Cellular immortality in brain tumours: An integration of the cancer stem cell paradigm

Brain tumours are a diverse group of neoplasms that continue to present a formidable challenge in our attempt to achieve curable intervention. Our conceptual framework of human brain cancer has been redrawn in the current decade. There is a gathering acceptance that brain tumour formation is a phenotypic outcome of dysregulated neurogenesis, with tumours viewed as abnormally differentiated neural tissue. In relation, there is accumulating evidence that brain tumours, similar to leukaemia and many solid tumours, are organized as a developmental hierarchy which is maintained by a small fraction of cells endowed with many shared properties of tissue stem cells. Proof that neurogenesis persists throughout adult life, compliments this concept. Although the cancer cell of origin is unclear, the proliferative zones that harbour stem cells in the embryonic, post-natal and adult brain are attractive candidates within which tumour-initiation may ensue. Dysregulated, unlimited proliferation and an ability to bypass senescence are acquired capabilities of cancerous cells. These abilities in part require the establishment of a telomere maintenance mechanism for counteracting the shortening of chromosomal termini. A strategy based upon the synthesis of telomeric repeat sequences by the ribonucleoprotein telomerase, is prevalent in ～ 90% of human tumours studied, including the majority of brain tumours. This review will provide a developmental perspective with respect to normal (neurogenesis) and aberrant (tumourigenesis) cellular turnover, differentiation and function. Within this context our current knowledge of brain tumour telomere/telomerase biology will be discussed with respect to both its developmental and therapeutic relevance to the hierarchical model of brain tumourigenesis presented by the cancer stem cell paradigm.     

Cancer stem cells in the mammalian central nervous system

Malignant tumours intrinsic to the central nervous system (CNS) are among the most difficult of neoplasms to treat effectively. The major biological features of these tumours that preclude successful therapy include their cellular heterogeneity, which renders them highly resistant to both chemotherapy and radiotherapy, and the propensity of the component tumour cells to invade, diffusely, the contiguous nervous tissues. The tumours are classified according to perceived cell of origin, gliomas being the most common generic group. In the 1970s transplacental administration of the potent neurocarcinogen, N-ethyl-N-nitrosourea (ENU), enabled investigation of the sequential development of brain and spinal neoplasms by electron microscopy and immunohistochemistry. The significance of the primitive cells of the subependymal plate in cellular origin and evolution of a variety of glial tumours was thereby established. Since then, the development of new cell culture methods, including the in vitro growth of neurospheres and multicellular tumour spheroids, and new antigenic markers of stem cells and glial/neuronal cell precursor cells, including nestin, Mushashi-1 and CD133, have led to a reappraisal of the histological classification and origins of CNS tumours. Moreover, neural stem cells may also provide new vectors in exciting novel therapeutic strategies for these tumours. In addition to the gliomas, stem cells may have been identified in paediatric tumours including cerebellar medulloblastoma, thought to be of external granule cell neuronal derivation. Interestingly, while the stem cell marker CD133 is expressed in these primitive neuroectodermal tumours (PNETs), the chondroitin sulphate proteoglycan neuronal/glial 2 (NG2), which appears to denote increased proliferative, but reduced migratory activity in adult gliomas, is rarely expressed. This is in contrast to the situation in the histologically similar supratentorial PNETs. A possible functional 'switch' between proliferation and migration in developing neural tumour cells may exist between NG2 and ganglioside GD3. The divergent pathways of differentiation of CNS tumours and the possibility of stem cell origin, for some, if not all, such neoplasms remain a matter for debate and continued research, but the presence of self-renewing neural stem cells in the CNS of both children and adults strongly suggests a role for these cells in tumour initiation and resistance to current therapeutic strategies.     

Endothelial cell differentiation of human breast tumour stem/progenitor cells

Breast tumour stem cells have been reported to differentiate in the epithelial lineage but a cross-lineage potential has not been investigated. We aimed to evaluate whether breast tumour stem cells were able to differentiate also into the endothelial lineage. We isolated and cloned a population of breast tumour stem cells, cultured as mammospheres that expressed the stem markers nestin and Oct-4 and not epithelial and endothelial differentiation markers, and formed serially transplantable tumours in SCID mice. When cultured in the presence of serum, mammosphere-derived clones differentiated in the epithelial lineage. When cultured in the presence of VEGF, the same clones were also able to differentiate in the endothelial lineage acquiring endothelial markers and properties, such as the ability to organize in Matrigel into capillary-like structures. In the transplanted tumours, originated from mammospheres, we demonstrate that some of the intratumour vessels were of human origin, suggesting an in vivo endothelial differentiation of mammosphere-derived cells. Finally, endothelial cell clones originated from mammospheres were able, when implanted in Matrigel in SCID mice, to form after 7 days a human vessel network and, after 3–4 weeks, an epithelial tumour suggesting that in the endothelial-differentiated cells a tumourigenic stem cell population is maintained. In conclusion, the results of the present study demonstrate that stem cells of breast cancer have the ability to differentiate not only in epithelial but also in endothelial lineage, further supporting the hypothesis that the tumour-initiating population possesses stem cell characteristics relevant for tumour growth and vascularization.     

