise to Lgr5⁺ cells, but both Lgr5 negative cells and Lgr5⁺ cells can form spheroids in culture and tumors in vivo with similar efficiency. This argues that at least some intestinal adenomas are not hierarchically organized into Lgr5⁺ tumorigenic cells and Lgr5 negative non-tumorigenic cells. Additional studies of the Lgr5⁺ and Lgr5 negative cell fractions from adenomas in more genetic backgrounds will be required to assess what fraction of adenomas exhibit hierarchical organization. It will also be important to test whether adenomas that do exhibit hierarchical organization continue to exhibit hierarchical organization after they progress to malignancies.

These lineage-tracing experiments thus provide limited support for the cancer stem cell model. Although the growth of benign skin papillomas was driven by a minor subpopulation of cells, a much larger fraction of cells contributed to the growth of squamous cell carcinomas²⁰. A key question now is whether some of the persistent clones that exhibit ongoing contribution to tumor growth might nonetheless have limited tumorigenic potential in transplantation assays. The data on intestinal adenomas also offer limited support for the cancer stem cell model as both Lgr5⁺ and Lgr5 negative cells have the ability to serve as the cell-of-origin and to propagate tumors upon transplantation, at least in certain genetic backgrounds. Ultimately, it will be necessary to integrate the data from both transplantation studies and fate mapping studies in significant numbers of human and mouse tumors to understand the biological diversity.

Testing fate through selective cell ablation

The selective ablation of genetically defined subsets of tumor cells is another approach to test which cells are fated to contribute to tumor growth or disease progression in the native tumor environment. A recent study addressed the role of Nestin positive cells in the maintenance of a mouse model of glioma by ablating these cells ¹⁹. GFP and the herpes simplex virus thymidine kinase (HSV-TK) were expressed under the control of the Nestin promoter such that HSV-TK positive cells could be selectively killed upon administration of ganciclovir. Nestin-expressing, GFP⁺ glioma cells were relatively quiescent and represented a minority of cells in the gliomas. Administration of ganciclovir modestly extended the lifespan of mice, indicating that the Nestin⁺ cells contribute to tumor growth. Importantly, when tumors were reduced in size by treatment with the chemotherapeutic temozolomide, pulse chase experiments suggested that tumor regrowth originated from the Nestin⁺ fraction of tumor cells. Co-administration of temozolomide and ganciclovir significantly slowed tumor re-growth; however, it was impossible to assess the long-term effects of eliminating the Nestin⁺ cells because mice independently developed lethal tumors unrelated to the original tumor. Consequently, it remains unclear if the Nestin⁺ cells are exclusively responsible for driving tumor growth and recurrence after therapy or if the Nestin negative cells also contribute. It would be particularly interesting to selectively ablate the Nestin negative cells to determine whether this also slows tumor growth and extends mouse lifespan.

Reversible plasticity among cancer cells

Some cancer cells reversibly transition among states that differ in their competence to contribute to tumor growth⁸⁵. For example, some cancer cells can reversibly transition between epithelial and mesenchymal states and there is evidence that breast cancer cells in the mesenchymal state are more competent to form tumors⁹. Melanoma cells reversibly turn on and off the JARID1B histone demethylase, and JARID1B-expressing cells are more competent to sustain tumor growth¹⁶. Many other markers are reversibly turned on and off within lineages of melanoma cells in a manner that does not correlate with the ability to form a tumor^15,17. Exposure of glioma cells to perivascular nitric oxide reversibly promotes their ability to form tumors¹³. The evidence that some cancer cells can undergo reversible changes in their competence to form tumors offers an alternative explanation for the increased tumorigenic potential of subsets of cancer cells independent of the differentiation of cancer stem cells.

Drug resistance is also a plastic property of some cancer cells, with rare subpopulations in cancer cell lines that exhibit resistance to a variety of therapeutics, reversibly forming sensitive or resistant progeny depending on whether the cells are passaged with or without the therapeutic¹⁸. This raises the possibility of intrinsic therapy resistance mechanisms that are not necessarily associated with a static hierarchy or an undifferentiated state.

