A comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies

doi:10.1016/j.yexcr.2010.12.022

Experimental Cell Research

Volume 317, Issue 7, 15 April 2011, Pages 1049-1059

https://doi.org/10.1016/j.yexcr.2010.12.022 Get rights and content

Abstract

Sphere forming assays have been useful to enrich for stem like cells in a range of tumors. The robustness of this system contrasts the difficulties in defining a stem cell population based on cell surface markers. We have undertaken a study to describe the cellular and organizational composition of tumorspheres, directly comparing these to neurospheres derived from the adult human subventricular zone (SVZ). Primary cell cultures from brain tumors were found to contain variable fractions of cells positive for tumor stem cell markers (CD133 (2–93%)/SSEA1 (3–15%)/CXCR4 (1–72%)). All cultures produced tumors upon xenografting. Tumorspheres contained a heterogeneous population of cells, but were structurally organized with stem cell markers present at the core of spheres, with markers of more mature glial progenitors and astrocytes at more peripheral location. Ultrastructural studies showed that tumorspheres contained a higher fraction of electron dense cells in the core than the periphery (36% and 19%, respectively). Neurospheres also contained a heterogeneous cell population, but did not have an organization similar to tumorspheres. Although tumorspheres clearly display irregular and neoplastic cells, they establish an organized structure with an outward gradient of differentiation. We suggest that this organization is central in maintaining the tumor stem cell pool.

Introduction

Glioblastoma is the most common and aggressive form of brain cancer. Through the combined efforts of surgery, radiotherapy and chemotherapy only a modest increase in overall survival has been accomplished since Bailey and Cushing's first systematic follow-up of these patients [1], [2]. Clearly, better understanding of the biology of these tumors is needed. To further understand and characterize the nature of this disease, good models are sought. In vitro culturing of brain tumor biopsies under serum free conditions has been shown by us [3], [4] and others [5], [6], [7], [8] to be a robust way to expand cells in vitro. Cells cultivated under these conditions proliferate as free floating cellular spheres (tumorspheres) while maintaining functional characteristics from the tumor of origin [3], [9], [10]. These spheres can give rise to tumors upon serial transplantation, thus containing the cellular material necessary to propagate tumor growth. Recently, the ability of a given tumor to generate tumorspheres has been shown to be an important prognostic factor [11], [12]. We have previously made direct comparisons of cellular properties in cells derived from tumorspheres and sphere forming cells derived from adult human subventricular zone (SVZ) (neurospheres). Cells from these different sources share abilities in differentiation and stem cell characteristics, but many of the properties are deranged in malignant cells [3]. We also showed that sphere growth rate correlates to tumor grade, further implying the biological relevance of the sphere assay.

Several attempts have been made to identify markers that could be used to isolate or deplete the tumor stem cell population from sphere cultures. Although a range of markers has been suggested (CD133/CD15/CXCR4/A2B5), none of these markers appears to be universal, and might even be of variable importance even within one tumor [13], [14], [15]. Also, the tumor stem cell hypothesis has been questioned, raising doubt whether a cellular hierarchy and the presence of “stem like” cells within tumors exist [16]. To further elucidate the stem cell nature of cells derived from brain tumor biopsies, as well as to identify possible phenotypic targets for selective targeting of the stem-like phenotype in tumor cells, we have studied the cellular content, organization, similarities, and differences between spheres derived from the adult human subventricular zone and glioblastoma tumor biopsies.

Section snippets

Biopsies and cell culturing

Tumor biopsy specimens from twelve patients undergoing surgery for glioblastoma and three SVZ biopsies from patients undergoing temporal lobe resection for epilepsy were obtained from informed and consenting patients. The tissue harvesting was approved by the Norwegian National Committee for Medical Research Ethics (07321b). The characteristics and CD133+ fraction of seven of these cultures have been described previously [4]. Biopsies were put in ice cold Leibowitz-15 medium (L-15, Invitrogen,

Primary cultures of brain tumor stem cells had high intersample variation of tumor stem cell marker positive populations

