cancer stem cell hypothesis definition

The cancer stem cell hypothesis: In search of definitions, markers, and relevance

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Introduction

The cancer stem cell hypothesis and its implications, cancer stem cells: an old idea reemerging at an important time, hematopoietic stem cells have led the way, definition: what is a cancer stem cell, cancer stem cells in solid tumors, clarifying the concepts and definitions, cancer stem cell assays, cancer stem cell markers, genetic and epigenetic signatures of “stemness”, implications for cancer therapy: opportunities and challenges, summary and future directions, appendix a. participant list, acknowledgments, cancer stem cells—perspectives on current status and future directions: aacr workshop on cancer stem cells.

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Michael F. Clarke , John E. Dick , Peter B. Dirks , Connie J. Eaves , Catriona H.M. Jamieson , D. Leanne Jones , Jane Visvader , Irving L. Weissman , Geoffrey M. Wahl; Cancer Stem Cells—Perspectives on Current Status and Future Directions: AACR Workshop on Cancer Stem Cells. Cancer Res 1 October 2006; 66 (19): 9339–9344. https://doi.org/10.1158/0008-5472.CAN-06-3126

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A workshop was convened by the AACR to discuss the rapidly emerging cancer stem cell model for tumor development and progression. The meeting participants were charged with evaluating data suggesting that cancers develop from a small subset of cells with self-renewal properties analogous to organ stem cells. Indeed, one critical question contemplated at the Workshop was whether tumors derive from organ stem cells that retain self-renewal properties but acquire epigenetic and genetic changes required for tumorigenicity or whether tumor stem cells are proliferative progenitors that acquire self-renewal capacity. Of course, both mechanisms may occur and may depend on the organ site. Either mechanism is different from the widely held notion that most cells in a tumor should be competent for tumor formation. If the cancer stem cell model is correct and if such cells retain the hallmarks of some tissue stem cells in being rare and entering the cell cycle infrequently, they could constitute a population that is intrinsically resistant to current therapies designed to kill cycling cells. The participants critically discussed the need for a precise definition of cancer stem cells, the requirement for new markers and more rapid and tractable in vitro and in vivo assays, and the need to develop drug screening strategies to selectively target cancer stem cells to generate therapeutics for this subpopulation of cells that could be resistant to classic treatments while possessing potent tumor-forming capacity.

In the cancer stem cell model of tumors, there is a small subset of cancer cells, the cancer stem cells, which constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and maintain the tumor. These cancer stem cells have the capacity to both divide and expand the cancer stem cell pool and to differentiate into the heterogeneous nontumorigenic cancer cell types that in most cases appear to constitute the bulk of the cancer cells within the tumor. If cancer stem cells are relatively refractory to therapies that have been developed to eradicate the rapidly dividing cells within the tumor that constitute the majority of the nonstem cell component of tumors, then they are unlikely to be curative and relapses would be expected. If correct, the cancer stem cell hypothesis would require that we rethink the way we diagnose and treat tumors, as our objective would have to turn from eliminating the bulk of rapidly dividing but terminally differentiated components of the tumor and be refocused on the minority stem cell population that fuels tumor growth. This explains why the cancer stem cell hypothesis is at the center of a rapidly evolving field that may play a pivotal role in changing how basic cancer researchers, clinical investigators, physicians, and cancer patients view cancer.

It has long been known from light microscopic studies that both normal tissues and the tumors that develop within them comprise a heterogeneous collection of cell types, frequently including immune cells, a stroma consisting of various mesenchymal and endothelial cells, and a variety of normal or malignant cells specific to the tissue. Cells within the tumor often seem to correspond to different stages of development. Epithelial cancers, for example, typically contain cells exhibiting divergent nuclear morphologies and differentiation features. Prevailing explanations for the observed tumor cell heterogeneity include influences of the microenvironment and genomic instability that generate the genetic and epigenetic changes, which prevent faithful and accurate replication and transmission of stable genotypes and phenotypes. Such instability could also explain why tumors typically contain a subset of cells that are refractory to most treatments. However, an alternative (or complementary) emerging concept is that malignant cell populations may reflect the continuing operation of perturbed differentiation processes. Inherent to such a model is the formation of malignant populations consisting of a developmentally defined hierarchy of heterogeneous phenotypes derived from a small subset of “cancer stem cells.”

Human cells fulfilling the properties expected of drug-resistant cancer stem cells were initially isolated from blood cancers. Tritium-labeling studies conducted on a variety of blood cancers in the 1960s showed the existence of a subset of primitive-appearing cells with cycling properties different from the majority of tumor cells. These early tritium-labeling studies, coupled with genetic studies suggesting that many leukemias contained an immature cell population capable of generating postmitotic progeny, predicted the existence of a leukemic stem cell.

Studies of acute myelogenous leukemia (AML) in the 1990s provide compelling evidence for the existence of a cancer stem cell subpopulation. Efforts to define the cell of origin of hematopoietic cancers were greatly enhanced by specific and quantitative assays, extensive lineage maps, and the availability of cell surface markers for distinct cell types comprising this system. For human AML, cancer stem cells were defined as those cells capable of regenerating human AML cell populations in irradiated transplanted nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. The AML stem cells possessing this property were found to display a CD34 + CD38 − cell surface phenotype, similar to that typical of normal human primitive hematopoietic progenitors. This suggested that the AML stem cells may have originated from normal stem cells rather than arising from more committed progenitors, although as will be discussed, this may not necessarily be the case for all cancer stem cells.

An accurate definition is critical to enable researchers working in the same or different systems to compare cells exhibiting a common set of properties. The consensus definition of a cancer stem cell that was arrived at in this Workshop is a cell within a tumor that possess the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Cancer stem cells can thus only be defined experimentally by their ability to recapitulate the generation of a continuously growing tumor. The implementation of this approach explains the use of alternative terms in the literature, such as “tumor-initiating cell” and “tumorigenic cell” to describe putative cancer stem cells.

It must be emphasized that proliferation is not synonymous with self-renewal. A self-renewing cell division results in one or both daughter cells that have essentially the same ability to replicate and generate differentiated cell lineages as the parental cell. Stem cells have the ability to undergo a symmetrical self-renewing cell division, causing identical daughter stem cells that retain self-renewal capacity, or an asymmetrical self-renewing cell division, resulting in one stem cell and one more differentiated progenitor cell. In addition, it is thought that stem cells may divide symmetrically to form two progenitor cells, which could lead to stem cell depletion. Promoting this form of division would be a way to deplete the cancer stem cell population and may constitute an alternative strategy to inducing cell death to treat cancer.

Evidence for the existence of cancer stem cells in solid tumors has been more difficult to obtain for several reasons. Cells within solid tumors are less accessible, and functional assays suitable for detecting and quantifying normal stem cells from many organs have not yet been developed. Therefore, the cell surface markers required to isolate such cells have not been identified. There has been some impressive work in this area recently, including the demonstration that single mouse mammary cells can be transplanted and reconstitute a complete mammary gland. Cells have also been isolated from human breast tumors that can cause breast cancer in NOD/SCID mice through serial transplantations, suggesting a capacity for self-renewal. These cells were CD44 + CD24 − /low in eight of nine patients and established tumors in recipient animals when as few as one hundred cells were transplanted, whereas tens of thousands of breast cancer cells with a different marker set failed to induce tumors. Brain tumor stem cells that can produce serially transplantable brain tumors in NOD/SCID mice have also been isolated from human medulloblastomas and glioblastomas. These cells can be enriched by sorting for CD133 + , a marker found on normal neural stem cells, and the transplantation of one hundred CD133 + tumor cells was sufficient to initiate the formation of a tumor in recipient animals. In contrast, no mice injected with the negative population developed brain tumors. More recently, cells have been isolated from human prostate cancer patients that can produce serially transplantable prostate tumors in NOD/SCID mice. Sorting for Hoechst dye–excluding side population (SP) cells and cells expressing CD44 allowed the isolation of a population enriched for cells with this ability. Together, these studies reveal that only a small subset of cells in several different tumor types is capable of tumor formation in such transplant assays. These data are consistent with the cancer stem cell hypothesis. Nonetheless, caution needs to be exerted when interpreting transplantation assays as described in the section on functional and phenotypic assays.

The term cancer stem cell has led to some confusion. Many interpret the term cancer stem cell to mean that such cells derive from the stem cells of the corresponding tissue. Cancer stem cells may indeed arise from normal stem cells by mutation of genes that make the stem cells cancerous, but this may not be the case in all tumors. For example, in blast crisis chronic myelogenous leukemia (CML), a committed granulocyte-macrophage progenitor may acquire self-renewal capacity and thus “reacquire” stem-like properties due to the effects of later mutations. It is conceivable that more differentiated cells can, through multiple mutagenic events, acquire the self-renewal capacity and immortality that typify cancer stem cells. In both of these examples, a differentiated cell, not the tissue stem cell, eventually evolves to become a full-blown cancer stem cell.

The term tumor-initiating cell can also cause confusion. In some of the seminal studies in this field, the term “cancer initiating” has been used to refer to the ability of these cells to initiate tumors when transplanted. The tumor-initiating cell is often used to mean the cell that causes a tumor (or leukemia) in xenograft models of human cancer. Some have extrapolated that the cell that initiates a tumor xenograft is the same as the cell that received the first oncogenic “hits” in the patient. It is clear that the cancer stem cell capable of forming a tumor at one point in time might change during the progression of the disease. Thus, a tumor may be initiated by a set of mutations leading to transformation of one cell type, but progressive mutations occurring during the evolution of the tumor may result in the acquisition of stem cell properties by a second cell type at a later time ( Fig. 1 ). The stem cells within an individual tumor may constitute a moving target, that is, the cells that drive growth at one point in time may not be identical to those doing so at another stage in tumor evolution or during metastasis. Furthermore, the genetic and epigenetic instability that are fundamental properties of tumor biology can induce cellular heterogeneity within the stem and nonstem cell populations of the tumor. Evidence was given that specific oncogenes or mutations could play a significant role in determining the target cell that eventually becomes malignant. Animal models will be useful for understanding the origins of cells with the properties expected of cancer stem cells and when and where they arise during cancer initiation and progression.

