Alternative models of cancer stem cells: The stemness phenotype model, 10 years later

The classical cancer stem cell (CSCs) theory proposed the existence of a rare but constant subpopulation of CSCs. In this model cancer cells are organized hierarchically and are responsible for tumor resistance and tumor relapse. Thus, eliminating CSCs will eventually lead to cure of cancer. This simplistic model has been challenged by experimental data. In 2010 we proposed a novel and controversial alternative model of CSC biology (the Stemness Phenotype Model, SPM). The SPM proposed a non-hierarchical model of cancer biology in which there is no specific subpopulation of CSCs in tumors. Instead, cancer cells are highly plastic in term of stemness and CSCs and non-CSCs can interconvert into each other depending on the microenvironment. This model predicts the existence of cancer cells ranging from a pure CSC phenotype to pure non-CSC phenotype and that survival of a single cell can originate a new tumor. During the past 10 years, a plethora of experimental evidence in a variety of cancer types has shown that cancer cells are indeed extremely plastic and able to interconvert into cells with different stemness phenotype. In this review we will (1) briefly describe the cumulative evidence from our laboratory and others supporting the SPM; (2) the implications of the SPM in translational oncology; and (3) discuss potential strategies to develop more effective therapeutic regimens for cancer treatment.     

Reprogramming strategies for the establishment of novel human cancer models

Cancer comprises heterogeneous cells, ranging from highly proliferative immature precursors to more differentiated cell lineages. The emergence of the “cancer stem cell” (CSC) hypothesis that they are the cells responsible for resistance, metastasis and secondary tumor appearance identifies these populations as novel obligatory targets for the treatment of cancer. CSCs, like their normal tissue-specific stem cell counterparts, are multipotent, partially differentiated, self-sustaining, yet transformed cells. To date, most studies on CSC biology have relied on the use of murine models and primary human material. In spite of much progress, the use of primary material presents several limitations that limit our understanding of the mechanisms underlying CSC formation, the similarities between normal stem cells and CSCs and ultimately, the possibility for developing targeted therapies. Recently, different strategies for controlling cell fate have been applied to the modeling of human cancer initiation and for the generation of human CSC models. Here we will summarize recent developments in the establishment and application of reprogramming strategies for the modeling of human cancer initiation and CSC formation.     

Cancer stem cells – the current status of an old concept: literature review and clinical approaches

As regards their morphology and biology, tumours consist of heterogeneous cell populations. The cancer stem cell (CSC) hypothesis assumes that a tumour is hierarchically organized and not all of the cells are equally capable of generating descendants, similarly to normal tissue. The only cells being able to self-renew and produce a heterogeneous tumour cell population are cancer stem cells. CSCs probably derive from normal stem cells, although progenitor cells may be taken into consideration as the source of cancer stem cells. CSCs reside in the niche defined as the microenvironment formed by stromal cells, vasculature and extracellular matrix. The CSC assays include FACS sorting, xenotransplantation to immunodeficient mice (SCID), incubation with Hoechst 33342 dye, cell culture in non-adherent conditions, cell culture with bromodeoxyuridine. CSCs have certain properties that make them resistant to anticancer therapy, which suggests they may be the target for potential therapeutic strategies.     

Aldehyde dehydrogenase: Its role as a cancer stem cell marker comes down to the specific isoform

Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of cancer stem cells (CSC) measurable by the aldefluor assay. ALDH1A1, one of 19 ALDH isoforms expressed in humans, was generally believed to be responsible for the ALDH activity of CSCs. More recently, experiments with murine hematopoietic stem cells, murine progenitor pancreatic cells, and human breast CSCs indicate that other ALDH isoforms, particularly ALDH1A3, significantly contribute to aldefluor positivity, which may be tissue and cancer specific. Therefore, potential prognostic application involving the use of CSC prevalence in tumor tissue to predict patient outcome requires the identification and quantification of specific ALDH isoforms. Herein we review the suggested roles of ALDH in CSC biology and the immunohistological studies testing the potential application of ALDH isoforms as novel cancer prognostic indicators.     