Cell cultures were established from brain tumor biopsies, before being submitted to analysis by flow cytometry. All samples contained a phenotypically heterogeneous cell population. While some suggested markers of tumor stem cells were positive in almost all cells, other markers varied in incidence from a very low to a very high fraction (Supplementary Fig. 1). Noteworthy, the most used marker, CD133, varied from sample to sample. Despite the difference in fractions of tumor stem cell markers,

Discussion

We have shown that there is a large variation in fractions of tumor stem cell marker positive cells between primary cultures from brain tumor biopsies. Tumorspheres are organized, and cells that are electron-dense, positive for stem cell antigens, and with high nuclear intensity staining are more frequent in the core than the periphery of spheres. This organization does not seem to be present in spheres derived from human SVZ. There are, however, clear similarities in the cellular phenotype and

Acknowledgments

We would like to thank Sissel Reinlie, Head of Department of Neurosurgery, and Professor Ansgar Aasen, Director of Institute for Surgical Research, Oslo University Hospital, for excellent working conditions. We are grateful for excellent technical assistance from Elin Kampenhaug and Emily Herrera Telmo, Institute for Surgical Research, Eli Gulliksen and Kristine Haug, Center for Eye Research, Goril Flatberg and Hela Soltani, Flow Cytometry Core Facility all at Oslo University Hospital and

References (54)

R. Stupp et al.
Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial
Lancet Oncol.
(2009)
A.L. Vescovi et al.
bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells
Neuron
(1993)
J. Lee et al.
Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines
Cancer Cell
(2006)
F. Besnard et al.
Multiple interacting sites regulate astrocyte-specific transcription of the human gene for glial fibrillary acidic protein
J. Biol. Chem.
(1991)
V.G. Kukekov et al.
Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain
Exp. Neurol.
(1999)
A. Bez et al.
Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization
Brain Res.
(2003)
C. Eliasson et al.
Intermediate filament protein partnership in astrocytes
J. Biol. Chem.
(1999)
B.H. Choi et al.
Radial glia in the human fetal cerebrum: a combined Golgi, immunofluorescent and electron microscopic study
Brain Res.
(1978)
T.Y. Wang et al.
Dynamic behavior of cells within neurospheres in expanding populations of neural precursors
Brain Res.
(2006)
H. Mori et al.
Image cytometry for analyzing regional distribution of cells inside human neurospheres
J. Biosci. Bioeng.
(2007)

O.N. Suslov et al.

RT-PCR amplification of mRNA from single brain neurospheres

J. Neurosci. Meth.

(2000)

M.C. Moe et al.

A comparison of epithelial and neural properties in progenitor cells derived from the adult human ciliary body and brain

Exp. Eye Res.

(2009)

J.B. Sneddon et al.

Location, location, location: the cancer stem cell niche

Cell Stem Cell

(2007)

A.J. Bredemeyer et al.

The gastric epithelial progenitor cell niche and differentiation of the zymogenic (chief) cell lineage

Dev. Biol.

(2009)

P. Bailey et al.

A Classification of the Tumors of the Glioma Group on a Histogenetic Basis With a Correlated Study of Prognosis

(1926)

M. Varghese et al.

A comparison between stem cells from the adult human brain and from brain tumors

Neurosurgery

(2008)

E.O. Vik-Mo et al.

Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum free conditions

Neuro Oncol.

(2010)

R. Galli et al.

Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma

Cancer Res.

(2004)

H.D. Hemmati et al.

Cancerous stem cells can arise from pediatric brain tumors

Proc. Natl Acad. Sci. USA

(2003)

T.N. Ignatova et al.

Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro

Glia

(2002)

X. Yuan et al.

Isolation of cancer stem cells from adult glioblastoma multiforme

Oncogene

(2004)

D.R. Laks et al.

Neurosphere formation is an independent predictor of clinical outcome in malignant glioma

Stem Cells

(2009)

R. Pallini et al.

Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme

Clin. Cancer Res.

(2008)

D. Beier et al.

CD133(+) and CD133(−) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles

Cancer Res.

(2007)

A.T. Ogden et al.