Some potential mechanisms by which tissue stem cells generate cancer. Several potential routes by which mutations can accumulate in stem cells to generate cancer, based on data presented at the AACR Cancer Stem Cell Workshop. A, normal cells divide within a niche ( green ) and can accumulate mutations over time. B, stem cells may have acquired mutations that enable them to survive and self-renew within an alternative niche. This may enable a population of mutant stem cells to expand in a new region and/or in the environment of different supporting cells (i.e., an alternate niche). Alternatively, mutant stem cells may have acquired the ability to induce the proliferation of niche cells to allow for expansion of the niche to accommodate the mutant stem cells. C, mutations within the stem cells may enable them to proliferate in the absence of a niche, but they would require additional mutations to undergo self-renewal ( D ). A related possibility is that mutated stem cells undergo a differentiation program but retain proliferative potential. Acquisition of additional mutations in the proliferative progenitors would then be required to enable them to self-renew. E, normal stem cells may be exposed to a niche that has itself undergone modifications. Self-renewing divisions in the aberrant niche may then select for specific types of mutations within the stem cells, which are the precursors of cancer. The cancer shown is composed of a heterogeneous cell population that could be generated by the self-renewing divisions of the mutated cancer stem cell along with its “differentiated” cell types that comprise the tumor. Various mutated proliferative progenitors could also contribute to the tumor along with the self-renewing, differentiation competent progenitor that is the cancer stem cell. Both models are compatible with clonal origin of most tumors as all cells shown derive from a common stem cell ancestor.

Normal tissue stem cells are dependent on interactions with adjacent stromal cells that comprise a specialized microenvironment or niche, which is necessary for the maintenance of stem cell identity and self-renewal capacity ( Fig. 1 ). Similarly, in some malignancies, tumor growth is also thought to depend on a dynamic interaction with adjacent stromal cells that compromise the tumor niche. As tumors grow, the niche may change. It is reasonable to surmise that the stem cells of a tumor may also evolve with changing cues in their microenvironment, including infiltration of immune cells and activation of inflammatory responses. In addition, from studies of the Drosophila germ cell niche shown at the Workshop, it is possible that cancer stem cells will signal to their niche to allow it to expand as the cancer stem cells proliferate.

One of the most frequent errors made in defining stem cells is generalizing results obtained in studies of stem cells in one organ. Although stem cells in different adult tissues share the fundamental properties of self-renewal and the ability to differentiate into a diversity of mature cell types, stem cells in different organs can differ significantly from one another. Thus, properties that are useful for the identification and characterization of stem cells in one tissue are frequently not shared with the stem cells in a different organ. This is likely to also be true for cancer stem cells isolated from different tumor types.

Self-renewal and lineage capacity are the hallmarks of any stem cell. Therefore, as with normal stem cells, assays for cancer stem cell activity need to be evaluated for their potential to show both self-renewal and tumor propagation. The gold standard assay that fulfills these criteria is serial transplantation in animal models, which, although imperfect, is regarded as the best functional assay for these two critical criteria.

In transplantation assays, cells are xenografted into an orthotopic site of immunocompromised (typically NOD/SCID) mice that are assayed at various time points for tumor formation. To show self-renewal, cells then must be isolated from the tumors and grafted into a second recipient animal. Issues complicating transplantation assays include potential effects of the grafting site. It is known that normal stem cells can be highly dependent on signals from the surrounding stroma for function, and it is not clear what the effect may be on separating cancer stem cells from any supporting cells during the course of the assay. Experiments using mixed populations of normal and breast tumor cells in mice have shown that combining tumor cells with normal fibroblasts increases latency and decreases tumor take, whereas combining them with carcinoma-associated fibroblasts has the opposite effect. Conversely, nontumor cells placed next to tumor stroma can become independently tumorigenic, possibly due to stroma-induced genetic or epigenetic instability. The number of cells needed to form a tumor can also be affected by the addition of irradiated feeder cells or the use of Matrigel; for feeder cells, by orders of magnitude.

Interpretation of transplantation assays is also complicated by the possibility that the cells that can recapitulate the tumor might have a greater ability to survive in the host; the extraordinarily high level of genetic and epigenetic changes that take place within most cancer cells, in some cases, as high as 1,000 daily, may allow some cells to generate diverse cell types not because they are stem cells per se but because of their genetic/epigenetic instability. Still, although serial transplantation assays remain the best developed method to date for identifying cells with the properties expected of cancer stem cells, more sophisticated, precise, and simpler assays are likely to emerge as the field develops.

In vivo assays are the gold standard for identifying stem cells; however, as serial transplantation experiments with animal models can take 6 months or more, high-throughput screens for lead compounds will be difficult, if not impossible, using animal models. Therefore, the development of reliable surrogate assays would significantly enhance drug development.

An ideal in vitro assay would be ( a ) quantitative; ( b ) highly specific, measuring only the cells of interest; ( c ) sufficiently sensitive to measure candidate stem cells when present at low frequency; and ( d ) rapid. Several in vitro assays have been used to identify stem cells, including sphere assays, serial colony-forming unit (CFU) assays (replating assays), and label-retention assays. Studies have also been done with the goal of determining genetic signatures that define cancer stem cells. However, each of these methods has potential pitfalls that complicate interpretation of the results. All three groups reporting the isolation of normal breast stem cells indicated that individual breast stem cells do not form spheres by themselves using conditions that are permissive for the formation of neurospheres or mammospheres. Furthermore, the rapidity of sphere development in many systems makes it unlikely that they arose from single cells solely through clonal expansion. The difficulty of distinguishing the relative contributions of aggregation and proliferation in sphere formation poses a major impediment to their use to construct lineage maps. Serial CFU assays have been used to identify cells with increased proliferative potential, but their activity must be confirmed by a clonal in vivo assay. Indeed, in the hematopoietic system, selecting for CFU in semisolid medium usually identifies progenitors and not stem cells. This may be due to the need for the niche to provide the requisite signals for both self-renewal and proliferation.

Additional technical issues make stem cell isolation and functional assays challenging. Although flow cytometry offers a sensitive, specific, and robust method of cell isolation and purification, some of its technical limitations make its application to stem cell purification challenging. For example, even when using advanced sorting techniques to distinguish single cells from aggregates, doublets (cells sticking to one another) can still occasionally sort together and need to be eliminated. Thus, microscopy is needed to show that single cells were indeed isolated. It is very difficult to make viable single-cell suspensions of solid tissues, such as brain and epithelial tissues cells. Thus, techniques for dissociating cells must be carefully developed. Most flow cytometers are typically set up to sort blood components using small diameter streams at high pressures. These conditions are often not tolerated by larger, more fragile cells found in many organs. Therefore, diameters of the liquid stream and sorting pressures frequently must be optimized for cells isolated from solid tissues. In addition, although phenotypes based on markers often use terms, such as high, middle, low, and nonexpression, to describe the properties of the sorted cells for each marker, these terms are subjective and can vary depending on the method used for cell preparation, how the gates are set, and the antibody preparation used. Thus, cells marked as one phenotype by one group may exhibit another phenotype in other hands. This could be remedied by availability of standardized antibodies and by consistent calibration of each batch of fluorescently labeled antibodies. At this point in time, such quality control needs to be done by the individual investigator. Because of these technical challenges, it takes the typical neophyte several months of training in an experienced stem cell laboratory to even begin to master flow cytometry isolation of cancer stem cells.

It is clearly not sufficient to define a stem cell based solely on surface markers in the absence of linking marker expression to a self-renewal assay. None of the markers used to isolate stem cells in various normal and cancerous tissues is expressed exclusively by stem cells. For example, CD133 was used to successfully enrich for brain tumor stem cells, but it is also present on normal brain stem cells and on many nonstem cells in various tumors and tissues. The same is true for other commonly used markers, such as CD44, Sca1, and Thy1. In fact, the vast majority of cells that express these markers are not stem cells. In addition, markers used to identify stem cells from one organ are frequently not useful for identifying stem cells in other tissues: Sca-1 is useful for the identification of murine blood stem cells, but it is not consistently expressed by murine mammary duct stem cells. Furthermore, just because a marker can be used to identify stem cells from a particular organ does not mean it will work in all other contexts. For example, placing stem cells in culture can drastically alter their marker expression. Thus, describing the markers presently used to identify stem cells from one tissue as a “stemness” marker when investigating a potential stem cell population in a different tissue is misleading. The marker may or may not be useful for identifying stem cells from the other organ or tumor type.

Another phenotype used to distinguish cells is their presence within the SP fraction defined by Hoechst dye efflux properties. However, as with cell surface markers, possession of a SP phenotype is not a universal property of stem cells, and in some tissues, the SP fraction may not contain the stem cells. Experiments that identified normal breast stem cells by their ability to generate mammary glands in cleared fat pads showed that the majority of these cells are not included within the SP fraction. Possible toxicity of the dye to cells that do not exclude it should also be considered as a caveat to interpreting functional assays of SP cells. As with other markers used to identify certain types of stem cells, marker expression must be linked with a functional assay. Because of this complicating factor, it is safer to first isolate stem cells using other methods and then ask whether that particular stem cell population is indeed included within the SP.

Label retention (bromodeoxyuridine incorporation) studies have also been proposed as way of identifying stem cells. This method is based on the assumption that normal or cancer stem cells either spend long periods not cycling or undergo an “immortal strand” DNA replication and therefore preserve the labeled state for an extended period. However, neither event is the case for stem cells in every organ or tissue. In fact, both normal and malignant breast stem cells appear to be cycling and so much more needs to be known about the regulation of cancer stem cell cycling behavior before assays based on this property can be relied on for purposes of identifying stem cells. Furthermore, it is known that not all stem cells are label retaining and not all label-retaining cells are stem cells. Thus, as with any other potential stem cell marker, one must link the property with a stem cell functional assay. In other words, label-retaining cells must be shown to regenerate the tissue in vivo .

Work has begun in several systems to develop genetic signatures that typify stem cells. Several genes and signaling pathways, including Bmi-1, Tie-2, Shh, Notch , and Wnt/β-catenin , have been shown to have important regulatory functions for some stem cells. However, as these genes frequently operate in other cell types, they cannot be called “stemness” genes. Microarray and genome-wide techniques can be applied to detect trends in genetic and epigenetic “blueprints” for cancer stem cells, but to identify true signatures, pure populations are necessary. This is especially true for cells expected to be rare, such as cancer stem cells, whose expression signature would be swamped by the majority of nonstem cells in a whole tumor sample. Even after a cancer stem cell signature from a particular type of tumor is identified, one cannot assume that a given signature is useful for identifying cancer stem cells in a different tumor type unless validated by a functional assay (such as an in vivo self-renewal assay as it is the most definitive at this point in time).