Targeting ROR1 inhibits the self‐renewal and invasive ability of glioblastoma stem cells

Glioblastoma is the most malignant of brain tumours and is difficult to cure because of interruption of drug delivery by the blood–brain barrier system, its high metastatic capacity and the existence of cancer stem cells (CSCs). Although CSCs are present as a small population in malignant tumours, CSCs have been studied as they are responsible for causing recurrence, metastasis and resistance to chemotherapy and radiotherapy for cancer. CSCs have self‐renewal characteristics like normal stem cells. The aim of this study was to investigate whether receptor tyrosine kinase‐like orphan receptor 1 (ROR1) is involved in stem cell maintenance and malignant properties in human glioblastoma. Knockdown of ROR1 caused reduction of stemness and sphere formation capacity. Moreover, down‐regulation of ROR1 suppressed the expression of epithelial‐mesenchymal transition‐related genes and the tumour migratory and invasive abilities. The results of this study indicate that targeting ROR1 can induce differentiation of CSCs and inhibit metastasis in glioblastoma. In addition, ROR1 may be used as a potential marker for glioblastoma stem cells as well as a potential target for glioblastoma stem cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

Brain tumour stem cells: implications for cancer therapy and regenerative medicine

The cancer relapse and mortality rate suggest that current therapies do not eradicate all malignant cells. Currently, it is accepted that tumorigenesis and organogenesis are similar in many respects, as for example, homeostasis is governed by a distinct sub-population of stem cells in both situations. There is increasing evidence that many types of cancer contain their own stem cells: cancer stem cells (CSC), which are characterized by their self-renewing capacity and differentiation ability. The investigation of solid tumour stem cells has gained momentum particularly in the area of brain tumours. Gliomas are the most common type of primary brain tumours. Nearly two-thirds of gliomas are highly malignant lesions with fast progression and unfortunate prognosis. Despite recent advances, two-year survival for glioblastoma (GBM) with optimal therapy is less than 30%. Even among patients with low-grade gliomas that confer a relatively good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells endowed with features of primitive neural progenitor cells and a tumour-initiating function. In general, this fraction is characterized for forming neurospheres, being endowed with drug resistance properties and often, we can isolate some of them using sorting methods with specific antibodies. The molecular characterization of these stem populations will be critical to developing an effective therapy for these tumours with very dismal prognosis. To achieve this aim, the development of a mouse model which recapitulates the nature of these tumours is essential. This review will focus on glioma stem cell knowledge and discuss future implications in brain cancer therapy and regenerative medicine.     

Characterization of brain cancer stem cells: a mathematical approach

Objective:  In recent years, support has increased for the notion that a subpopulation of brain tumour cells in possession of properties typically characteristic of stem cells is responsible for initiating and maintaining the tumour. Unravelling details of the brain tumour stem cell (BTSC) hierarchy, as well as interactions of these cells with various therapies, will be essential in the design of optimal treatment strategies.
Materials and methods:  Motivated by this, we have developed a mathematical model of the BTSC hypothesis that may aid in characterization of brain tumours, as well as in prediction of effective therapeutic strategies, which can be further validated in experimental and clinical studies. At the level of a small number of cells, the model developed herein is stochastic. For larger populations of cancer cells, the model is handled from a deterministic approach.
Results and conclusions:  In the stochastic regime, importance of a relationship between the likelihoods of two distinct types of symmetric BTSC divisions in determining BTSC survival rates becomes apparent, consequently emphasizing the need for a set of biomarkers that are able to better characterize the BTSC hierarchy. At the large scale, we predict the importance of the aforementioned symmetric division rates in dictating brain tumour composition. Furthermore, we demonstrate possible therapeutic benefits of considering combination treatments of radiotherapy and putative BTSC inhibitors, such as bone morphogenetic proteins, while reinforcing the importance of developing novel treatment strategies that specifically target the BTSC subpopulation.     

Isolation and characterization of human mammary stem cells

Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.     