Bladder cancer stem cells

Stem cells are undifferentiated cells that renew themselves while simultaneously producing differentiated tissue- or organspecific cells through asymmetric cell division. The appreciation of the importance of stem cells in normal tissue biology has prompted the idea that cancers may also develop from a progenitor pool (the "cancer stem cell (CSC) hypothesis"), and this idea is gaining increasing acceptance among scientists. CSCs are sub-populations of cancer cells responsible for tumor initiation, differentiation, recurrence, metastasis, and drug resistance. First identified in the hematopoietic system, CSCs have also been discovered in solid tumors of the breast, colon, pancreas, and brain. Recently, the tissue-specific stem cells of the normal urothelium have been proposed to reside in the basal layer, and investigators have isolated phenotypically similar populations of cells from urothelial cancer cell lines and primary tumors. Herein, we review the CSC hypothesis and apply it to explain the development of the two different types of bladder cancer: noninvasive ("superficial") carcinoma and invasive carcinoma. We also examine potential approaches to identify CSCs in bladder cancer as well as therapeutic applications of these findings. While exciting, the verification of the existence of CSCs in bladder cancer raises several new questions. Herein, we identify and answer some of these questions to help readers better understand bladder cancer development and identify reasonable therapeutic strategy for targeting stem cells.     

Basal/HER2 breast carcinomas

High rates of inherent primary resistance to the humanized monoclonal antibody trastuzumab (Herceptin) are frequent among HER2 gene-amplified breast carcinomas in both metastatic and adjuvant settings. The clinical efficacy of trastuzumab is highly correlated with its ability to specifically and efficiently target HER2-driven populations of breast cancer stem cells (CSCs). Intriguingly, many of the possible mechanisms by which cancer cells escape trastuzumab involve many of the same biomarkers that have been implicated in the biology of CS-like tumor-initiating cells. In the traditional, one-way hierarchy of CSCs in which all cancer cells descend from special self-renewing CSCs, HER2-positive CSCs can occur solely by self-renewal. Therefore, by targeting CSC self-renewal and resistance, trastuzumab is expected to induce tumor shrinkage and further reduce breast cancer recurrence rates when used alongside traditional therapies. In a new, alternate model, more differentiated non-stem cancer cells can revert to trastuzumab-refractory, CS-like cells via the activation of intrinsic or microenvironmental paths-to-stemness, such as the epithelial-to-mesenchymal transition (EMT). Alternatively, stochastic transitions of trastuzumab-responsive CSCs might also give rise to non-CSC cellular states that lack major attributes of CSCs and, therefore, can remain “hidden” from trastuzumab activity. Here, we hypothesize that a better understanding of the CSC/non-CSC social structure within HER2-overexpressing breast carcinomas is critical for trastuzumab-based treatment decisions in the clinic. First, we decipher the biological significance of CSC features and the EMT on the molecular effects and efficacy of trastuzumab in HER2-positive breast cancer cells. Second, we reinterpret the genetic heterogeneity that differentiates trastuzumab-responders from non-responders in terms of CSC cellular states. Finally, we propose that novel predictive approaches aimed at better forecasting early tumor responses to trastuzumab should identify biological determinants that causally underlie the intrinsic flexibility of HER2-positive CSCs to “enter” into or “exit” from trastuzumab-sensitive states. An accurate integration of CSC cellular states and EMT-related biomarkers with the currently available breast cancer molecular taxonomy may significantly improve our ability to make a priori decisions about whether patients belonging to HER2 subtypes differentially enriched with a “mesenchymal transition signature” (e.g., luminal/HER2 vs. basal/HER2) would distinctly benefit from trastuzumab-based therapy ab initio.     

Tracing and targeting cancer stem cells: New venture for personalized molecular cancer therapy

Tumors consist of a mixture of heterogeneous cell types. Cancer stem cells(CSCs) are a minor sub-population within the bulk cancer fraction which has been foundto reconstitute and propagate the disease and to be frequently resistant to chemotherapy, irradiation, cytotoxic drugs and probably also against immune attack. CSCs are considered as the seeds of tumor recurrence, driving force of tumorigenesis and metastases. This underlines the urgent need for innovative methods to identify and target CSCs. However, the role and existence of CSCs in therapy resistance and cancer recurrence remains a topic of intense debate. The underlying biological properties of the tumor stem cells are extremely dependent on numerous signals, and the targeted inhibition of these stem cell signaling pathways is one of the promising approaches of the new antitumor therapy approaches. This perspective review article summarizes the novel methods of tracing CSCs and discusses the hallmarks of CSC identification influenced by the microenvironment or by having imperfect detection markers. In addition, explains the known molecular mechanisms of therapy resistance in CSCs as reliable and clinically predictive markers that could enable the use of new targeted antitumor therapy in the sense of personalized medicine.     

