The brain tumor microenvironment

High-grade brain tumors are heterogeneous with respect to the composition of bona fide tumors cells and with respect to a range of intermingling parenchymal cells. Glioblastomas harbor multiple cell types, some with increased tumorigenicity and stem cell-like capacity. The stem-like cells may be the cells of origin for tumor relapse. However, the tumor-associated parenchymal cells-such as vascular cells, microglia, peripheral immune cells, and neural precursor cells-also play a vital role in controlling the course of pathology. In this review, we describe the multiple interactions of bulk glioma cells and glioma stem cells with parenchymal cell populations and highlight the pathological impact and signaling pathways known for these types of cell-cell communication. The tumor-vasculature not only nourishes glioblastomas, but also provides a specialized niche for these stem-like cells. In addition, microglial cells, which can contribute up to 30% of a brain tumor mass, play a role in glioblastoma cell invasion. Moreover, non-neoplastic astrocytes can be converted into a reactive phenotype by the glioma microenvironment and can then secrete a number of factors which influences tumor biology. The young brain may have the capacity to inhibit gliomagenesis by the endogenous neural stem and progenitor cells, which secrete tumor suppressive factors. The factors, pathways, and interactions described in this review provide a new prospective on the cell biology of primary brain tumors, which may ultimately generate new treatment modalities. However, our picture of the multiple interactions between parenchymal and tumor cells is still incomplete. ? 2011 Wiley-Liss, Inc.     

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc⁺/SV40Tag⁺/Tet-on⁺) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.     

Multimodal single-cell analysis reveals distinct radioresistant stem-like and progenitor cell populations in murine glioma

Radiation therapy is part of the standard of care for gliomas and kills a subset of tumor cells, while also altering the tumor microenvironment. Tumor cells with stem-like properties preferentially survive radiation and give rise to glioma recurrence. Various techniques for enriching and quantifying cells with stem-like properties have been used, including the fluorescence activated cell sorting (FACS)-based side population (SP) assay, which is a functional assay that enriches for stem-like tumor cells. In these analyses, mouse models of glioma have been used to understand the biology of this disease and therapeutic responses, including the radiation response. We present combined SP analysis and single-cell RNA sequencing of genetically-engineered mouse models of glioma to show a time course of cellular response to radiation. We identify and characterize two distinct tumor cell populations that are inherently radioresistant and also distinct effects of radiation on immune cell populations within the tumor microenvironment.     

恶性胶质瘤中干细胞分离与细胞周期检测

目的从多形性胶质母细胞瘤组织标本中分离、培养、鉴定胶质瘤干细胞,并检测其所处的细胞周期。方法采用逐步递减培养基中血清含量的方式从多形性胶质母细胞瘤组织标本中获得悬浮生长的肿瘤球,应用免疫荧光染色检测胶质瘤干细胞及其分化细胞表面标志物的表达,免疫组化检测裸鼠颅内移植瘤表型。免疫磁珠分选多形性胶质母细胞瘤组织标本中的CD133阳性细胞后立即检测细胞周期。结果在多形性胶质母细胞瘤组织标本中成功分离出胶质瘤干细胞,在无血清培养液中呈悬浮生长,具有很强的自我更新与繁殖能力,免疫荧光染色显示该细胞表达CD133和巢蛋白,诱导分化后可分化成为神经元、星形胶质细胞与少突胶质细胞,裸鼠颅内移植后可重现亲本肿瘤表型。位于其中的胶质瘤干细胞大多处于G0～G1期。结论多形性胶质母细胞瘤组织中存在胶质瘤干细胞,相对处于静止状态。     

Aberrant signaling pathways in medulloblastomas: a stem cell connection

Medulloblastoma is a highly malignant primary tumor of the central nervous system. It represents the most frequent type of solid tumor and the leading cause of death related to cancer in early childhood. Current treatment includes surgery, chemotherapy and radiotherapy which may lead to severe cognitive impairment and secondary brain tumors. New perspectives for therapeutic development have emerged with the identification of stem-like cells displaying high tumorigenic potential and increased radio- and chemo-resistance in gliomas. Under the cancer stem cell hypothesis, transformation of neural stem cells and/or granular neuron progenitors of the cerebellum are though to be involved in medulloblastoma development. Dissecting the genetic and molecular alterations associated with this process should significantly impact both basic and applied cancer research. Based on cumulative evidences in the fields of genetics and molecular biology of medulloblastomas, we discuss the possible involvement of developmental signaling pathways as critical biochemical switches determining normal neurogenesis or tumorigenesis. From the clinical viewpoint, modulation of signaling pathways such as TGFβ, regulating neural stem cell proliferation and tumor development, might be attempted as an alternative strategy for future drug development aiming at more efficient therapies and improved clinical outcome of patients with pediatric brain cancers.     