The development of in vitro assays, although critical, is at an early stage, and the results of all in vitro work must be examined rigorously and ultimately validated in vivo . Therefore, any method used to identify putative stem cells must be verified and followed by functional assays, preferably a gold standard in vivo assay, before claims about “stemness” can be made. It should be a goal of the field to develop cell surface marker and gene activity profiles that can be used reliably to identify cancer stem cells. At this time, the presence of particular markers or gene expression signatures associated with other stem cell populations, normal or cancer derived, is not sufficient to label a given population of cells as cancer stem cells without confirmation by functional assays. In addition, analysis and interpretation of these data are limited by the purity of the cell population in a given system; results from a mixed population constitute an average and not a specific signature. Furthermore, use of gene inactivation to eliminate “stemness” or gene activation to engender “stemness” would be required to functionally link any marker to stem cell identity. Although the isolation of markers correlated with stem cells can aide in stem cell isolation, the identification and isolation of genes that are functionally significant for “stemness” would constitute an important step forward and could provide valuable targets for drug development.

The cancer stem cell hypothesis posits that cancer stem cells are a minority population of self-renewing cancer cells that fuel tumor growth and remain in patients after conventional therapy has been completed. The hypothesis predicts that effective tumor eradication will require obtaining agents that can target cancer stem cells while sparing normal stem cells. Experimental evidence in human AML suggests that, compared with the bulk population of leukemic blasts, the leukemia stem cells are relatively resistant to conventional chemotherapeutic agents. Although it has been speculated in solid tumors that conventional agents kill the nontumorigenic cancer cells while sparing the cancer stem cells, this has not been proven. There are other models of drug resistance consistent with the existence of cancer stem cells that could explain relapse, including the classic view of mutation and selection.

The moving target nature of cancer stem cells may present a challenge in the clinic. To achieve effective implementation of new therapies, physicians will require methods of determining the type (or types) of cancer stem cells present in a given patient's tumor. Work involving 150 CML patient peripheral blood and bone marrow samples is encouraging in that patients in blast crisis all exhibited an expansion of the granulocyte-macrophage progenitor population, which included the fraction displaying stem cell properties. Therefore, it seems reasonable to expect that tumors sharing a similar pathology may also share common features in their cancer stem cell populations, which would facilitate diagnosis and the application of appropriate treatments. This point, however, needs to be borne out by further study.

It is important that agents directed against cancer stem cells discriminate between cancer stem cells and normal stem cells. This will require identification of realistic drug targets unique to cancer stem cells. The identification of such targets and the development of anticancer agents will require a fuller understanding of normal stem cell biology as well as the genetics and epigenetics of tumor progression. There is some indication that such an approach can be successful. For example, stem cells isolated from AML patients display differences from normal hematopoietic stem cells.

There has also been some success identifying agents effective against leukemia stem cells. Conventional anthracycline agents show synergy with proteasome inhibitors against AML stem cells, reducing viability in vitro dramatically. The novel agent parthenolide, isolated from Mexican medicinal plants and shown to be a potent nuclear factor-κB inhibitor, promotes apoptosis of AML stem cells and inhibits tumor development in NOD/SCID mice. Mutation of the Janus-activated kinase 2 (JAK2) kinase is found in many patients with the blood disorder Polycythemia Vera, and JAK2 inhibitors display efficacy against the cancer stem cells from these patients, although individual responses vary significantly.

Participants in the AACR Workshop agreed that, to move the cancer stem cell field forward, multiple assays need to be validated for as many putative stem cell populations as possible. Cells should be interrogated by multiple methods, including functional assays, marker analysis, and analysis of genetic and epigenetic signatures. More accurate and standardized reagents are needed, particularly for the cell surface markers used for sorting.

Participants also expressed the frequent difficulties in obtaining sufficient quantities of patient tissues. It was noted that patients are often willing to supply tissue, but the current regulatory climate presents an obstacle for many. Joint efforts by the research and patient advocacy communities are needed to overcome these regulatory barriers.

Clearly, there is much excitement and momentum in this important field. Investigation of cancer stem cells offers the possibility of generating novel targets that could overcome issues of drug resistance, improve therapeutic efficacy, and make cancer treatment more successful and perhaps even curative while obviating systemic toxicity. The AACR will form a task force to discuss developments in this field to help identify and eliminate bottlenecks and to expedite progress in this promising area through focused scientific meetings and other mechanisms.

Note: The AACR Cancer Stem Cells Workshop was held on February 2-4, 2006 in Lansdowne, Virginia.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Cancer Stem Cell (CSC) Hypothesis

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Cancer cells are considered to be aberrant versions of their tissues of origin. However, their ability to proliferate and resistance to therapy led to the theory that cancers contain a heterogeneous population of cells. One theory that has gained traction since a hallmark paper was published in 2001 is the cancer stem cell (CSC) hypothesis.

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The first assumption of the CSC hypothesis is that the cells in any given cancer mass are unequal, or heterogeneous. This describes a tumor in which cancer cells are hierarchically organized, and contain a small subpopulation of cancer stem cells (CSCs) that can proliferate extensively to sustain the progression and growth of the tumor.

The rationale behind this theory is that stem cells possess two properties, or hallmarks; (1) indefinite self-renewal and (2) pluripotency. The latter refers to the differentiation of the stem cell to become a specific cell type which is unipotent (can only produce daughter cells of the same type).

The second assumption of the CSC hypothesis is that CSCs are physically identifiable, possessing distinctive surface markers which differentiate them from other cancer cells.

Thirdly, the CSCs in a tumor comprises a mixture of cells which are either tumor-promoting (tumorigenic) or benign (non-tumorigenic). The tumorigenic population of CSCs is often considered to be tumor-initiating cells (TICs), aiding tumor relapse and resistance to therapy. For this reason, TICs are important therapeutic targets in drug development.

Evidence for CSCs

The most persuasive evidence for the existence of stem cells within a tumor comes from the analysis of cell surface markers. In 2003, Al-Hajj and colleagues identified that, in 8/9 primary breast tumor samples, the tumorigenic cells were CD44 + /CD24 -/low /ESA + /lin - i.e. positive for marker CD44, negative or low for CD24, positive for the marker Epithelial Specific Antigen (ESA), and negative for the lineage (lin) marker. This identified the breast cancer stem cells.

Singh et al . revealed that the tumorigenic brain cancer stem cell population was characterized by CD133 + . These cells could produce new tumor colonies termed neurospheres and could be reproduced in human brain tumors when injected into mice. Similarly, human colorectal cancer cells possessed the same qualities of tumor colony initiation and tumor reproduction. This evidence, whilst robust, has been contested.

Evidence Against CSCs?

The CSC hypothesis has been directly refuted by work that has demonstrated that tumors can grow rapidly; if CSCs existed, and were drivers of formation, they should be infrequent and identifiable by surface markers.

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• Researchers identify possible candidates for multipotent stem cells in the sea anemone

In 2007, Andreas Strasser found that a high proportion (1 in 10) of leukemia cells transplanted into mice could initiate lymphomas. Similarly, Sean Morrison found 25% of unselected melanoma cells (that is, without any surface marker) could form tumors in mice. These findings have been refuted since, with an increasing body of evidence pointing to the existence of CSCs possessing the qualities of infrequency, isolation by surface markers, the ability to form tumors following xenotransplantation.

Origins of CSCS: the connection between Epithelial-Mesenchymal Transition (EMT) and CSCs

The Epithelial-Mesenchymal Transition (EMT) is the process in which differentiated cells de-differentiate to acquire stem cell properties. Oncogenic mutations are posited to drive this process and promote self-renewal.

This mechanistic link is palpable, as it suggests that a large cell population could possess tumorigenic potential (as found by Strasser and Morrison), but only a small subset would initiate the tumor. The evidence for this comes from the work of Thomas Brabletz (2005) and Mani, which was published in Cell in 2008.

More specifically, factors that control gene expression called transcription factors, were found to be upregulated in CSCs. These transcription factors activated transcriptional programs that are seen in stem cells; the factors that are known to cause EMT, SNAIL and TWIST were identified.

Furthermore, extrinsic drivers of CSC production were found to promote the transcription of factors needed for the EMT. This was demonstrated by Scheel et al., who found that mammary cells underwent EMT by extrinsic signaling molecules TGF-β and Wnt.

The EMT program in mammary cells subsequently induced migration and sphere formation. In agreement, inhibition of extrinsic factors in transformed cells prevented tumorigenesis and metastatic spread. Thus, circulating tumor cells have features of both EMT and cancer stem cells and play a substantial role in resistance and metastasis .

Implications for current and emerging therapies

The notion that EMT induction leads to the formation of CSCs has been supported by research in more recent years and suggested that targeting the EMT process can represent a potential therapeutic target to treat cancer. Conventional chemotherapy primarily targets the numerous highly proliferative cells to reduce the tumor size, promoting the increased frequency of CSCs that arise through EMT.

This subsequently replenishes these lost cells, and tumor recurrence results. Direct target of CSCs, through interference with signaling pathways and transcription factors implicated in EMT, and thus CSC production - termed ablation - would prevent cancer stem cells from providing differentiated cells to populate the tumor mass.

Such inhibition of tumor regeneration would ultimately lead to the regression of the tumor. Efforts are currently underway and include blocking stem cell self-renewal pathways, targeting the microenvironment that sustains CSCs, inducing CSC programmed cell death, as well as forcing CSCs to differentiate into non-proliferative cell types amongst targeting the EMT pathway.

• https://www.nature.com/articles/35102167
• https://www.ncbi.nlm.nih.gov/pubmed/9212098
• http://www.pnas.org/content/100/7/3983.long
• https://www.ncbi.nlm.nih.gov/pubmed/18485877
• https://www.nature.com/articles/nrc1694
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• http://science.sciencemag.org/content/317/5836/337.long
• https://www.nature.com/articles/nature07567
• https://www.nature.com/articles/nature03128

Last Updated: Dec 6, 2018

Hidaya Aliouche

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The Cancer Stem Cell Hypothesis: Failures and Pitfalls

Rahman, Maryam MD * ; Deleyrolle, Loic PhD * ; Vedam-Mai, Vinata PhD * ; Azari, Hassan PhD * ; Abd-El-Barr, Muhammad MD, PhD *† ; Reynolds, Brent A PhD *

*Department of Neurosurgery, University of Florida, Gainesville, Florida; †Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Received, January 15, 2010.

Accepted, June 5, 2010.