Cancer stem cells: current status and evolving complexities

The cancer stem cell (CSC) model has been established as a cellular mechanism that contributes to phenotypic and functional heterogeneity in diverse cancer types. Recent observations, however, have highlighted many complexities and challenges: the CSC phenotype can vary substantially between patients, tumors may harbor multiple phenotypically or genetically distinct CSCs, metastatic CSCs can evolve from primary CSCs, and tumor cells may undergo reversible phenotypic changes. Although the CSC concept will have clinical relevance in specific cases, accumulating evidence suggests that it will be imperative to target all CSC subsets within the tumor to prevent relapse.     

Stem cells: The root of prostate cancer?

The cancer stem cell (CSC) model states that tumors contain a reservoir of self-renewing cells that maintain the heterogeneous cell population of the tumor. These cells appear to be resistant to therapy and can therefore survive to repopulate the tumor during progression to therapy resistant disease. The biology of CSCs is still not definitive since it is difficult to isolate them from solid tumors and analyze their characteristics in vitro. Another challenge is to correlate these characteristics with tumor development and progression in vivo. Using the prostate CSC as a model, this review presents the CSC hypothesis, reviews the origin, identification and functions of prostate CSCs, and discusses the clinical implications and therapeutic challenges CSCs have for cancer therapy.     

Designing precision medicine panels for drug refractory cancers targeting cancer stemness traits

Cancer is one amongst the major causes of death today and cancer biology is one of the most well researched fields in medicine. The driving force behind cancer is considered to be a minor subpopulation of cells, the cancer stem cells (CSCs). Similar to other stem cells, these cells are self-renewing and proliferating but CSCs are also difficult to target by chemo- or radio-therapies. Cancer stem cells are known to be present in most of the cancer subgroups such as carcinoma, sarcoma, myeloma, leukemia, lymphomas and mixed cancer types. There is a wide gamut of factors attributed to the stemness of cancers, ranging from dysregulated signaling pathways, and activation of enzymes aiding immune evasion, to conducive tumor microenvironment, to name a few. The defining outcome of the increased presence of CSCs is tumor metastasis and relapse. Predictive medicine approach based on the plethora of CSC markers would be a move towards precision medicine to specifically identify CSC-rich tumors. In this review, we discuss the cancer subtypes and the role of different CSC specific markers in these varying subtypes. We also categorize the CSC markers based their defining trait contributing to stemness. This review thus provides a comprehensive approach to catalogue a predictive set of markers to identify the resistant and refractory cancer stem cell population within different tumor subtypes, so as to facilitate better prognosis and targeted therapeutic strategies.     

Cancer stem cell marker glycosylation: Nature,function and significance

Glycans are essential for the maintenance of normal biological function, with alterations in glycan expression being a hallmark of cancer. Cancer stem cells (CSCs) are a subset of cells within a tumour capable of self-renewal, cellular differentiation and resistances to conventional therapies. As is the case with stem cells, marker proteins present on the cell surface are frequently used to identify and enrich CSCs, with the expression of these markers statistical correlating with the likelihood of cancer recurrence and overall patient survival. As such CSC markers are of high clinical relevance. The majority of markers currently used to identify CSC populations are glycoproteins, and although the diverse biological roles for many of these markers are known, the nature and function of the glycan moiety on these glycoproteins remains to be fully elucidated. This mini-review summarises our current knowledge regarding the types and extent of CSC marker glycosylation, and the various roles that these glycans play in CSC biology, including in mediating cell adhesion, metastasis, evading apoptosis, tear shear resistance, tumour growth, maintaining pluripotency, self-renewal, trafficking, maintaining stability, maintaining enzymatic activity and aiding epithelial mesenchymal transitioning. Given that CSCs markers have multiple diverse biological functions, and are potentially of significant diagnostic and therapeutic benefit the search for new markers that are uniquely expressed on CSCs is vital to selectively target/identify this subset of cancer cells. As such we have also outlined how high-throughput lectin microarrays can be used to successfully profile the glycosylation status of CSC and to identify glyco-markers unique to CSCs.     