原发和复发脑肿瘤干细胞的生物学特征观察

目的 比较原发和复发脑肿瘤干细胞的牛物学特征.方法 源于同一患者的原发和复发脑肿瘤干细胞在干细胞培养液中长期传代培养、冻存和复苏,观察其生物学特性.结果 在于细胞培养液中培养出了悬浮的干细胞球,加血清培养时能贴壁分化为多形性瘤细胞.两者都具有分化抑制特性,染色体为异倍体.肿瘤球裸小鼠原位移植,原发为局部侵袭性生长,复发为广泛浸润、播散性生长.结论 对同一患者原发和复发脑肿瘤干细胞生物学特征比较,可为进一步研究肿瘤发生、发展、侵袭和播散等拓展思路.     

干细胞样抗原提高树突状细胞介导胶质瘤免疫治疗的体外研究

目的 比较胶质瘤干细胞样抗原与非干细胞样抗原致敏树突状细胞(DCs)疫苗对胶质瘤的体外作用.方法 体外用无血清法和血清法培养恶性胶质瘤细胞,有限稀释法检测其克隆形成率;冻融法获取相应胶质瘤抗原;GM-CSF、IL-4体外诱导人外周血单核细胞获取DCs,与抗原孵育后再激活T淋巴细胞,同位素法检测效应细胞对胶质瘤细胞的杀伤率;流式细胞术检测DCs表面分子变化以及非贴壁细胞中T细胞比例的变化.结果 无血清培液中的胶质瘤细胞具有更高的克隆形成率(P<0.01);DCs经不同抗原刺激后HLA-A、HLA-DR、CD80、CD86表达上调(P<0.01);非贴壁细胞与DCs共培养后T细胞比例明显增高;干细胞样抗原激活的效应细胞对非干细胞样细胞和干细胞样细胞杀伤率分别为(70.2±5.13)%和(56.7±7.81)%(P<0.001),而非干细胞样抗原活化的效应细胞相应的杀伤率为(36.6±6.45)%和(9.05±3.49)%(P<0.001).结论 无血清培养液中的胶质瘤细胞具有更强的增殖能力,胶质瘤干细胞样抗原较传统抗原具有更强的抗原性,为进一步提高胶质瘤DCs疫苗疗效奠定基础.     

Immortalized neural stem cells differ from nonimmortalized cortical neurospheres and cerebellar granule cell progenitors

Pluripotent neural stem cells (NSCs) have been used as replacement cells in a variety of neurological disease models. Among the many different NSCs that have been used to date, most robust results have been obtained with the immortalized neural stem cell line (C17.2) isolated from postnatal cerebellum. However, it is unclear if other NSCs isolated from different brain regions are similar in their potency as replacement therapies. To assess the properties of NSC-like C17.2 cells, we compared the properties of these cells with those reported for other NSC populations identified by a variety of different investigators using biological assays, microarray analysis, RT-PCR, and immunocytochemistry. We show that C17.2 cells differ significantly from other NSCs and cerebellar granule cell precursors, from which they were derived. In particular, they secrete additional growth factors and cytokines, express markers that distinguish them from other progenitor populations, and do not maintain karyotypic stability. Our results provide a caution on extrapolating results from C17.2 to other nonimmortalized stem cell populations and provide an explanation for some of the dramatic effects that are seen with C17.2 transplants but not with other cells. We suggest that, while C17.2 cells can illustrate many fundamental aspects of neural biology and are useful in their own right, their unique properties cannot be generalized.     

Apoptosis in glioma-bearing rats after neural stem cell transplantation

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of gJioblastoma treatment by neural stem ceiJ trans- plantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in sus- pension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly in-creased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.     

Isolation, cultivation and identification of brain glioma stem cells by magnetic bead sorting

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening.Furthermore,the proliferation,differentiation and self-renewal biological features of brain glioma stem cells were identified.Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated.Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability,and expressed the stem cell-specific markers CD133 and nestin.After incubation with fetal bovine serum,the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly,indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons.Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells.These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.     