It is critical to distinguish models in which intrinsic differences in tumorigenic capacity reflect reversible changes in cell state as compared to irreversible differentiation as these models make very different experimental and clinical predictions (Figure 3). If the heterogeneity within tumors reflects cells that reversibly and efficiently transition between tumorigenic and non-tumorigenic states^9,85 it may not be possible to experimentally identify any population that lacks tumorigenic potential (Figure 3f). Furthermore, it would remain necessary to eliminate all cancer cells during therapy, as even the non-tumorigenic cells could drive disease recurrence by giving rise to tumorigenic cells (Figure 3i). In contrast, if heterogeneity reflects hierarchical organization in which cancer stem cells irreversibly differentiate into non-tumorigenic cells, then therapies that eliminate cancer stem cells should be necessary and sufficient to cure disease (Figure 3a, g). This distinction is thus critical to understand the underlying biology and to develop more effective therapies. Importantly, almost all of the existing evidence for reversible transitions between tumorigenic and non-tumorigenic states comes from studies of cells in culture, often cell lines, so it remains uncertain to what extent this is observed within spontaneously arising cancers in vivo.

"VSports app下载" Genetic heterogeneity within tumors

The conclusion that cancer stem cells can recapitulate the heterogeneity of the tumors from which they derive has consistently been based on analyses of small numbers of surface markers, calling into question the degree to which there is genetic heterogeneity within tumors that is not recapitulated after the transplantation of tumorigenic cells⁸⁶. If genetic heterogeneity within tumors is low, then the differentiation of cancer stem cells into non-tumorigenic progeny could be the major driver of heterogeneity (Figure 4). Conversely, if genetic heterogeneity is extensive, every tumorigenic cell could form a genetically distinct tumor rather than recapitulating the tumor from which it derives. In tumors with extensive genetic heterogeneity, phenotypic and functional differences among cells cannot be assumed to reflect the differentiation of cancer stem cells - they could reflect genetic differences.

With these questions as a backdrop it is interesting to consider the implications of recent deep sequencing studies. Deep sequencing has been used to examine the genetic heterogeneity within tumors, the subclonal composition of tumors, and the evolutionary relationships of mutations during disease progression. Deep sequencing cannot directly test the cancer stem cell model. However, the frequencies of allelic variants in bulk tumor cells can be used to quantify the relative contribution of different clones to tumors. Even neutral passenger mutations can be informative because by following the contribution of the cells bearing these mutations to tumor growth and disease progression we gain insights into the fates of individual cancer cells and their progeny. Data from AML^25,87, CLL⁸⁸, breast cancer ^27,89,90, renal cell carcinoma ^28,91, and pancreatic cancers ^92–94 show surprisingly extensive genetic heterogeneity. Extensive genetic heterogeneity provides many opportunities for genetic changes to confer phenotypic and functional heterogeneity within tumors that is not addressed by the cancer stem cell model (and that may complicate the testing of the model; Figure 3c, f, i).

It has long been understood that cancer cells undergo clonal evolution in which mutations occur stochastically in individual cancer cells and then are subject to positive or negative selection depending on whether they confer a competitive advantage or disadvantage^12,95. Cancer stem cells are no exception. Leukemogenic ALL cells obtained from the same patient exhibit genetic heterogeneity and undergo genetic changes over time when passaged in mice ^24,44. It is not clear whether these ALLs follow the cancer stem cell model as leukemogenic cells are common in some ALLs and it has proven difficult to identify any clear hierarchical organization ^61,96. Human colorectal tumors have also been serially transplanted in mice and their genetic heterogeneity assessed ⁸². Only a small number of de novo genetic variants were detected in serially transplanted tumors as compared to primary patient samples. Since there is compelling evidence that colorectal cancers are hierarchically organized into tumorigenic and non-tumorigenic components ^34–36, these results show that genetic changes do occur in colon cancer stem cells, though the paucity of such changes raises the possibility that the rate of mutagenesis may be suppressed in those cells. There is no inherent inconsistency between the cancer stem cell model and the clonal evolution model⁴ (Figure 5).