Turning Cancer Stem Cells Inside Out: An Exploration of Glioma Stem Cell Signaling Pathways

Tumors are complex collections of heterogeneous cells with recruited vasculature, inflammatory cells, and stromal elements. Neoplastic cells frequently display a hierarchy in differentiation status. Recent studies suggest that brain tumors have a limited population of neoplastic cells called cancer stem cells with the capacity for sustained self-renewal and tumor propagation. Brain tumor stem cells contribute to therapeutic resistance and tumor angiogenesis. In this minireview, we summarize recent data regarding critical signaling pathways involved in brain tumor stem cell biology and discuss how targeting these molecules may contribute to the development of novel anti-glioma therapies.Cancers can be considered organ systems with aberrant activation of developmental and wound response pathways. Recent evidence suggests that within some tumors there is a cell subpopulation with the special capacity for sustained self-renewal and tumor propagation in vivo. Cells fulfilling these criteria were originally reported in acute myeloid leukemia (1), but similar populations were soon successively identified within various solid tumors (2). The proper terminology regarding these cells remains unsettled, with most groups using terms such as CSCs,² tumor-initiating/propagating cells, and stem-like cancer cells. Although CSCs are a source of controversy, the concept recognizes the well described heterogeneity of tumor cells. Many critics contest the hypothesis on the grounds of a potential stem cell origin, challenge of current markers, or CSC frequency, none of which are implicit requirements of the CSC hypothesis (3).Malignant gliomas are essentially universally lethal despite conventional therapy, with surgical resection and chemoradiation limited to palliation. Glioma CSCs were among the first solid tumor CSCs described (4) and remain one of the most widely used CSC models. Glioma CSCs share significant similarities with normal NSCs, including the expression of stem cell markers (CD133, Nestin, Musashi, and Sox2) and the capacity to differentiate into multiple lineages (5), but the overlap is incomplete. Notably, glioma CSCs are also highly resistant to chemoradiotherapies (5, 6), underscoring the importance of developing more efficient therapies against CSCs and prompting researchers to elucidate the molecular mechanisms regulating CSCs. Here, we summarize recent findings regarding the signaling pathways that are critical to glioma CSC biology.     

Targeting cancer stem cells expressing an embryonic signature with anti-proteases to decrease their tumor potential

Cancer stem cells (CSCs) are a specific subset of cancer cells that sustain tumor growth and dissemination. They might represent a significant treatment target to reduce malignant progression and prevent tumor recurrence. In solid tumors, several hierarchically organized CSC clones coexist, even within a single tumor. Among them, CSCs displaying an embryonic stem cell ‘stemness'' signature, based on the expression of Oct-4, Nanog and Sox2, are present in distinct high-grade tumor types associated with poor prognosis. We previously designed a model to isolate pure populations of these CSCs from distinct solid tumors and used it to screen for molecules showing selective toxicity for this type of CSC. Here we show that human immunodeficiency virus (HIV)-protease inhibitors (HIV-PIs) specifically target CSCs expressing an embryonic signature derived from tumors with distinct origins. They reduced proliferation in a dose-dependent manner with a higher specificity as compared with the total population of cancer cells and/or healthy stem cells, and they were efficient in inducing cell death. Lopinavir was the most effective HIV-PI among those tested. It reduced self-renewal and induced apoptosis of CSCs, subsequently impairing in vivo CSC-induced allograft formation. Two key pharmacophores in the LPV structure were also identified. They are responsible for the specificity of CSC targeting and also for the overall antitumoral activity. These results contribute to the identification of molecules presenting selective toxicity for CSCs expressing an embryonic stemness signature. This paves the way to promising therapeutic opportunities for patients suffering from solid cancer tumors of poor prognosis.     

Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.     

Ionizing radiation induces stemness in cancer cells

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.     

综述: 肿瘤干细胞微环境与靶向干预策略

肿瘤干细胞具有自我更新和可塑性的潜能,能够维持肿瘤生长和异质性的能力．肿瘤干细胞是肿瘤产生、转移、耐药和复发的根源,肿瘤干细胞学说逐渐被肿瘤研究者所接受,因此,对肿瘤干细胞的深入理解有重大的科学和临床意义．肿瘤干细胞的微环境是肿瘤微环境的组成部分,包括细胞-细胞接触、分泌型因子等．肿瘤非干细胞和肿瘤干细胞本身都可以作为肿瘤干细胞的微环境．肿瘤干细胞的微环境可以维持肿瘤干细胞的可塑性,保护肿瘤干细胞免受免疫系统攻击,也可以促进其转移．肿瘤干细胞对其微环境的塑造、肿瘤干细胞的微环境对肿瘤干细胞自我更新的影响,以及针对肿瘤干细胞微环境的靶向干预等问题,已成为肿瘤干细胞研究的前沿问题．本文就肿瘤干细胞的发现、自我更新维持机制、肿瘤干细胞的微环境,及其肿瘤干细胞及微环境的干预策略等研究进展进行了综述．     

One renegade cancer stem cell?