Cancer stem cells: implications for the progression and treatment of metastatic disease

Metastasis is the major cause of death for cancer patients with solid tumours, due mainly to the ineffectiveness of current therapies once metastases begin to form. Further insight into the biology of metastasis is therefore essential in order to gain a greater understanding of this process and ultimately to develop better cancer therapies. Metastasis is an inefficient process, such that very few cells that leave a tumour successfully form macrometastases in distant sites. This suggests that only a small subset of cells can successfully navigate the metastatic cascade and eventually re-initiate tumour growth to form life-threatening metastases. Recently, there has been growing support for the cancer stem cell (CSC) hypothesis which stipulates that primary tumours are initiated and maintained by a small subpopulation of cancer cells that possess "stem-like" characteristics. Classical properties of normal stem cells are strikingly reminiscent of the observed experimental and clinical behaviour of metastatic cancer cells, including an unlimited capacity for self renewal; the requirement for a specific 'niche' or microenvironment to grow; use of the stromal cell-derived factor 1 (SDF-1)/chemokine receptor 4 (CXCR4) axis for migration; enhanced resistance to apoptosis and an increased capacity for drug resistance. Therefore, in addition to playing a role in primary tumour formation, we believe that CSCs are also key players in the metastatic process. We will review the current evidence supporting this idea and discuss the potential implications of the CSC hypothesis with regards to experimental investigation and treatment of metastatic disease.     

Cortical network from human embryonic stem cells

The connection of embryonic stem cell technology and developmental biology provides valuable tools to decipher the mechanisms underlying human brain development and diseases, especially among neuronal populations, that are not readily available in primary cultures. It is obviously the case of neurons forming the human cerebral cortex. In the images that are presented, the neurons were generated in vitro from human embryonic stem cells via forebrain-like progenitors. Maintained in culture for prolonged time, they acquired a mainly glutamatergic phenotype and morphological characteristics of cortical pyramidal neurons, including dendritic spines, and formed spectacular networks.     

Detection and characterization of CD133+ cancer stem cells in human solid tumours

Background

Osteosarcoma is the most common primary tumour of bone. Solid tumours are made of heterogeneous cell populations, which display different goals and roles in tumour economy. A rather small cell subset can hold or acquire stem potentials, gaining aggressiveness and increasing expectancy of recurrence. The CD133 antigen is a pentaspan membrane glycoprotein, which has been proposed as a cancer stem cell marker, since it has been previously demonstrated to be capable of identifying a cancer initiating subpopulation in brain, colon, melanoma and other solid tumours. Therefore, our aim was to observe the possible presence of cells expressing the CD133 antigen within solid tumour cell lines of osteosarcoma and, then, understand their biological characteristics and performances.

Methodology and Principal Findings

In this study, using SAOS2, MG63 and U2OS, three human sarcoma cell lines isolated from young Caucasian subjects, we were able to identify and characterize, among them, CD133⁺ cells showing the following features: high proliferation rate, cell cycle detection in a G2\M phase, positivity for Ki-67, and expression of ABCG2 transporters. In addition, at the FACS, we were able to observe the CD133⁺ cell fraction showing side population profile and forming sphere-clusters in serum-free medium with a high clonogenic efficiency.

Conclusions

Taken together, our findings lead to the thought that we can assume that we have identified, for the first time, CD133⁺ cells within osteosarcoma cell lines, showing many features of cancer stem cells. This can be of rather interest in order to design new therapies against the bone cancer.     

Role of nuclear receptors in breast cancer stem cells

The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells,capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells(CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs(BCSCs) are likely to sustain the growth of the primary tumour mass, as wellas to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and proinflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the antiinflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.     

hDlk-1: a cell surface marker common to normal hepatic stem/progenitor cells and carcinomas

Advances in stem cell biology have clarified that a tumour is a collection of heterogeneous cell populations, and that only a small fraction of tumour cells possesses the potential to self-renew. Delta-like 1 protein (Dlk-1) is a surface antigen present on foetal hepatic stem/progenitor cells but absent from mature hepatocytes in neonatal and adult rodent liver. Using a monoclonal antibody (mAb) against hDlk-1, Yanai et al. (Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J. Biochem. 2010;148:85-92) have shown that human (h) Dlk-1 is expressed in human foetal, but not adult, liver and that 20% of all hepatocellular carcinomas (HCCs) are hDlk-1(+). Importantly, an even higher percentage of HCCs in younger patients are hDLK-1(+). These authors also found that hDlk-1 is present at high frequency in colon adenocarcinomas, pancreatic islet carcinomas and small cell lung carcinomas. Here, I discuss the implications of the expression of foetal hepatic stem/progenitor cell antigens on carcinoma cells.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