Correspondence: Brent Reynolds, PhD, or Maryam Rahman, MD, Box 100265, Department of Neurosurgery, University of Florida, Gainesville, FL 32610. E-mail: [email protected] or [email protected]

Based on the clonal evolution model and the assumption that the vast majority of tumor cells are able to propagate and drive tumor growth, the goal of cancer treatment has traditionally been to kill all cancerous cells. This theory has been challenged recently by the cancer stem cell (CSC) hypothesis, that a rare population of tumor cells, with stem cell characteristics, is responsible for tumor growth, resistance, and recurrence. Evidence for putative CSCs has been described in blood, breast, lung, prostate, colon, liver, pancreas, and brain. This new hypothesis would propose that indiscriminate killing of cancer cells would not be as effective as selective targeting of the cells that are driving long-term growth (ie, the CSCs) and that treatment failure is often the result of CSCs escaping traditional therapies.

The CSC hypothesis has gained a great deal of attention because of the identification of a new target that may be responsible for poor outcomes of many aggressive cancers, including malignant glioma. As attractive as this hypothesis sounds, especially when applied to tumors that respond poorly to current treatments, we will argue in this article that the proposal of a stemlike cell that initiates and drives solid tissue cancer growth and is responsible for therapeutic failure is far from proven. We will present the point of view that for most advanced solid tissue cancers such as glioblastoma multiforme, targeting a putative rare CSC population will have little effect on patient outcomes. This review will cover problems with the CSC hypothesis, including applicability of the hierarchical model, inconsistencies with xenotransplantation data, and nonspecificity of CSC markers.

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Cancer stem cell hypothesis: a brief summary and two proposals

• Review Paper
• Published: 19 December 2012
• Volume 65 , pages 505–512, ( 2013 )

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• Shuhua Zheng 1 , 2 ,
• Longzuo Xin 3 ,
• Aihua Liang 1 &
• Yuejun Fu 1  

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The investigation and development of the cancer stem cell (CSC) model has received much focus during these years. CSC is characterized as a small fraction of cancer cells that have an indefinite ability for self-renewal and pluripotency and are responsible for initiating and sustaining of the bulk of cancer. So, whether current treatment strategies, most of which target the rapid division of cancer cells, could interfere with the slow-cycling CSCs is broadly questioned. Meanwhile, however, the new understanding of tumorigenesis has led to the development of new drug screening strategies. Both stem cells and mesenchymal stem cells have been vigorously used in pre-clinical studies of their anti-tumor potential, mainly due to their inherent tumoritropic migratory properties and their ability to carry anti-tumor transgenes. Here, based on the tumorigenic and tumoritropic characteristics of CSCs, we proposed two hypotheses exploring possible usage of CSCs as novel anti-tumor agents and potential sources for tissue regeneration. Further experimental validation of these hypotheses may unravel some new research topics.

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Acknowledgments

This work was supported partially by “National Natural Science Foundation of China” (No.31272100 and No.30901774), the “National High Technology Research and Development Program of China (863 Program)” (No.2012AA020809), the Innovative Research Program for Graduates of Shanxi Province (20113018) and the Program for the Top Young Academic Leaders of Higher Learning Institutions of Shanxi.

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Shuhua Zheng, Aihua Liang & Yuejun Fu

University of Miami Miller School of Medicine, Miami, FL, 33136, USA

Shuhua Zheng

School of Agriculture and Forest Science, Hebei North University, Zhangjiakou, 075000, People’s Republic of China

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Zheng, S., Xin, L., Liang, A. et al. Cancer stem cell hypothesis: a brief summary and two proposals. Cytotechnology 65 , 505–512 (2013). https://doi.org/10.1007/s10616-012-9517-3

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Issue Date : August 2013

DOI : https://doi.org/10.1007/s10616-012-9517-3

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What Are Cancer Stem Cells?

They influence how a tumor grows

Role in Cancer Growth

Resistance to therapy, importance of cancer stem cell research.

Cancer stem cells are a small subpopulation of cells found within tumors that are tumorigenic, meaning they can create a cancerous tumor. Self-renewal and the ability to differentiate into diverse cell types are hallmark features of cancer stem cells. They can reproduce themselves and sustain cancer in the body. They are therefore hypothesized to be the primary driver of cancer growth and metastasis . This is called the stem cell theory of cancer. Effective cancer treatment then must target and attack these cells. Doing so can improve the chances of cancer remission.

Cancer stem cells have been identified in brain , breast , colon , ovarian , pancreatic , and prostate tumors, as well as in melanoma , multiple myeloma , nonmelanoma skin cancer , and leukemia .

Cancer stem cell research is ongoing, and new studies are emerging frequently.

What Are Stem Cells?

Stem cells are undifferentiated (or only partly differentiated) human cells that can turn into different types of cells in the body, from nerve cells (neurons) to brain cells. They can also fix damaged tissues. They must possess two major qualities: self-renewal and the capacity to differentiate. Stem cell-based therapies are also being studied to treat serious illnesses such as paralysis and Alzheimer's disease .

There are two types of stem cells: embryonic and adult stem cells. Embryonic stem cells come from unused embryos and are created from an in vitro fertilization process. They are pluripotent, meaning they can turn into more than one cell type. Within adult stem cells, there are two different types: one type comes from fully developed tissues such as the brain, skin, and bone marrow, and the other is induced pluripotent stem cells, which have been changed in the lab to be more like embryonic stem cells.

luismmolina / Getty Images

The stem cell theory of cancer hypothesizes that cancer stem cells are thought to drive tumor initiation and may be responsible for therapeutic resistance and cancer recurrence.

Like many areas of biomedical research, cancer stem cells are an evolving field of study. Multiple studies have indicated that insufficient evidence exists to confirm the existence of cancer stem cells. A review of 1,000 Web of Science publications revealed that only 49% supported the cancer stem cell hypothesis.

Cell surface markers can be used to identify cancer stem cells, as has been done in research that supports the hypothesis that these stem cells do not respond to traditional therapies such as chemotherapy . This research also supports the idea that cancer stem cells are the source of cancer metastasis.

Like all stem cells, cancer cells must have the following characteristics:

• Self-renewal: When stem cells divide into more stem cells, this process is referred to as cell renewal.
• Cell differentiation: Cell differentiation is when a cell changes from a less differentiated to a more differentiated cell type.

Cancer stem cells use specific signaling pathways. It is hypothesized that cancer stem cells can also act as a reservoir of cancer cells, which may cause a relapse after surgery, radiation, or chemotherapy has eliminated all observable signs of cancer. Targeting these cells would thus highly improve the chances of a patient's remission if cancer stem cells are the origin of the tumor.

Cancer stem cells have the capacity to change into more specialized cell types, so they can potentially lead to tumor cell heterogeneity. Due to this quality, they are cited as a major factor of chemoresistance. Their highly resistant nature can lead to tumors metastasizing and tumor regrowth. As such, the developing research on cancer stem cells could dramatically change the prognosis of multiple cancer types.

Also, many new anticancer therapies are evaluated based on their ability to shrink tumors, but if the therapies are not killing the cancer stem cells, the tumor will soon grow back, often with resistance to the previously used therapy.

Cancer stem cell research is critical because it addresses the potential root cause of cancer proliferation and can lead to the development of more effective and safer treatments. Treatments targeting cancer stem cells will likely have fewer side effects compared with existing options because they will leave other kinds of cells untouched.  

Understanding these cells can also help modify current treatments for maximum effect. Research has shown that cancer stem cells are resistant to the ionizing radiation used to treat cancer. Understanding this resistance may in the future help researchers find compounds that undermine this process and make cancer stem cells vulnerable to radiation damage.  

A Word From Verywell

Cancer stem cell research offers promising hope for the continually evolving field of cancer therapeutics, but more research needs to be done to confirm the stem cell theory of cancer. Cancer stem cell research has the potential to generate better treatments for cancer with fewer side effects, as well as to improve the efficacy of current treatment options. If the theory is proven, therapies targeting cancer stem cells may even be able to lower the rate of cancer recurrence. While its existence is still up for debate, it represents an exciting opportunity to advance cancer care and improve cancer survival.

Yu Z, Pestell TG, Lisanti MP, Pestell RG. Cancer stem cells . Int J Biochem Cell Biol . 2012 Dec;44(12):2144-51. doi: 10.1016/j.biocel.2012.08.022

Ayob AZ, Ramasamy TS. Cancer stem cells as key drivers of tumour progression . J Biomed Sci . 2018 Mar 6;25(1):20. doi: 10.1186/s12929-018-0426-4

Stanford Children's Health. What are stem cells?

Walcher L, Kistenmacher AK, Suo H, Kitte R, Dluczek S, Strauß A, Blaudszun AR, Yevsa T, Fricke S, Kossatz-Boehlert U. Cancer stem cells-origins and biomarkers: perspectives for targeted personalized therapies . Front Immunol . 2020 Aug 7;11:1280. doi: 10.3389/fimmu.2020.01280

Bartram I, Jeschke JM. Do cancer stem cells exist? A pilot study combining a systematic review with the hierarchy-of-hypotheses approach . PLoS One . 2019 Dec 13;14(12):e0225898. doi: 10.1371/journal.pone.0225898

Dawood S, Austin L, Cristofanilli M. Cancer stem cells: implications for cancer therapy .  Oncology (Williston Park) . 2014;28(12):1101-1110.

Stanford Medicine. The stem cell theory of cancer .

Nassar D, Blanpain C. Cancer stem cells: basic concepts and therapeutic implications.   Annu Rev Pathol . 2016;11:47-76. doi:10.1146/annurev-pathol-012615-044438

Barbato L, Bocchetti M, Di Biase A, Regad T. Cancer stem cells and targeting strategies.   Cells . 2019;8(8):926. doi:10.3390/cells8080926

Ayob AZ, Ramasamy TS. Cancer stem cells as key drivers of tumour progression. J Biomed Sci. 2018;25(1):20. doi.org/10.1186/s12929-018-0426-4

Stanford Medicine. What CSCs mean for cancer treatment .