The CSC compartment represents the subpopulation of tumor cells with clonogenic potential and the ability to initiate new tumors. Besides self renewal, one of their main features is their ability to differentiate into variety of cells within the tumor. The question remains whether this potential resides within the single CSC or whether many different CSCs are necessary to generate a heterogeneous population of tumor cells. There is an increasing amount of evidence showing that single CSC indeed has the potential to reconstitute complete tumor phenotype. This is likely to be a general phenomenon and it has been demonstrated in many tumors so far. Here we show that single GBM CSCs have multilineage potential, although not exclusively. Furthermore, our results show that CSCs originating from same tumor are not necessarily uniform in respect to their differentiation potential.     

Identification of cancer stem cells from human glioblastomas: growth and differentiation capabilities and CD133/prominin-1 expression

CD133 can be a marker of tumorigenic CSCs (cancer stem cells) in human GBM (glioblastoma multiforme), although tumorigenic CD133-negative CSCs have been also isolated. Additional evidence indicates that CSCs from GBM exhibit different phenotypes, with increasing interest in the potential significance of the different CSCs with respect to diagnosis, prognosis and the development of novel targets for treatment. We have analysed the expression of CD133 in freshly isolated cells from 15 human GBM specimens. Only 4 of them contained cells positive for AC133 by FACS analysis, and all of them yielded distinct CSC lines, whereas only 6 CSC lines were obtained from the other 11 GBMs. Of these 10 CSCs lines, we further characterized 6 CSC lines. Three CSCs grew as fast-growing neurospheres with higher clonogenic ability, whereas the remaining 3 grew as slow-growing semi-adherent spheres of lower clonogenicity. In addition, the former CSC lines displayed better differentiation capabilities than the latter ones. PCR and Western blot analysis showed that all 6 GBM CSC lines expressed CD133/prominin-1, suggesting that cells negative by FACS analysis may actually represent cells expressing low levels of CD133 undetected by FACS. Nevertheless, all the 6 CSC lines were tumorigenic in nude mice. In conclusion, CSCs from human primary GBMs show different phenotypes and variable levels of CD133 expression, but these parameters did not directly correlate with the tumorigenic potential.     

Cancer stem cells and angiogenesis

Cancer stem cells (CSCs) or tumor initiating cells were identified and characterized as a unique subpopulation with stem cell features in many types of cancer. Current CSC studies provide novel insights regarding tumor initiation, progression, angiogenesis, resistance to therapy and interplay with the tumor micro-environment. A cancer stem cell niche has been proposed based on these findings. The niche provides the soil for CSC self-renewal and maintenance, stimulating essential signaling pathways in CSCs and leading to secretion of factors that promote angiogenesis and long term growth of CSCs. We present evidence which has emerged over the past 5 years indicating interaction of CSCs with angiogenesis in the proposed "vascular niche". Based on these findings, targeting the "cancer stem cell niche" by combining an individualized anti-CSC approach with treatment of their microenvironment may represent a novel therapeutic strategy against solid tumor systems.     

Apoptotic and autophagic pathways with relevant small‐molecule compounds,in cancer stem cells

Accumulating evidence demonstrates existence of cancer stem cells (CSCs), which are suspected of contributing to cancer cell self‐renewal capacity and resistance to radiation and/or chemotherapy. Including evasion of apoptosis and autophagic cell death, CSCs have revealed abilities to resist cell death, making them appealing targets for cancer therapy. Recently, molecular mechanisms of apoptosis and of autophagy in CSCs have been gradually explored, comparing them in stem cells and in cancer cells; distinct expression of these systems in CSCs may elucidate how these cells exert their capacity of unlimited self‐renewal and hierarchical differentiation. Due to their proposed ability to drive tumour initiation and progression, CSCs may be considered to be potentially useful pharmacological targets. Further, multiple compounds have been verified as triggering apoptosis and/or autophagy, suppressing tumour growth, thus providing new strategies for cancer therapy. In this review, we summarized regulation of apoptosis and autophagy in CSCs to elucidate how key proteins participate in control of survival and death; in addition, currently well‐studied compounds that target CSC apoptosis and autophagy are selectively presented. With increasing attention to CSCs in cancer therapy, researchers are now trying to find responses to unsolved questions as unambiguous as possible, which may provide novel insight into future anti‐cancer regimes.