The discovery of neural stem cells (NSCs) in the mammalian brain has raised many expectations as these unique cells might recapitulate different neurological diseases, including brain tumors, both ...from a functional and molecular perspective. Proper in vitro culturing of NSCs has emerged as a critical methodological issue, given that it should preserve the in vivo features of NSCs, with particular emphasis on cell heterogeneity. At the same time, the methodology for NSC culturing should allow the production of large amounts of cells to be exploited not only for prospective clinical applications, but also for drug screening. Direct in vitro selection of NSCs and, very recently, cancer stem cells (CSCs) by means of defined serum-free conditions represents the most reliable methodology to obtain long-term expanding SC lines. Here we describe the methods currently employed to enrich for NSCs/CSCs based on the NeuroSphere Assay (NSA) and their adaptation to specific assays for testing the efficacy of neuroactive compounds.
The mammalian target of rapamycin (mTOR) pathway is a central controller of growth and homeostasis, and, as such, is implicated in disease states where growth is deregulated, namely cancer, metabolic ...diseases, and hamartoma syndromes like tuberous sclerosis complex (TSC). Accordingly, mTOR is also a pivotal regulator of the homeostasis of several distinct stem cell pools in which it finely tunes the balance between stem cell self-renewal and differentiation. mTOR hyperactivation in neural stem cells (NSCs) has been etiologically linked to the development of TSC-associated neurological lesions, such as brain hamartomas and benign tumors. Animal models generated by deletion of mTOR upstream regulators in different types of NSCs reproduce faithfully some of the TSC neurological alterations. Thus, mTOR dysregulation in NSCs seems to be responsible for the derangement of their homeostasis, thus leading to TSC development. Here we review recent advances in the molecular dissection of the mTOR cascade, its involvement in the maintenance of stem cell compartments, and in particular the implications of mTOR hyperactivation in NSCs in vivo and in vitro.
The poor transport of molecular and nanoscale agents through the blood-brain barrier together with tumour heterogeneity contribute to the dismal prognosis in patients with glioblastoma multiforme. ...Here, a biodegradable implant (μMESH) is engineered in the form of a micrometre-sized poly(lactic-co-glycolic acid) mesh laid over a water-soluble poly(vinyl alcohol) layer. Upon poly(vinyl alcohol) dissolution, the flexible poly(lactic-co-glycolic acid) mesh conforms to the resected tumour cavity as docetaxel-loaded nanomedicines and diclofenac molecules are continuously and directly released into the adjacent tumour bed. In orthotopic brain cancer models, generated with a conventional, reference cell line and patient-derived cells, a single μMESH application, carrying 0.75 mg kg
of docetaxel and diclofenac, abrogates disease recurrence up to eight months after tumour resection, with no appreciable adverse effects. Without tumour resection, the μMESH increases the median overall survival (∼30 d) as compared with the one-time intracranial deposition of docetaxel-loaded nanomedicines (15 d) or 10 cycles of systemically administered temozolomide (12 d). The μMESH modular structure, for the independent coloading of different molecules and nanomedicines, together with its mechanical flexibility, can be exploited to treat a variety of cancers, realizing patient-specific dosing and interventions.
Transformed stem cells have been isolated from some human cancers. We report that, unlike other brain cancers, the lethal glioblastoma multiforme contains neural precursors endowed with all of the ...critical features expected from neural stem cells. Similar, yet not identical, to their normal neural stem cell counterpart, these precursors emerge as unipotent (astroglial) in vivo and multipotent (neuronal-astroglial-oligodendroglial) in culture. More importantly, these cells can act as tumor-founding cells down to the clonal level and can establish tumors that closely resemble the main histologic, cytologic, and architectural features of the human disease, even when challenged through serial transplantation. Thus, cells possessing all of the characteristics expected from tumor neural stem cells seem to be involved in the growth and recurrence of adult human glioblastomas multiforme.
Purpose: Cancer stem cells (CSC) have been isolated from human tumors, including glioblastoma multiforme (GBM). The aims of this study
were the immunobiological characterization of GBM CSCs and the ...assessment of whether these cells represent suitable targets
for immunotherapy.
Experimental Design: GBM CSC lines and their fetal bovine serum (FBS)–cultured non-CSC pair lines were generated and examined by flow cytometry
for expression of known tumor antigens, MHC-I and MHC-II molecules, antigen-processing machinery components, and NKG2D ligands.
In addition, immunogenicity and immunosuppression of such cell lines for autologous or allogeneic T lymphocytes were tested
by cytokine secretion (ELISPOT) or proliferation (carboxyfluorescein diacetate succinimidyl ester) assays, respectively.
Results: Both GBM CSC and FBS lines were weakly positive and negative for MHC-I, MHC-II, and NKG2D ligand molecules, respectively.
Antigen-processing machinery molecules were also defective in both cell types. Upregulation of most molecules was induced
by IFNs or 5-Aza deoxycytidine, although more efficiently in FBS than in CSCs. Patient T-cell responses, mediated by both
TH1 and the TH2 subsets, against autologous CSC could be induced in vitro . In addition, CSC but not their paired FBS tumor lines inhibited T-cell proliferation of healthy donors. Notably, a differential
gene signature that was confirmed at the protein levels for some immunologic-related molecules was also found between CSC
and FBS lines.