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The Concept of “Cancer Stem Cells” in the Context of Classic Carcinogenesis Hypotheses and Experimental Findings

In this Commentary , the operational definition of cancer stem cells or cancer initiating cells includes the ability of certain cells, found in a heterogeneous mixture of cells within a tumor, which are able to sustain growth of that tumor. However, that concept of cancer stem cells does not resolve the age-old controversy of two opposing hypotheses of the origin of the cancer, namely the stem cell hypothesis versus the de-differentiation or re-programming hypothesis. Moreover, this cancer stem concept has to take into account classic experimental observations, techniques, and concepts, such as the multi-stage, multi-mechanism process of carcinogenesis; roles of mutagenic, cytotoxic and epigenetic mechanisms; the important differences between errors of DNA repair and errors of DNA replication in forming mutations; biomarkers of known characteristics of normal adult organ-specific stem cells and of cancer stem cells; and the characteristics of epigenetic mechanisms involved in the carcinogenic process. In addition, vague and misleading terms, such as carcinogens, immortal and normal cells have to be clarified in the context of current scientific facts. The ultimate integration of all of these historic factors to provide a current understanding of the origin and characteristics of a cancer stem cell , which is required for a rational strategy for prevention and therapy for cancer, does not follow a linear path. Lastly, it will be speculated that there exists evidence of two distinct types of cancer stem cells , one that has its origin in an organ-specific adult stem cell that is ‘initiated’ in the stem cell stage, expressing the Oct4A gene and not expressing any connexin gene or having functional gap junctional intercellular communication (GJIC). The other cancer stem cell is derived from a stem cell that is initiated early after the Oct4A gene is suppressed and the connexin gene is expressed, which starts early differentiation, but it is blocked from terminal differentiation.

“ The biochemistry of cancer is a problem that obligates the investigator to combine the reductionalistic approaches of the molecular biologists with the holistic requirements of hierarchies within the organism. The cancer problem is not merely a cell problem, it is a problem of cell interactions, not only within tissues but also with distal cells in other tissues ” [ 1 ] . “Some would argue that the search for the origin and treatment of this disease will continue over the next quarter century in much the same manner as it already has in the recent past, by adding further layers of complexity to a scientific literature that is already complex beyond measure. But we anticipate otherwise: those researching the cancer [or any other human disease] problem will be practicing a dramatically different type of science than we have experience over the last 25 years. Surely much of this change will be apparent on the technical level. But ultimately the more fundamental change will be conceptual” [ 2 ]. “Personalized medicine is the latest promise of a gene-centered biomedicine to provide custom-tailored to the specific needs of patients. Although surrounded by much hype, personalized medicine lacks the empirical and theoretical foundations necessary to render it a long-term perspective . In particular, the role of genetic data and the relationship between causal understanding, prediction, prevention and treatment of a disease need clarifying” [ 3 ].

1. Introduction: How Some Historic Experimental Findings and Hypotheses of Cancer Shaped Today’s Concept of Cancer Stem Cells

These three introductory quotes embody much of my concern in this Commentary as it concerns the concept of cancer stem cells . They span decades of cancer research by different disciplinarians, involving years of experimental research and philosophical reflection of their experiences of their field.

In this Commentary , I rely on those giants of cancer research and my own research experience of 50 years, to try to make sense of both great discoveries and ideas, as well as confusing and often contradictory uses of terms and concepts. With that as a framework of what is to follow in a non-linear historical fashion, I will try to use both experimental findings and concepts, as well as my own historic and philosophical musings, to generate a view of cancer stem cells that is testable.

Because it has long been known that cells derived from a patient’s cancer could outlast the patient, from whom they were derived, and could be perpetuated either in vitro or in experimental animals, it was assumed to have developed the property of immortality during the initiation of the carcinogenic process. Take for example, the HeLa cell line, derived from Henrietta Lang, which has been studied in laboratories all over the world and has been subject of thousands of research papers [ 4 ]. Consequently, one objective of many studies was to induce immortality in normal cells, in vitro, in order to determine the mechanism by which this happens and to use this protocol to determine if any physical, chemical or biological agent might be a carcinogen . Although there was some initial success using rodent cells for this purpose [ 5 ], trying to immortalize normal human cells met with failure [ 6 , 7 , 8 ]. When the new concept of oncogenes was introduced, genetically-engineering normal rodent cells with specific DNA sequences led the way to get insights on the immortalizing normal human cells [ 9 , 10 , 11 ].

However, before the next breakthrough that seemed to provide another view of the immortalizing process, the concept of cancer stem cells has to be viewed from the perspective of many classical experimental animal cancer studies and those from epidemiology. In many of today’s studies of cancer stem cells , these classic studies have been largely ignored. When Percivall Potts correlated the unusually high frequency of scrotal cancers in chimney sweepers and the soot from the combustion of fuel [ 12 ], a link was formed to chemicals in the soot that seemed to be carcinogen. One school of thought was that these chemicals must have an irreversible effect on the genome (DNA) and later studies did show some of these chemicals could attack the DNA [ 13 ]. However, at that time, animal experiments [ 14 , 15 ], in general, seemed to indicate that the carcinogenic process was not a one-hit process, by which a single normal cell could be irreversibly altered to become an invasive, metastatic cancer cell [ 16 , 17 , 18 ]. The new concept emerged suggesting that carcinogenesis was a multi-step, multi-mechanism process, consisting of an initiation of a single “normal” cell to become “immortal”, followed by a promotion event over a long period of regular exposures at a threshold level to clonally amplify this single “initiated” cell into a benign tumor, which then transitioned to become an invasive and metastatic cell by the progression process.

Another important concept that added to this classic understanding of this initiation/promotion/progression process was that, even though all the cells in the tumor appeared to be heterogeneous in terms of their genotypes and phenotypes, they were derived from a single common “normal” ancestor [ 19 , 20 ].

Added to this new concept were two opposing hypotheses as to which single cell gave rise to these cancer cells. The stem cell hypothesis [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ], and the de-differentiation or re-programming hypothesis [ 29 ] emerged. The late Dr. Van R. Potter conceptualized the stem cell hypothesis as: “Oncogeny as partially-blocked ontogeny” [ 1 ].

2. The Stem Cell versus the De-Differentiation Hypotheses: The Origin of the “Cancer Stem Cell”

Now back to how the concept of cancer stem cells emerged and how the current controversy of the origin of the cancer stem cells seems to be unresolved. In the history of science, the intellectual journey from the starting idea of any explanation for a disease causation to the current hypothesis is never a linear journey. Ideas that ultimately get to the current state come by jumps, starts, rejection or refinement of disproven paradigms, parallel experimental, methodological or conceptual advances in different disciplines, and of course, serendipity. To finally merge all these disconnected ideas, it takes what was once said: “ Research is to see what everyone has seen and think what nobody has thought” [ 30 ]. To make clear, several words have been used in the history of trying to understand the mechanisms of carcinogenesis, namely, normal cells, carcinogens and immortal , are now seen as very misleading. As will be shown later, what is the normal cell that is being converted to ultimately become a cancer stem cell ? What is a carcinogen, a concept that implies an agent that can induce all three phases of the multi-step, multi-mechanism process of carcinogenesis, even after a single exposure? Last, is a cell being classified as immortal due to an induced change in the phenotype caused by some irreversible process, such as a mutation, or by this mutation causing the cell to stay in its natural immortal state. These are important issues that need to be resolved.

While this brief review of the history of carcinogenesis is not linear in time, in retrospect, the early events now make sense in view of recent characterizations of stem cells. Recall the early concept that stem cells might be the target cells for the start of the carcinogenic process; when this concept was developed, no one had even isolated or characterized a real stem cell. The idea of stem cells in developmental biology and embryology was a reasonable logical concept. After it was shown that in vitro experimental approaches, using soft agar growth, might help us to understand the mechanisms of carcinogenesis, the induction of apparent tumors that appeared when normal cells were exposed to some agent (physical, chemical, biological). To test if these abnormal looking clones of cells are really tumorigenic, they were placed in soft agar or injected back into an immune-deficient rodent. If tumors appeared after this procedure, the agent that brought about the in vitro and in vivo tumors was assumed to be a carcinogen and probably a mutagen. However, if only a few cells of the soft agar clones were used to be injected in the immune-deficient rodent, no tumors were seen in vivo, unless large numbers were injected. At the time of these early experiments, no one had any idea why this was the case. In addition, several in vitro assays to determine if an agent that contributed to this conversion of a “normal” cell to one that gave rise to a tumor in an immune-deficient rodent, it was assumed to be a mutagen or genotoxicant. The Ames assay and many other so-called mutation assays were shown to be non-consistent with one another for a number of reasons. Even later, when real mutations at the molecular level were found, both in vitro and in vivo, in the cells shown to be tumorigenic, problems of interpretation again arose, since the DNA studies were not determined to be either the genomic DNA or mitochondrial DNA or that the methods of isolating the DNA might have contributed to the measurements. In brief, major challenges to the interpretation of these mutation assays have been discussed [ 31 , 32 ].

One early observation was that of discrepancies in these in vitro assays to find transformed cells, few of which actually cite this paper; a study was designed to determine why, from lab to lab, or day to day in the same lab using identical chemicals and cells, as well as protocols, dramatically different results were found [ 33 ]. To make this long story short, it was shown, using a pool of Syrian embryo cells to test the presumptive chemical carcinogen, that if the population of normal cells had a few cells that seemed to have no contact-inhibition, one could obtain transformed cells after the application of the presumptive carcinogen . If the population from this pool of Syrian hamster embryo cells did not have in its population the type of no-contact inhibited cells, no amount of the presumptive carcinogen would induce transformed cells. The clue was that only the few cells that did not have contact inhibition gave rise to transformed cells. The clue was contact inhibition [ 34 ].

In another disciplinary field, the work of Werner Loewenstein and Kanno [ 35 ], as well as the freeze fracture pioneer Dr. J.P. Revel [ 36 ], fused the fields of electrophysiology and electron microscopy to identify a structure of cell membranes (gap junctions) to a physiological function of this structure to synchronizing both metabolic and electrotonic functions of cells in tissues. Later, Borek and Sachs [ 37 ] and Borek et al. [ 38 ] noted that normal cells, which had gap junctions, could contact inhibit or have growth control and differentiate, as well as have the potential to become senescent. On the other hand, cancer cells that do not contact-inhibit or have growth control, cannot terminally differentiate, but were immortal , and also had no functional gap junctional intercellular communication [ 39 ].

The terms, senescent , immortal and normal cells appear, again and again, in the cancer literature. However, it has now been shown that normal primary human fibroblasts cells would, through replicative replication, senesce after about 50 cell passages in vitro [ 40 ]. However, later it was shown that, given the manner by which human primary biopsies that gave rise to these fibroblasts were grown at ambient oxygen levels, they followed Hayflick’s observation. Yet, if these primary fibroblasts were grown at very low oxygen levels, they could be passaged much longer [ 41 , 42 , 43 , 44 , 45 ]. It was as though oxygen was a toxic agent to some cells in the population that were needed for their sustained growth. Even later, it was shown in our laboratory that early passages of skin fibroblasts contained adult stem cells [ 46 , 47 ]. Our results, which are currently unpublished, have shown that very early primary human fibroblasts express a key stem cell marker, Oct4A. Could oxygen levels affect the stem cell state of stemness?