Brain tumor stem cells

The concept of brain tumor stem cells is gaining increased recognition in neuro-oncology. Until recently, the paradigm of a tumor-initiating stem cell was confined to hematopoietic malignancies where the hierarchical lineages of stem progenitor cells are well established. The demonstration of persistent stem cells and cycling progenitors in the adult brain, coupled with the expansion of the cancer stem cell concept to solid tumors, has led to the exploration of “stemness” within gliomas. Emerging data are highly suggestive of the subsistence of transformed multipotential cells within a glioma, with a subfraction of cells exhibiting increased efficiency at tumor initiation. However, data in support of true glioma stem cells are inconclusive to date, particularly with respect to functional characterization of these cells. Ongoing work aims at the identification of unique pathways governing self-renewal of these putative stem cells and at their validation as ultimate therapeutic targets.     

The pathological characteristics of glioma stem cell niches

Brain tumor stem cells (BTSC) are predicted to be critical drivers of tumor progression due to their self-renewal capacity and limitless proliferative potential. Recent studies suggest that stem cells are controlled by a particular microenvironment known as a “niche”. We therefore analysed human glioma tissues and found that the CD133⁺ and nestin⁺ niches are perivascularly localized in all glioma tissues. Furthermore, there is a positive correlation between the CD133⁺ niches and CD133⁺ blood vessels, which is similar to the correlation between the nestin⁺ niches and nestin⁺ blood vessels. We demonstrate that both CD133⁺ blood vessels and nestin⁺ blood vessels have an important role in maintaining the structure of the glioma stem cell niche. Moreover, the abundance of CD133⁺ niches and nestin⁺ niches increases significantly as tumor grade increases. These findings provide a new insight into the biology of BTSC and open a new perspective for targeted therapy against the brain tumors.     

Acute injury directs the migration, proliferation, and differentiation of solid organ stem cells: evidence from the effect of hypoxia-ischemia in the CNS on clonal "reporter" neural stem cells

Clonal neural cells with stem-like features integrate appropriately into the developing and degenerating central and peripheral nervous system throughout the neuraxis. In response to hypoxic-ischemic (HI) injury, previously engrafted, integrated, and quiescent clonal neural stem cells (NSCs) transiently re-enter the cell cycle, migrate preferentially to the site of ischemia, and differentiate into neurons and oligodendrocytes, the neural cell types typically lost following HI brain injury. They also replenish the supply of immature uncommitted resident stem/progenitor cells. Although they yield astrocytes, scarring is inhibited. These responses appear to occur most robustly within a 3-7 day "window" following HI during which signals are elaborated that upregulate genetic programs within the NSC that mediate proliferation, migration, survival, and differentiation, most of which appear to be terminated once the "window closes" and the chronic phase ensues, sending the NSCs into a quiescent state. These insights derived from using the stem cell in a novel role--as a "reporter" cell--to both track and probe the activity of endogenous stem cells as well as to "interrogate" and "report" the genes differentially induced by the acutely vs. chronically injured milieu. NSCs may be capable of the replacement of cells, genes, and non-diffusible factors in both a widespread or more circumscribed manner (depending on the therapeutic demands of the clinical situation). They may be uniquely responsive to some types of neurodegenerative conditions. We submit that these various capabilities are simply the normal expression of the basic homeostasis-preserving biologic properties and attributes of a stem cell which, if used rationally and in concert with this biology, may be exploited for therapeutic ends.     

Wnt3a mediated activation of Wnt/β-catenin signaling promotes tumor progression in glioblastoma

Presence of a distinct population of cells that drives tumor progression supports the hierarchical model of tumor development in Glioblastoma (GBM) and substantiates the cancer stem cell hypothesis. Amongst the various developmental signaling pathways that are aberrantly activated, we here show that activated Wnt/β-catenin signaling pathway plays a critical role in malignant transformation and tumor progression in gliomas. We demonstrate that Wnt ligands — Wnt1 and Wnt3a are expressed in a graded manner in these tumors as well as over-expressed in glioma stem cell-lines. A selective inhibition of Wnt signaling pathway by selective knock-down of its ligands Wnt1 and Wnt3a in glioma-derived stem-like cells led to decreased cell proliferation, cell migration and chemo-resistance. Furthermore, Wnt silencing in glioma cells reduced the capacity to form intra-cranial tumors in vivo. Taken together, our study indicates Wnt/β-catenin signaling pathway as an essential driver of glioma tumorigenesis, recognizing role of Wnt3a as an oncogene and thereby offering novel therapeutic strategies for management of these tumors.     