IL-4-mediated drug resistance in colon cancer stem cells

Cancer stem cells are defined as cells able to both extensively self-renew and differentiate into progenitors. Cancer stem cells are thus likely to be responsible for maintaining or spreading a cancer, and may be the most relevant targets for cancer therapy. The CD133 glycoprotein was recently described as a reliable cancer stem-like cell marker in colon carcinoma. CD133+ cells are both necessary and sufficient to initiate tumour growth in animal models. The CD133+ cell population and spheroid cultures contain cells expressing the stem cell marker Musashi-1 which is involved in maintenance of stem cell fate in several tissues and importantly, this expression is maintained in stem-like cells derived from xenografted tumours. Here we discuss the potential use of the CD133 antigen in concert with Musashi-1 as markers to identify the colon cancer stem cell population. Since the up-regulation of IL-4 cytokine was recently demonstrated to constitute an important mechanism that protects the tumorigenic CD133+ cells from apoptosis, the potential benefits of standard chemotherapeutic treatments in combination with IL-4 inhibitors in the context of human colon carcinoma, are also discussed.     

The effect of tissue-specific growth patterns of target stem cells on the spectrum of tumours resulting from multistage tumorigenesis

A multistage mathematical model of tumorigenesis has been developed to explore the effects of target cell growth pattern on the proportions of tumours deriving from different tissues (the tumour spectrum). Analytical modelling techniques have shown that the effect of the target cell growth pattern on the tumour spectrum also depends on the number of stages (gene mutations) necessary for malignant change in cells of each tissue type. This suggests the existence of temporal "windows of opportunity" for tumours of different types in relation to stage number and growth kinetics. Models of this kind are applicable to cancer-prone transgenic (e.g. p53 deficient) mice, where homozygotes and heterozygotes differ in one carcinogenic stage, and differ also in the spectrum of tumours observed. Generally, tumours deriving from target stem cells which are developmentally short-lived will arise more frequently in homozygotes than heterozygotes. Such models may also be applicable to human syndromes (e.g. Li-Fraumeni) in which susceptibility to cancer is inherited.     

Neural stem cells: balancing self-renewal with differentiation

Stem cells are captivating because they have the potential to make multiple cell types yet maintain their undifferentiated state. Recent studies of Drosophila and mammalian neural stem cells have shed light on how stem cells regulate self-renewal versus differentiation and have revealed the proteins, processes and pathways that all converge to regulate neural progenitor self-renewal. If we can better understand how stem cells balance self-renewal versus differentiation, we will significantly advance our knowledge of embryogenesis, cancer biology and brain evolution, as well as the use of stem cells for therapeutic purposes.     

Spermatogonial stem cells, infertility and testicular cancer

The spermatogonial stem cells (SSCs) are responsible for the transmission of genetic information from an individual to the next generation. SSCs play critical roles in understanding the basic reproductive biology of gametes and treatments of human infertility. SSCs not only maintain normal spermatogenesis, but also sustain fertility by critically balancing both SSC self-renewal and differentiation. This self-renewal and differentiation in turn is tightly regulated by a combination of intrinsic gene expression within the SSC as well as the extrinsic gene signals from the niche. Increased SSCs self-renewal at the expense of differentiation result in germ cell tumours, on the other hand, higher differentiation at the expense of self-renewal can result in male sterility. Testicular germ cell cancers are the most frequent cancers among young men in industrialized countries. However, understanding the pathogenesis of testis cancer has been difficult because it is formed during foetal development. Recent studies suggest that SSCs can be reprogrammed to become embryonic stem (ES)-like cells to acquire pluripotency. In the present review, we summarize the recent developments in SSCs biology and role of SSC in testicular cancer. We believe that studying the biology of SSCs will not only provide better understanding of stem cell regulation in the testis, but eventually will also be a novel target for male infertility and testicular cancers.     

An adult tissue-specific stem cell molecular phenotype is activated in epithelial cancer stem cells and correlated to patient outcome

Recent studies have shown that embryonic stem cell-like molecular phenotypes are commonly activated in human epithelial primary tumors and are linked to adverse patient prognosis. However it remains unclear whether these correlations to outcome are linked to the differentiation status of the human primary tumours1 or represent molecular reminiscences of epithelial cancer stem cells. In addition, while it has been demonstrated that leukemic cancer stem cells re-acquire an embryonic stem cell-like phenotype, the molecular basis of stem cell function in epithelial cancer stem cells has not been investigated. Here we show that a normal adult tissue-specific stem cell molecular phenotype is commonly activated in epithelial cancer stem cells and for the first time provide evidence that enrichment in cancer stem cells-specific molecular signatures are correlated to highly aggressive tumor phenotypes in human epithelial cancers.