3. Clues to Prove the Stem Cell versus De-Differentiation Hypotheses of the Origin of Cancer Stem Cells

Having these published observations in several disciplines in mind, in the context of my laboratory wanting to resolve the issue as to whether the Stem Cell hypothesis or the De-differentiation hypothesis was correct as being the origin of the “cancer stem cell” hypothesis, the question was: “ How could a normal stem cell in any tissue/organ survive without being forced to differentiate by close differentiated offspring that had functional gap junctions?” Only two possible explanations seemed reasonable. First, the stem cells were sequestered by some physical barrier that prevents contact with these gap junction-expressing cells, or second, these stem cells did not express their connexin, or gap junction, genes. We then designed what we called the kiss of death assay [ 48 ].

This assay was based on disassociating all the cells of a normal human organ biopsy, which contained three kinds of cells, namely, a few rare organ-specific adult stem cells, the large numbers of progenitor cells and the terminally differentiated cells. We assumed that the stem cells did not have either expressed connexin genes or have any functional gap junctions. The progenitor cells would have functional gap junctions, while the terminally differentiated cells might or might not have gap junctions, but they could not divide. Next, with approximately a million of these disassociated cells, they were placed on a confluent mat of normal human fibroblast, which were lethally irradiated and were unable to proliferate. Once the progenitor cells attached to the confluent mat of fibroblasts, they formed gap junctions with the proliferative- disabled fibroblasts and eventually died. The terminally differentiated cells never formed any clones on the mat. Since they either died by apoptosis or because they could not proliferate. On the other hand, after a week, a few small clones of cells appeared to be proliferating. After these clones were removed, they were tested for expressed connexins and functional gap junctions. None were found. These cells were then exposed to various differentiating agents, and then they expressed connexin genes, had functional gap junctions and ultimately differentiated (see Figure 3 in [ 48 ]).

Later, when it was shown that one of the biomarker genes of embryonic stem cells was the Oct4 gene [ 49 , 50 , 51 ], our lab had several organ-specific adult stem cells (kidney, breast, pancreas, and later, liver). We tested them for expressed Oct4 and non-expressed connexin genes [ 52 ]. This confirmed our hypothesis that the clones we isolated via the kiss of death assay and later other techniques [ 53 ] were, in fact, true adult stem cells. We decided that only two of our 20,000+ genes needed to be tested as biomarkers for any stem cells. Oct4 is required for maintaining stemness and the connexin genes and functional gap junctions are required for differentiation, growth control, apoptosis [ 54 ] and senescence.

4. A Test for Stem Cell and De-Differentiation Hypotheses for the Origin of the “Cancer Stem Cells”

Even though speculations and experimental tests were reported to support the stem cell hypothesis [ 22 , 23 , 24 , 25 , 26 , 27 ], no reports used a single isolated human adult stem cell to put these hypotheses to a test. Using normal human adult stem cells as the target cell of the initiation/promotion/progression carcinogenic in vitro process, we tested these cells for the expression of Oct4A and for functional gap junctional intercellular communication [ 23 ]. Oct4A was expressed but no connexin43 was expressed and there was no functional gap junctions in the human breast stem cells. We then tested whether these human breast adult stem cells could be differentiated into breast epithelial cells and whether Oct4A was still expressed and whether the connexin43 was expressed, and if they had functional gap junctions. These differentiated breast epithelial cells had no expressed Oct4A, but did express connexin43 and had functional gap junctions (see Figure 1 in [ 23 ]).

Next, the normal human adult breast stem cells and the differentiated breast epithelial cells were transfected with the large T gene of the SV-40 virus; only a few clones of proliferating breast stem cells were obtained. These cells still expressed the Oct4A gene and they had no functional gap junctions. These cells were apparently immortal , but not tumorigenic when tested in immune-suppressed mice. No immortalized cells were derived from those differentiated breast epithelial cells, confirming what many previous studies had shown, that to immortalize normal differentiated cells was either difficult or impossible [ 6 , 7 , 8 ].

The immortalized human breast stem cells were now X ray-irradiated, and a few clones that formed soft agar clones were isolated. These were tested for tumorigenicity in immune-deficient mice and they formed slowly or weakly growing tumors. Next, these X-irradiated cloned cells were treated with the ErB2/Neu gene and several clones that had significant rapid growth in soft agar were tested for tumorigenicity in immune-suppressed mice. In this case, the tumors were very tumorigenic and still expressed Oct4A gene and did not have functional gap junctional intercellular communication.

The take-home message of this experiment on a clonally-derived series of adult human breast stem cells showed that the tumorigenic breast cell line was directly derived from the normal adult stem cell that expressed the Oct4A gene and did not have functional gap junctions. In other words, the biomarker gene, Oct4A, was not induced by the carcinogenic process, but remained expressed from the start of the initiating event. Moreover, the initiating event blocked the differentiation process, as the late Dr. Potter predicted (“Oncogeny as partially blocked ontogeny” [ 1 ]). In addition, since the original stem cells are naturally “immortal” until they are terminally differentiated or become mortal, the so-called immortalizing viruses, e.g., SV40, are not immortalizing a normal mortal cell, they are blocking mortalization . These types of immortalizing viruses should be re-named. In other words, this experiment adds to those speculated hypotheses and actual direct experiments that strongly suggest the stem cell hypothesis is the correct hypothesis for the origin of cancers.

The recent demonstration of the isolation of induced pluripotent stem cells has given renewed support for re-programming or the de-differentiation of normal differentiated somatic cells to become “immortal”, or embryonic-like [ 55 ]. While no one can doubt that genetically-engineering a population of normal differentiated somatic cells with the Yamanaka embryonic genes, includingOct4, cannot produce “iPS” cells. However, there is now a legitimate reason to challenge the interpretation that re-programming took place. Clearly, that original population contained a few fibroblast stem cells [ 46 , 47 ]. These few adult fibroblast stem cells that, when transfected with these embryonic genes, now had both their own endogenous Oct4 gene expressed, but also those of the exogenous Oct4 that were introduced in its genome. That gave these few fibroblast adult stem cells the growth advantage over the somatic differentiated non-Oct4 expressing cells. Therefore, those using “iPS” cells for all kinds of experiments are really using normal fibroblast stem cells with their genome altered by the exogenous embryonic genes. In effect, these “iPS” cells are not really the result of “re-programming”, but rather the selection of pre-existing adult stem cells of the original primary culture of human tissue.

Now, one has to demonstrate if these tumorigenic cancer cells that were derived from a single normal immortal adult specific stem cell contain the cancer stem cells.

5. Characteristics of the Cancer Stem Cells

Today, these terms, cancer stem cell or cancer-initiating cell , are defined, operationally, as the cell that has the ability to sustain the long-term growth of a tumor, having all the characteristics of a tumor from which it was derived. One of the first clues came from a creative experiment, using Hoechst dye to stain cells of a tumor [ 56 ]. When these cells were placed in a cell sorter, two populations of cells were obtained, one fluorescing and a small population that did not incorporate the dye, hence not fluorescing. These latter population were classified as side population cells. These side population cells were shown to develop into tumors, manifesting the same characteristics as the tumor from which they were derived. Hence this procedure to isolate cancer-initiating or cancer stem cells became the operational definition of the cancer stem cells . These cells did not retain the Hoechst dye because they expressed functional drug transporter genes. Therefore, the Hoechst dye-containing cells or the cancer non-stem cells did not express those genes; hence, the dye, which binds to DNA, indicated that toxic chemicals could enter these types of cells and be killed. On the other hand, the side population cells would be resistant to potential toxic chemicals. Hence, this is a significant clue as to why most anti-cancer drugs, designed to kill cancer cells, can only kill the cancer non-stem cells but not the cancer stem cells .

At the time, one had to view this finding from an evolutionary vantage point. If during early evolution of the multi-cellular organism, when the stem cell appeared, that organism, with its few stem cells and many progenitor and terminally differentiated cells, if they were exposed to some toxic agent, and all cell types were equally susceptible to the toxic agent, the organism would not survive. The stem cells are needed for growth wound repair, tissue damage and the natural attrition of dying cells. Evolution selected for the stem cells to be able to be maintained in a low oxygen micro-environment, have various anti-oxidant systems to protect against free radicals, and to have both a nuclear membrane to act as another barrier to free radical production of the mitochondria of its neighboring differentiated daughters [ 57 , 58 ] and DNA repair enzymes [ 59 ].

6. Are All “Cancer Stem Cells” Identical?

The cancer stem cell of the breast, colon, liver, pancreas, etc. would have specific organ-specific markers. However, do they share some common markers that make them a cancer stem cell ? As pointed out previously, if the cancer stem cell is derived from a normal organ-specific adult stem cell, and if one marker, namely, the Oct4A gene, is shared by all cancer stem cells , as seems to be indicated by many published papers [ 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 ], then it would seem that some strategy should be developed to find ways to either shut down this gene in order to force the cells to differentiate or apoptose. In addition, if the connexin genes and functional gap junctions are not found in either the normal stem cells or cancer stem cells , agents that might induce the connexin genes to be expressed could induce either differentiation or apoptosis. One such example of this happening has been demonstrated [ 78 ].

An additional series of observations have, however, complicated this simple solution. In a series of many canine tumor types, Oct4 was detected in over 95% of these tumors [ 79 ]. Yet, there were a few canine tumors where no Oct4 marker was detected in the tumor cell population. Again, to complicate the situation, virtually all tumors are found in two states, namely, the embryonic-like phenotype, or in the quasi-differentiated phenotype (basal cell skin carcinoma or squamous skin carcinoma; polyp-type colon carcinoma and the flat-type or embryonic-type colon carcinoma; lung small cell carcinoma or the non-small cell carcinoma, etc.). To my knowledge, no one has examined the cancer stem cell of either type of tumors of any organ to determine if the Oct4A is not expressed, but some connexin gene is expressed in the partially differentiated stem cell, and if cancer stem cells of the embryonic-type organ tumor have the Oct4A gene expressed and no connexin gene or functional gap junctional intercellular communication is found. If that prediction is confirmed, then only these two genes (possibly the drug transporter genes) need to be monitored.