Identification of A2B5+CD133− tumor-initiating cells in adult human gliomas

Neurosurgery

(2008)

J. Wang et al.

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

Int. J. Cancer

(2008)

E. Quintana et al.

Efficient tumour formation by single human melanoma cells

Nature

(2008)

Cited by (26)

Functional temozolomide sensitivity testing of patient-specific glioblastoma stem cell cultures is predictive of clinical outcome
2022, Translational Oncology
Citation Excerpt :
The serum-free technique enriches the cell culture for the subpopulation of glioblastoma stem cells (GSCs) [3]. The GSCs possess important qualities as a model system in GBM that includes the ability to preserve key genetic alterations of the parent tumor [4,5], to maintain a range of individual clones from the same tumor [6,7], and preserve the invasive phenotype in vivo [8,9]. Furthermore, patient-derived GSC cultures have clinical predictive relevance, as tumorsphere formation of GSCs in vitro and a GSC gene signature both are independent negative predictors of patient survival [10,11].
Serum-free culturing of patient-derived glioblastoma biopsies enrich for glioblastoma stem cells (GSCs) and is recognized as a disease-relevant model system in glioblastoma (GBM). We hypothesized that the temozolomide (TMZ) drug sensitivity of patient-derived GSC cultures correlates to clinical sensitivity patterns and has clinical predictive value in a cohort of GBM patients. To this aim, we established 51 individual GSC cultures from surgical biopsies from both treatment-naïve primary and pretreated recurrent GBM patients. The cultures were evaluated for sensitivity to TMZ over a dosing range achievable in normal clinical practice. Drug efficacy was quantified by the drug sensitivity score. MGMT-methylation status was investigated by pyrosequencing. Correlative, contingency, and survival analyses were performed for associations between experimental and clinical data. We found a heterogeneous response to temozolomide in the GSC culture cohort. There were significant differences in the sensitivity to TMZ between the newly diagnosed and the TMZ-treated recurrent disease (p <0.01). There was a moderate correlation between MGMT-status and sensitivity to TMZ (r=0.459, p=0.0009). The relationship between MGMT status and TMZ efficacy was statistically significant on multivariate analyses (p=0.0051). We found a predictive value of TMZ sensitivity in individual GSC cultures to patient survival (p=0.0089). We conclude that GSC-enriched cultures hold clinical and translational relevance by their ability to reflect the clinical heterogeneity in TMZ-sensitivity, substantiate the association between TMZ-sensitivity and MGMT-promotor methylation status and appear to have a stronger predictive value than MGMT-promotor methylation on clinical responses to TMZ.
Synergistic drug combinations for a precision medicine approach to interstitial glioblastoma therapy
2020, Journal of Controlled Release
Citation Excerpt :
Unlike single lineage cells, neurospheres contain a mix of GBM stem cells and differentiated cells [53]. This is more reflective of the composition of human GBM tumors [53], and as such is an asset for drug screening. Lastly, a non-germline genetically engineered murine cell line, TRP, was evaluated.
Glioblastoma (GBM) is a highly aggressive and heterogeneous form of brain cancer. Genotypic and phenotypic heterogeneity drives drug resistance and tumor recurrence. Combination chemotherapy could overcome drug resistance; however, GBM's location behind the blood-brain barrier severely limits chemotherapeutic options. Interstitial therapy, delivery of chemotherapy locally to the tumor site, via a biodegradable polymer implant can overcome the blood-brain barrier and increase the range of drugs available for therapy. Ideal drug candidates for interstitial therapy are those that are potent against GBM and work in combination with both standard-of-care therapy and new precision medicine targets. Herein we evaluated paclitaxel for interstitial therapy, investigating the effect of combination with both temozolomide, a clinical standard-of-care chemotherapy for GBM, and everolimus, a mammalian target of rapamycin (mTOR) inhibitor that modulates aberrant signaling present in >80% of GBM patients. Tested against a panel of GBM cell lines in vitro, paclitaxel was found to be effective at nanomolar concentrations, complement therapy with temozolomide, and synergize strongly with everolimus. The strong synergism seen with paclitaxel and everolimus was then explored in vivo. Paclitaxel and everolimus were separately formulated into fibrous scaffolds composed of acetalated dextran, a biodegradable polymer with tunable degradation rates, for implantation in the brain. Acetalated dextran degradation rates were tailored to attain matching release kinetics (~3% per day) of both paclitaxel and everolimus to maintain a fixed combination ratio of the two drugs. Combination interstitial therapy of both paclitaxel and everolimus significantly reduced GBM growth and improved progression free survival in two clinically relevant orthotopic models of GBM resection and recurrence. This work illustrates the advantages of synchronized interstitial therapy of paclitaxel and everolimus for post-surgical tumor control of GBM.