Therapy resistance

Tumorigenic cells in certain cancers have been observed to be intrinsically resistant to certain therapies. For example, tumorigenic glioblastoma ⁶ and breast cancer ⁷ cells were enriched after irradiation of xenografts. A similar enrichment of tumorigenic cells was observed in cyclophosphamide-treated colorectal tumors ⁹⁷. Activation of ATM-dependent DNA-damage repair in tumorigenic glioblastoma cells ⁶ and decreased reactive oxygen species in tumorigenic breast cancer cells ⁷ may explain the therapy resistance of these cells.

Although it is sometimes suggested that cancer stem cells can be defined by therapy resistance, this is not true in any general sense. Differentiation therapies specifically target cancer stem cells by exploiting their capacity to differentiate. Acute promyelocytic leukaemia (APL) is treated with arsenic trioxide and trans-retinoic acid to induce terminal differentiation, growth arrest, and apoptosis by clonogenic APL cells ⁹⁸. Mouse glioblastoma stem cells can be induced to differentiate into glia by treating with bone morphogenetic protein 4 (BMP4), reducing proliferation, tumor growth, and tumorigenic cell frequency ⁹⁹. BMP4 also promotes glial differentiation by normal CNS stem cells ¹⁰⁰ suggesting that tumorigenic cancer cells sometimes inherit differentiation pathways from normal stem cells in the same tissue. Cis-retinoic acid improves survival in high-risk neuroblastoma patients ^101,102 by inducing the differentiation of undifferentiated neuroblastoma cells. Thus, tumorigenic cells are more sensitive to some therapies and less sensitive to other therapies as compared to non-tumorigenic cells.

Genetic changes clearly confer therapy resistance in some circumstances¹⁰³. Sequential genetic analysis of cancers before therapy and after relapse have been consistent with this. In ALL ^24,26, AML ²⁵, and CLL ^88,104, minor subclones prior to therapy often become dominant after therapy. If the inherent therapy resistance of cancer stem cells were the major determinant of survival during therapy then dominant clones prior to therapy would be likely to remain dominant after therapy. The observation that therapy selects for minor subclones suggests that either survival is stochastic (many cells have a similar low probability of surviving), non-cell-autonomously determined, or determined by genetic differences among subclones.

When the dominant pre-therapy clone remains dominant after relapse, the dominant clone often gains de novo mutations ^25,26,88. Relapse-specific mutations may confer therapy resistance. For example, recurrent relapse-specific mutations were identified in the gene NTC52 in 10–20% of T-ALLs^105,106. NT5C2 is a 5′ nucleotidase that can inactivate nucleoside analogs, such as the chemotherapy drugs 6-mercaptopurine and 6-thioguanine used to treat ALL. The relapse-specific NTC52 mutations are predicted to be gain-of-function mutations that enhance enzymatic activity^105,106. Certain genetic changes are likely sufficient for therapy resistance.

The intrinsic resistance of certain cancer stem cells to certain therapies likely collaborates with genetic change to enable disease progression (Figure 6). CML follows a cancer stem cell model as CML stem cells form terminally differentiated myeloid cells during chronic phase ¹⁰⁷. CML stem cells are inherently resistant to imatinib ^54,108–110. Cells with features of CML stem cells persist after therapy, even in patients who achieve a complete cytogenetic remission ¹¹¹. Imatinib is thought to restore the health of CML patients primarily by eliminating differentiated CML cells. This profoundly reduces leukemia burden but leaves CML stem cells lurking in the patients, ready to re-establish the disease upon discontinuation of imatinib^112,113. As long as patients are maintained on imatinib they remain healthy, until a mutation arises in BCR-ABL that confers imatinib resistance ^114,115. In this way, the inherent imatinib-resistance of CML stem cells allows the disease to smolder along in remission, but a genetic change is required for more robust imatinib resistance and disease progression in the face of therapy.