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson’s disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [18 F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.     

Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis

Astroglial cells support or restrict the migration and differentiation of neural stem cells depending on the developmental stage of the progenitors and the physiological state of the astrocytes. In the present study, we show that astroglial cells instruct noncommitted, immortalized neuroectodermal stem cells to adopt a neuronal fate, while they fail to induce neuronal differentiation of embryonic stem cells under similar culture conditions. Astrocytes induce neuron formation by neuroectodermal progenitors both through direct cell-to-cell contacts and via short-range acting humoral factors. Neuron formation takes place inside compact stem cell assemblies formed 30- 60 h after the onset of glial induction. Statistical analyses of time-lapse microscopic recordings show that direct contacts with astrocytes hinder the migration of neuroectodermal progenitors, while astroglia-derived humoral factors increase their motility. In non-contact co-cultures with astrocytes, altered adhesiveness prevents the separation of frequently colliding neural stem cells. By contrast, in contact co-cultures with astrocytes, the restricted migration on glial surfaces keeps the cell progenies together, resulting in the formation of clonally proliferating stem cell aggregates. The data indicate that in vitro maintained parenchymal astrocytes (1) secrete factors, which initiate neuronal differentiation of neuroectodermal stem cells; and (2) provide a cellular microenvironment where stem cell/stem cell interactions can develop and the sorting out of the future neurons can proceed. In contrast to noncommitted progenitors, postmitotic neuronal precursors leave the stem cell clusters, indicating that astroglial cells selectively support the migration of maturing neurons as well as the elongation of neurites.     

Glutamate and the biology of gliomas

Several important and previously unrecognized roles for the neurotransmitter glutamate in the biology of primary brain tumors have recently been elucidated. Glutamate is produced and released from glioma cells via the system x(c) (-) cystine glutamate transporter as a byproduct of glutathione synthesis. Glutamate appears to play a central role in the malignant phenotype of glioma via multiple mechanisms. By binding to peritumoral neuronal glutamate receptors, glutamate is responsible for seizure induction and similarly causes excitotoxicity, which aids the expansion of tumor cells into the space vacated by destroyed tissue. Glutamate also activates ionotropic and metabotropic glutamate receptors on glioma cells in a paracrine and autocrine manner. α-Amino-3-hydroxy-5-methyl-4-isoaxazolepropionate acid (AMPA) glutamate receptors lack the GluR2 subunit rendering them Ca(2+) permeable and capable of activating the AKT and MAPK pathways. Furthermore, these receptors are critical in aiding the invasion of glioma cells into normal brain. AMPA-Rs accumulate at focal adhesion sites where they may indirectly mediate interactions between the extracellular matrix and integrins. Glutamate receptor stimulation results in activation of focal adhesion kinase, which is critical to the regulation of growth factor and integrin-stimulated cell motility and invasion. The multitude of effects of glutamate on glioma biology supports the rationale for pharmacological targeting of glutamate receptors and transporters. Several ongoing and recently completed clinical trials are exploring the therapeutic potential of interrupting glutamate-mediated brain tumor growth.     

免疫磁珠法分离、培养人脑胶质瘤干细胞

目的建立免疫磁珠法分离，并培养人脑胶质瘤干细胞的方法。方法将术中取得的脑胶质瘤标本，通过剪切、消化和吹打成单细胞悬液，筛网过滤，免疫磁珠分选试剂盒分选出CD133^＋细胞，用神经干细胞无血清培养法培养出具有单细胞克隆能力的细胞球，取第3代进行诱导分化，分化前后用免疫细胞荧光化学方法鉴定肿瘤干细胞及分化后细胞。结果免疫磁珠分选出的CD133^＋细胞，可悬浮生长并形成神经干细胞样细胞球，有较强的增殖能力，干细胞标志物巢蛋白（nestin）阳性，分化后细胞表达神经元小管相关蛋白β-3（β-tubulin3）和星形胶质细胞胶质纤维酸性蛋白质（GFAP）特异性抗原，而巢蛋白、CD133^＋阴性，并具有肿瘤的核型。结论免疫磁珠分选法可避免原代培养中众多细胞混杂生长的发生，能够从大量肿瘤细胞中分离出只占极少比例的肿瘤干细胞，细胞结合磁珠后在体外可以长期培养和传代，进一步证实了肿瘤干细胞的存在，并为胶质瘤干细胞的研究奠定基础。