In a recent paper, it has been predicted, based on many arguments presented here, that there exist two cancer stem cells [ 80 , 81 ]. Therefore, using any cancer therapy protocol against one type will not work against the other type in the same organ. Currently, there is no explanation as to why some cancer stem cells do not express the Oct4A gene, yet are still derived from the normal organ-specific adult stem cell, as are the Oct4A expressing cancer stem cells . One potential explanation is that, as an adult stem cell starts to differentiate, it starts to turn off the expression of Oct4A; it is initiated in this transition state. The connexin gene is expressed and it starts to differentiate, but does not achieve the ability to terminally differentiate (i.e., asymmetrical cell division is inhibited, but the symmetrical cell division is still functioning). In these cases, since it was shown that gap junction can be inhibited if some oncogene is also expressed, thus blocking the gap junction function. Only future experiments to test this hypothesis will affirm or deny its validity.

Last, one of the new insights that has emerged in the search for ways to target the cancer stem cells in a cancer patient is to find ways to minimize the unintended consequences of the toxic effects of the therapies being used. One of the side effects of killing cancer cells by radiation or chemotherapeutic cytotoxicants is that it triggers a cytokine storm [ 82 , 83 ], which is a natural response for an organism to repair tissue or the loss of dying cells. These various cytokines have been shown to modulate gap junctional intercellular communication [ 84 ].

Since the drug metformin has already been shown to protect against chemicals that can inhibit gap junctional intercellular communication to cause enhanced cell proliferation [ 85 ], and since metformin has already been shown to target cancer stem cells in a three dimensional human breast organoid [ 86 ], we predicted that using it together with any anti-cancer therapy to minimize both the side consequences of the therapy on the non-cancer cells, as well as to help sensitize targeting the cancer stem cells, would seem to be a rational strategy. Several reviews of the literature seems to have provided mixed results, with some studies showing no effects, others some negative effects, and some demonstrating positive effects [ 87 , 88 , 89 , 90 ]. Since metformin seems to act in a similar, but not identical, way to other chemicals that protect cells from agents that can inhibit gap junction intercellular communication, such as resveratrol, CAPE, green tea components, licorice components, caffeine, lycopene [ 91 ], as well as lovastatin [ 92 ], melatonin [ 86 ] and others, understanding all the different mechanisms by which agents can modulate gap junctional intercellular communication is critical. Phorbol esters, a powerful gap junction inhibitor, work by a very different biochemical cellular mechanism than does DDT. There are both receptor- and receptor-independent mechanisms by which inhibitors of gap junction function, for example low dose estrogen verses high dose estrogen, have different effects of triggering intracellular signaling. The mixed clinical trials might simply be due to not understanding the specific factors involved, namely, the individual’s genetics, gender, development stage, the specific intra-cellular signaling pathways that are triggered by the agents being used, dose to be used, timing with the anti-cancer therapy, knowing which of the two types of cancer stem cells is in the patient’s tumors, as well as the time of day the therapy is administered. If ever there was a support for precision medicine or personalized medicine, this could be an example. This is seen in the context of the definition of personalized or precision medicine which refers to a medical approach in which diseases are diagnosed, prevented, and treated according to the context of each patient’s unique genetics, history, and lifestyle. Treatments are optimized and side effects are reduced, and this drastically reduces the overall cost of healthcare to society.

7. The Ultimate Problem of Designing an Anti-Cancer Agent That Targets These Cancer Stem Cells

Strategically, one would like to prevent any future cancer over the treatment of an existing tumor or the wide-spread metastatic cancer. However, there are many obstacles that have to be overcome. The first is the problem of the initiation of a single normal cell (in this case, it is assumed that a normal cell is an adult organ-specific adult stem cell), because initiation, while preventable to a degree, it is not possible to eliminate all initiating events or a mutation in a critical cancer-associated gene responsible for blocking asymmetrical cell division of the organ-specific stem cell. One needs to not be exposed to too much UV light from the sun or to not sit on a uranium pile. However, mutations can be produced not only by the genomic DNA being damaged ( errors in DNA repair ) [ 93 , 94 , 95 , 96 , 97 ], but also by errors in DNA replication [ 98 ]. Consequently, even in the absence of genomic DNA damage, every time a stem cell replicates during a growth spurt, hormone growth factor, or cytokine stimulation during wound healing, cell death of a tissue and tissue removal, there is a finite probability that a “spontaneous” mutation in one of those initiating genes would occur.

Probably one of the most convincing proofs that a specific oncogene mutation in a tumor associated with lung cancer is the demonstration that mutations in non-smokers’ lung cancer cell’s Ha-ras oncogene were identical to the mutation in that gene found in lung tumor cells of smokers [ 99 ]. As an important side observation, it was a classic chemical found in cigarette smoke that was shown not to be a genotoxicant or mutagen, but rather an agent that increased the transformation frequency of baby hamster embryonic cells [ 100 , 101 , 102 ]. Later, studies on any of the predominant aromatic hydrocarbons in cigarette smoke showed them to be tumor promoters, but not initiators [ 103 , 104 ]. When using assays to detect agents that could reversibly inhibit gap junctional intercellular communication and shown that they were epigenetic-acting chemicals, the most predominate aromatic hydrocarbon was a non-mutagenic, 1-methyl anthracene [ 105 ]. Therefore, tying this set of observations together, it seems that lung cancers of non-smokers and smokers might be the result of a spontaneous mutation, caused by an error of DNA replication in a gene that blocks asymmetric cell division of a stem cell of the lung, which was promoted by endogenous or exogenous epigenetic chemicals.

Returning to the major problem of killing the cancer stem cells , how can an agent be designed to be given in vivo to target the cancer stem cell of a benign or malignant cancer? It is now accepted that all tumors are a heterogeneous mixture of non-cancer stem cells [ 106 ] and a few cancer stem cells, together with normal stromal cells and invasive immune cells. In general, there is a very wide range of genotypes (both chromosomal and gene-wise) in the cancer non-stem cells. To date, there is no solid evidence that the deviation chromosomal/gene mutations and chromosome instability, let alone epigenetic deviations of the initiated cells during its evolution, were responsible for the origin of the cancer stem cell.

First, as was pointed out before, we will never eliminate the origin of the initiated adult stem cell because it is usually surrounded by, and communicating with, its normal differentiated progenitor or differentiated daughters. Under those conditions, these communicating normal cells are sending signals to induce a normal phenotype in the initiated cell, in spite of having a critical mutation in a cancer-associated gene. This allows the “initiated cell” to escape the immune system, as it appears to be “normal” or self-like.

However, when these cell–cell communication-coupled cells are exposed to agents and conditions that can inhibit this communication process, these initiated cells can clone multiply to form those benign lesions, such as a papilloma of the skin, enzyme altered foci of the liver, polyp of the colon or nodule in the breast. Not all of these lesions will go on to form an invasive and metastatic tumor. In fact, some might even be blocked from further development or even regress [ 107 ]. However, a few of these lesions will have a cell within the benign lesions that will have acquired the “hallmarks of cancer” [ 2 , 108 ].

Faced with the problem of targeting any agent that might induce differentiation, apoptosis or cytotoxicity in vivo of any organ-specific tumor, one has to deal with a tumor that is a heterogeneous mixture of cancer non-stem cells, cancer stem cells and various normal stromal and immune cells. Moreover, a chemical anti-cancer drug or inducer of an immune response has to find its way to those cancer stem cells. In addition, any cancer stem cell -targeted drug must not attack the normal stem cells of the body. One can imagine the complex cell–cell interactions between all of these different cell types, changing the normal gene expressions and phenotypes of those cells that are absent of those interactions. The apparent strategy is to design at least a multiple approach, first to kill the sensitive cancer non-stem cells, and then to eliminate at least some of the barriers to any cancer stem cell -targeted agent. However, remember that the death of those non-cancer stem cells will be releasing various cytokine-like chemicals that could cause the stimulation of the resistant and surviving cancer stem cells. Using stem cells grown in three-dimensions to mimic tissue organization, both tumor promoters and anti-cancer agents have been used to target the cancer stem cells [ 81 , 109 ].

In this Commentary, an examination of the origin of the cancer stem cell concept was developed by viewing historical experimental observations, techniques and concepts that have led to the operational concept that these cancer stem cells are those responsible for sustaining the growth of any tumor. Challenges to the understanding of what is the cancer stem cell , and from which cell it originated, have come from terms, such as normal cells, immortal, and carcinogens . These terms were examined in the context of several major classic concepts, such as the multi-stage, multi-mechanism process of carcinogenesis, and the demonstration of epigenetic agents as being the driver of this multi-stage, multi-mechanism process. A major challenge to the concept that re-programming of somatic differentiated cells exists during the immortalization of a normal cell. Characterization of several markers of isolated stem cells, such as Oct4A and connexin genes, in both normal adult stem cells and in cancer stem cells , has led to the potential strategy of targeting the cancer stem cells in a heterogeneous mixture of cancer cells in all tumors. Last, it has been speculated that there exist two different kinds of cancer stem cells, which suggests two very different kinds of anti-cancer drug strategy must be developed.

Acknowledgments

While I fully acknowledge all the work, discussions and intellectual contributions of my former students, postdoctoral fellows, and international Visiting Scholars and collaborating colleagues during my 50 years of research, I take full responsibility of all the speculative claims made in this manuscript.

As a Professor Emeritus, I received no grants (governmental, Foundation, or institutional) that supported the development of this manuscript.

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Not applicable.

Informed Consent Statement

Conflicts of interest.

The author has no conflict of interest.

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The company they keep

Neighboring cells influence whether tumors grow or perish

By Krista Conger

Illustration by John Hersey

In 2020, Everett Moding , MD, PhD, an assistant professor in Stanford Medicine’s radiation oncology department, noticed that some people with a rare cancer called soft-tissue sarcomas were cured with surgery and radiation while others saw their cancers quickly recur. “Two patients could have the same diagnosis and be treated the same way, but their cancers would respond very differently,” he said. “And there was no effective way to predict who would have a poorer prognosis.”

Around the same time, Magdalena Matusiak , PhD, then a postdoctoral student in the laboratory of professor of pathology Matt van de Rijn , MD, PhD, the Sabine Kohler, MD, Professor in Pathology, was growing frustrated with the traditional methods of predicting cancer cells’ growth based primarily on mutations in their DNA.

“In many instances, we can’t explain tumor biology just by looking at mutations or gene expression,” Matusiak said. “Ductal carcinoma in situ, a common early breast cancer, is not usually life threatening. But in about 1 in 4 patients, these cancers will become invasive for reasons we can’t explain with conventional methods.”

Both young researchers turned for answers to a rapidly growing field defined by leaps in technology and machine learning that allow a close-up look at the thousands of interactions between cancer cells and the healthy cells and tissues in which they reside. This three-dimensional neighborhood is broadly defined as the tumor microenvironment, and our growing understanding of its importance relies heavily on studies of what’s been called spatial biology.