"V体育官网入口" Perspective

Cancer stem cell properties have been suggested to explain diverse unsolved clinical problems yet these predictions have often not been carefully tested. The roles played by other sources of heterogeneity (such as genetic) in disease progression have often not been factored into such claims. Moreover, difficulties confirming solid cancer stem cell markers have undermined efforts to confirm their existence in some cancers and to study their biology. It remains unclear to what extent these difficulties reflect variation in marker expression by cancer stem cells from different patients versus misguided efforts to apply the stem cell model to cancers that lack hierarchical organization. The tendency not to publish data that are difficult to interpret or inconsistent with the model exaggerates the extent to which cancer stem cell markers are conserved among patients. It is time to critically test the model and its predictions, to acknowledge when the data don’t fit the model, and to integrate the data with other sources of heterogeneity when they do.

Tumorigenic cells are rare and phenotypically distinct in some cancers. In other cancers, tumorigenic cells are common and phenotypically diverse with no clear hierarchical organization. We do not yet know what fraction of cancers follow the stem cell model.

As we develop a more complete understanding of genetic heterogeneity within tumors there may be some cancers in which genetic heterogeneity is the major driver of phenotypic and functional heterogeneity. In some cancers with pervasive genetic heterogeneity it may not be possible to rigorously test the cancer stem cell model as genetic differences within and between tumors may make it impossible to identify any reproducible hierarchical organization, even if there is differentiation to post-mitotic progeny. Some cancers may have epigenetic heterogeneity that is not well described by the cancer stem cell model. Indeed, a general question concerns the extent to which the phenotypic and functional properties of cancer cells undergo reversible changes. New models of cancer heterogeneity/plasticity may emerge.

In cancers that do not follow the stem cell model it will be important to demonstrate this, to avoid fruitlessly focusing on small subpopulations of cancer cells that have no more capacity to drive disease progression or therapy resistance than other cancer cells. In cancers that do follow the stem cell model, it will be important to clarify the markers that can be used to identify these cells and the contexts in which they work. It will also be critical to integrate our understanding of the biology of these cells with our understanding of other sources of heterogeneity to develop a realistic view of how each contributes to disease progression. For example, it is possible in some cancers that clones with a hierarchy of tumorigenic and non-tumorigenic cells may co-exist in the same tumors with clones that have lost their hierarchical organization as a consequence of additional mutations. This would profoundly complicate the testing of the model and undermine the extent to which it can explain clinical behavior.

• Cancer stem cell markers will have to be tested in larger numbers of patients^15,40,65 to account for heterogeneity among patients and to determine whether certain markers are more reliable in certain subsets of patients. This will provide insight into whether only certain subtypes or stages of disease follow the cancer stem cell model. Studies should not assume that markers that distinguish tumorigenic from non-tumorigenic cells in one tumor will do so in other tumors.

• To date, there is no evidence that any combination of cancer stem cell markers isolates any cancer stem cell population to a high degree of purity. The inability to purify any cancer stem cell is a profound impediment to characterizing the biology of these cells with precision. Until a high degree of purity can be demonstrated, claims related to the cell cycle distribution and gene expression profiles of “cancer stem cells” will be of uncertain validity.

• It will be informative to perform lineage tracing^20,21 and selective cell ablation experiments¹⁹ in more cancers and at more stages of disease to assess whether many, or few, cells are fated to contribute to tumor growth and disease progression.

• Studies of genetic heterogeneity should be integrated with studies of tumorigenic potential to develop a more realistic understanding of the extent to which individual tumorigenic cells recapitulate the heterogeneity of the tumor from which they derive. In many cancers that follow the stem cell model, tumorigenic cells may reproduce the cellular hierarchy but not the genotype of the tumor-of-origin.