Conclusions: These results indicate lower immunogenicity and higher suppressive activity of GBM CSC compared with FBS lines. The immunogenicity,
however, could be rescued by immune modulation leading to anti-GBM T cell–mediated immune response. Clin Cancer Res; 16(3);
800–13
Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by hamartomatous neurological lesions that exhibit abnormal cell proliferation and differentiation. Hyperactivation of ...mTOR pathway by mutations in either the
Tsc1 or
Tsc2 gene underlies TSC pathogenesis, but involvement of specific neural cell populations in the formation of TSC-associated neurological lesions remains unclear. We deleted
Tsc1 in
Emx1-expressing embryonic telencephalic neural stem cells (NSCs) and found that mutant mice faithfully recapitulated TSC neuropathological lesions, such as cortical lamination defects and subependymal nodules (SENs). These alterations were caused by enhanced generation of SVZ neural progeny, followed by their premature differentiation and impaired maturation during both embryonic and postnatal development. Notably, mTORC1-dependent Akt inhibition and STAT3 activation were involved in the reduced self-renewal and earlier neuronal and astroglial differentiation of mutant NSCs. Thus, finely tuned mTOR activation in embryonic NSCs may be critical to prevent development of TSC-associated brain lesions.
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Tsc1 deletion in eNSCs deregulates their self-renewal and differentiation ► mTOR hyperactivation in eNSCs disarranges postnatal neurogenesis in the SVZ and SGZ ► mTOR-dependent Akt inhibition impairs eNSC self-renewal in vitro ► Premature differentiation of eNSCs in vitro requires mTOR-dependent STAT3 activation
Bone marrow-derived cells contribute to tumor angiogenesis. Here, we demonstrate that monocytes expressing the Tie2 receptor (Tie2-expressing monocytes TEMs) (1) are a distinct hematopoietic lineage ...of proangiogenic cells, (2) are selectively recruited to spontaneous and orthotopic tumors, (3) promote angiogenesis in a paracrine manner, and (4) account for most of the proangiogenic activity of myeloid cells in tumors. Remarkably, TEM knockout completely prevented human glioma neovascularization in the mouse brain and induced substantial tumor regression. Besides TEMs and endothelial cells (ECs), Tie2 expression distinguished a rare population of tumor stroma-derived mesenchymal progenitors representing a primary source of tumor pericytes. Therefore, Tie2 expression characterizes three distinct cell types required for tumor neovascularization: ECs, proangiogenic cells of hematopoietic origin, and pericyte precursors of mesenchymal origin.
Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological ...significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFR(pos) and EGFR(neg) TIC subpopulations within the same tumor might affect clinical response to treatment.
Tuberous sclerosis complex (TSC) is a dominantly inherited disease caused by hyperactivation of the mTORC1 pathway and characterized by the development of hamartomas and benign tumors, including in ...the brain. Among the neurological manifestations associated with TSC, the tumor progression of static subependymal nodules (SENs) into subependymal giant cell astrocytomas (SEGAs) is one of the major causes of morbidity and shortened life expectancy. To date, mouse modeling has failed in reproducing these 2 lesions. Here we report that simultaneous hyperactivation of mTORC1 and Akt pathways by codeletion of Tsc1 and Pten, selectively in postnatal neural stem cells (pNSCs), is required for the formation of bona fide SENs and SEGAs. Notably, both lesions closely recapitulate the pathognomonic morphological and molecular features of the corresponding human abnormalities. The establishment of long-term expanding pNSC lines from mouse SENs and SEGAs made possible the identification of mTORC2 as one of the mediators conferring tumorigenic potential to SEGA pNSCs. Notably, in spite of concurrent Akt hyperactivation in mouse brain lesions, single mTOR inhibition by rapamycin was sufficient to strongly impair mouse SEGA growth. This study provides evidence that, concomitant with mTORC1 hyperactivation, sustained activation of Akt and mTORC2 in pNSCs is a mandatory step for the induction of SENs and SEGAs, and, at the same time, makes available an unprecedented NSC-based in vivo/in vitro model to be exploited for identifying actionable targets in TSC.
Achaete-scute homolog 1 gene (ASCL1) is a gene classifier for the proneural (PN) transcriptional subgroup of glioblastoma (GBM) that has a relevant role in the neuronal-like differentiation of GBM ...cancer stem cells (CSCs) through the activation of a PN gene signature. Besides prototypical ASCL1 PN target genes, the molecular effectors mediating ASCL1 function in regulating GBM differentiation and, most relevantly, subgroup specification are currently unknown. Here we report that ASCL1 not only promotes the acquisition of a PN phenotype in CSCs by inducing a glial-to-neuronal lineage switch but also concomitantly represses mesenchymal (MES) features by directly downregulating the expression of N-Myc downstream-regulated gene 1 (NDRG1), which we propose as a novel gene classifier of MES GBMs. Increasing the expression of ASCL1 in PN CSCs results in suppression of self-renewal, promotion of differentiation and, most significantly, decrease in tumorigenesis, which is also reproduced by NDRG1 silencing. Conversely, both abrogation of ASCL1 expression in PN CSCs and enforcement of NDRG1 expression in either PN or MES CSCs induce proneural-to-mesenchymal transition (PMT) and enhanced mesenchymal features. Surprisingly, ASCL1 overexpression in MES CSCs increases malignant features and gives rise to a neuroendocrine-like secretory phenotype. Altogether, our results propose that the fine interplay between ASCL1 and its target NDRG1 might serve as potential subgroup-specific targetable vulnerability in GBM; enhancing ASCL1 expression in PN GBMs might reduce tumorigenesis, whereas repressing NDRG1 expression might be actionable to hamper the malignancy of GBM belonging to the MES subgroup.
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