It turns out that the company that cancer cells keep — and the way that company reacts to their presence — is critical to determining whether a new cancer grows, thrives and metastasizes to other parts of the body or is pounced upon and eliminated by the immune system.

“Cells don’t exist in isolation,” said assistant professor of biomedical data science Aaron Newman , PhD. “A cell’s identity, its behavior, its characteristics depend on what other cells are around it in three-dimensional space and what those cells are doing. But even five years ago we didn’t have a good way to identify these interactions. Now we can begin to assess aspects of this nuanced, community-specific biology.”

“Cells don’t exist in isolation. A cell’s identity, its behavior, its characteristics depend on what other cells are around it in three-dimensional space and what those cells are doing.” Aaron Newman, PhD, assistant professor of biomedical data science

Newman, a member of the Stanford Cancer Institute and a Chan Zuckerberg Biohub Investigator, is one of several Stanford Medicine scientists developing tools and techniques to collect and interpret dizzying amounts of data from human tumors to identify, on a cellular communications level, exactly who says what to whom, as well as where, when and why. It’s a daunting task when you consider that a tumor the size of a small grape contains something on the order of 1 billion cells.

Some heavy hitters back this research, among them the National Cancer Institute, which in 2016 named the Human Tumor Atlas Network as one of the key research initiatives of its Cancer Moonshot — a program created to focus on areas of research deemed most likely to benefit cancer patients. The tumor atlas network aims to detail the evolution of the cellular and molecular interactions among healthy and diseased cells as a precancerous growth develops into full-blown cancer.

“It’s really clear that a tumor is not just a collection of cancer cells,” said Sylvia Plevritis , PhD, chair of Stanford Medicine’s Department of Biomedical Data Science, the head of the Stanford Center for Cancer Systems Biology and the Stanford Cancer Institute’s associate director of cancer AI.

“In fact, some of the most difficult tumors to treat, like pancreatic tumors, are mostly noncancer cells. Techniques to study the spatial biology of tumors, like those developed in Aaron’s lab and several others at Stanford including mine, are changing our understanding of cancer. Now, we can not only see what cell types are in the tumor but who their neighbors are and the molecular interactions that allow them to communicate and sustain each other.”

In just a few years, researchers have gone from deciphering flat, stained slices of tumor tissue highlighting the gross anatomy of a tumor to parsing not just the precise cellular composition of small tumor samples but even identifying specific cellular neighborhoods and interactions that can determine health or disease. The insights are providing important clues to medical mysteries, like those puzzling Moding and Matusiak: Why do some patients with what seem to be very similar cancers have better outcomes than others?

Proving the link between cancer cells and their surroundings

The idea that the cells and tissue surrounding a cancer cell may be as important as the cancer cell itself for determining whether the cancer cell thrives, divides and — eventually — metastasizes was first floated in 1863 when German physician Rudolf Virchow, MD, noted a connection between inflammation and cancer. In 1889, English surgeon Stephen Paget, FRCS, advanced his “seed and soil” hypothesis that the cellular environment within which a metastasizing cancer cell landed influenced whether it would flourish or die in its new location.

At that time, there were few ways to prove these hypotheses on a cellular level. Aspiring investigators pored over microscope slides holding thin slices of tissue stained a dull purple to delineate individual cells and structures. Researchers could only infer relationships among cells from a snapshot in time frozen on a two-dimensional grid — a bit like trying to predict how occupants of a high-rise spend their time by looking at the building’s blueprints.

Decades later, in the late 1960s, scientists devised a way to attach color-changing proteins to antibodies that recognize and bind to specific cellular structures — vastly increasing the amount of information that could be garnered from a single slide. Now they could see the arrangement of furniture in individual rooms and predict the function of each space. But still, there was no inkling of how the cells communicated, or didn’t, with one another in living tissue.

“Many times, if you just look at tumors as a bag of cells, your ability to predict a patient’s prognosis is not great, even if you know how many of each cell type is in the sample. But if you can incorporate where those cells are in the tumor, those predictions become much better.” Michael Angelo , MD, PhD, associate professor of pathology

The floodgates started to open when genomic sequencing took off in the early 2000s. Soon researchers learned how to infer the cellular composition of a tumor by identifying the relative levels of RNA messages, or transcripts, expressed by the cells — first in bulk and then, almost incomprehensibly, at the level of individual cells. Suddenly, the high-rise blueprint shows not just rooms and furniture but also people and what was on their minds.

That’s because, although most cells share a common vocabulary in the form of the genes encoded by their DNA, RNA messages are the genetic words a cell mutters to itself to accomplish a certain goal at a particular time. Single-cell RNA sequencing allows researchers to eavesdrop on these internal conversations.

Newman and his peers at Stanford Medicine have developed technologies that build on these earlier advances. One, CIBERSORTx, functions like an eerily accurate fortune teller, predicting the various cell types in a bulk tissue sample based on the relative abundance and patterns of RNA messages in the sample. Another, EcoTyper, builds on this prediction to determine what the cell types are up to (a condition called cell state) and which other cells they are interacting with. The information allows researchers to build a picture of complex cellular neighborhoods called ecotypes within tumor tissue that hint at how the tumor is (or isn’t) thriving.

“Spatial transcriptomics is a new technology that gives us information about gene expression and spatial location so we can understand the modular architecture of healthy and cancerous tissue,” said Newman, the Institute for Stem Cell Biology and Regenerative Medicine Faculty Scholar. “In ecology, a species changes its characteristics and behavior in response to its local environment. Cells do this as well.”

Most recently, another tool, CytoSPACE, developed in Newman’s lab, maps these neighborhoods to precise locations in the tumor tissue, while also assessing the activity of all of each cell’s 20,000 genes.

“Techniques to study the spatial biology of tumors … are changing our understanding of cancer. Now, we can not only see what cell types are in the tumor but who their neighbors are and the molecular interactions that allow them to communicate and sustain each other.” Sylvia Plevritis, PhD, associate director of cancer AI at the Stanford Cancer Institute

“Many times, if you just look at tumors as a bag of cells, your ability to predict a patient’s prognosis is not great, even if you know how many of each cell type is in the sample,” said associate professor of pathology Michael Angelo , MD, PhD. Angelo developed a way to visualize the locations of up to 50 individual proteins in a cell using a technique called MIBI-TOF. “But if you can incorporate where those cells are in the tumor, those predictions become much better. And they don’t seem to have a whole lot to do with the tumor cells themselves,” Angelo said. “The much more important angle is how the nontumor cells are responding to the presence of the cancer.”

Importantly, the machine learning that drives each of these advances has no preconceptions about what it might find. By simply looking for patterns — this type of cell is likely to be found rubbing membranes with this other type of cell, but only when both are in a particular cell state, for example — the computers can identify interactions that defy expectations.

“When my lab started working with single-cell data of tumors, we kept finding fibroblasts coming up as really important,” said Plevritis, the William M. Hume Professor in the School of Medicine. “Fibroblasts are most known for creating part of the skeleton that cells sit in and are one of the most understudied parts of a tumor, so it is very interesting and exciting to study this association.”

Further studies in Plevritis’ lab found that fibroblasts at the leading edge of a lung tumor had properties that stimulated cancer cells to invade surrounding tissue, while the fibroblasts in the interior appear to be more tumor suppressive.

New tools allow for deeper probes of archived cancer tissues and types

Taken together, these technologies have given researchers, including Matusiak and Moding, valuable insight as to why people with the same type and stage of cancers can have such different outcomes.

Matusiak compared the location and activity of immune cells called macrophages in breast and colon cancers with healthy tissue. Prior to her study, researchers identified macrophages in tumor tissue by the presence of a protein that appears universally on all macrophages. Matusiak used single-cell RNA sequencing data to identify additional proteins that appear on only a subset of macrophages. She then found antibodies to these subset-specific proteins and used them to probe slides of tissue from colorectal and breast tumors.

She learned that macrophages are found in five distinct and very different cellular neighborhoods, or niches, within the tumors and that the macrophages were acting differently in each location.

“This was a big surprise,” Matusiak said. “We were definitely not expecting to see such distinct and separate spatial regions.”

For example, macrophages with a protein called IL4I1 on their surfaces were found in regions of high cellular turnover in both healthy and cancerous tissue — gobbling dead or dying cells. T

he presence of this class of macrophages correlated with a good response to immunotherapy in breast cancer patients and more favorable outcomes in people with colorectal cancers. In contrast, although macrophages with a protein called SPP1 were associated with tumor cell death, their presence in colorectal tumors correlated with poor outcomes.

“Now we have the first tools to really investigate macrophage biology in different tissues and cancer types in archived human tissue, including ductal carcinomas in situ,” Matusiak said.

Krista Conger

Krista Conger is a Senior Science Writer in the Office of Communications. Email her at [email protected] .

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Cancer stem cell hypothesis 2.0 in glioblastoma: Where are we now and where are we going?

Affiliations.

• 1 Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
• 2 Case Comprehensive Cancer Center, Cleveland, Ohio, USA.
• 3 Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
• 4 Department of Pediatrics, Section of Hematology and Oncology, Baylor College of Medicine, Houston, Texas, USA.
• 5 Texas Children's Cancer Center, Texas Children's Hospital, Houston, Texas, USA.
• 6 Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center, Cleveland Clinic, Cleveland, Ohio, USA.
• PMID: 38394444
• PMCID: PMC11066900 (available on 2025-02-23 )
• DOI: 10.1093/neuonc/noae011

Over the past 2 decades, the cancer stem cell (CSC) hypothesis has provided insight into many malignant tumors, including glioblastoma (GBM). Cancer stem cells have been identified in patient-derived tumors and in some mouse models, allowing for a deeper understanding of cellular and molecular mechanisms underlying GBM growth and therapeutic resistance. The CSC hypothesis has been the cornerstone of cellular heterogeneity, providing a conceptual and technical framework to explain this longstanding phenotype in GBM. This hypothesis has evolved to fit recent insights into how cellular plasticity drives tumor growth to suggest that CSCs do not represent a distinct population but rather a cellular state with substantial plasticity that can be achieved by non-CSCs under specific conditions. This has further been reinforced by advances in genomics, including single-cell approaches, that have used the CSC hypothesis to identify multiple putative CSC states with unique properties, including specific developmental and metabolic programs. In this review, we provide a historical perspective on the CSC hypothesis and its recent evolution, with a focus on key functional phenotypes, and provide an update on the definition for its use in future genomic studies.

Keywords: cancer stem cell; glioblastoma; heterogeneity; proliferation.

© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected].

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Conflict of interest statement

None declared.

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