Role of the calcium toolkit in cancer stem cells
2019, Cell Calcium
Citation Excerpt :
This culture medium promotes the formation of colonies developing as spheres [24–26] and in most cases, the cells that form spheres are also the ones that are the most effective for spreading tumors in serial xenotransplantation [14,24,27,28]. Analysis of the structure and composition of glioma tumor spheres showed a concentration of CSCs in the core and an outward differentiation gradient [29]. Because CSCs form colonies, sphere assays along with limit dilution assays, which are both based on the ability of CSC to form colonies, are commonly used to detect CSCs and estimate their amounts.
Cancer stem cells are a subpopulation of tumor cells that proliferate, self-renew and produce more differentiated tumoral cells building-up the tumor. Responsible for the sustained growth of malignant tumors, cancer stem cells are proposed to play significant roles in cancer resistance to standard treatment and in tumor recurrence. Among the mechanisms dysregulated in neoplasms, those related to Ca²⁺ play significant roles in various aspects of cancers. Ca²⁺ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are tightly controlled by channels, pumps, exchangers and Ca²⁺ binding proteins. These components support the genesis of Ca²⁺ signals with specific spatio-temporal characteristics that define the cell response. Being involved in the coupling of extracellular events with intracellular responses, the Ca²⁺ toolkit is often hijacked by cancer cells to promote notably their proliferation and invasion. Growing evidence obtained during the last decade pointed to a role of Ca²⁺ handling and mishandling in cancer stem cells. In this review, after a general overview of the concept of cancer stem cells we analyse and discuss the studies and current knowledge regarding the complex roles of Ca²⁺ toolkit and signaling in these cells. We highlight that numbers of Ca²⁺ signaling actors promote cancer stem cell state and are associated with cell resistance to current cancer treatments and thus may represent promising targets for potential clinical applications.
Time-lapse phenotyping of invasive glioma cells ex vivo reveals subtype-specific movement patterns guided by tumor core signaling
2016, Experimental Cell Research
Citation Excerpt :
The first hypothesis is supported by our observation of the transition between different morphological subtypes and the localization of different cell morphology according to their localization in the rodent brain in the in vivo experiments; the majority of invading cells in the corpus callosum were Type I, whereas outside this anatomical structure, the GBM cells had predominantly heterogeneous morphology. The second hypothesis is supported by our previous report of the cellular heterogeneity within single tumorspheres [39]. In the present study we demonstrate the stability of the individualized invasive properties for any given cell culture in both in vivo and ex vivo conditions as well as the conserved proportions of the morphological subpopulations of cells observed in replicate grafts of the same culture.
The biology of glioblastoma invasion and its mechanisms are poorly understood. We demonstrate using time-lapse microscopy that grafting of glioblastoma (GBM) tumorspheres into rodent brain slices results in experimental ex vivo tumors with invasive properties that recapitulate the invasion observed after orthotopic transplantation into the rodent brain.
The migratory movements and mitotic patterns were clearly modified by signals extrinsic to the invading cells. The cells migrated away from the tumorspheres, and removal of the spheres reduced the directed invasive movement. The cell cultures contained different populations of invasive cells that had distinct morphology and invasive behavior patterns. Grafts of the most invasive GBM culture contained 91±8% cells with an invasive phenotype, characterized by small soma with a distinct leading process. Conversely, the majority of cells in less invasive GBM grafts were phenotypically heterogeneous: only 6.3±4.1% of the cells had the invasive phenotype. Grafts of highly and moderately invasive cultures had different proportions of cells that advanced into the brain slice parenchyma during the observation period: 89.2±2.2% and 23.1±6.8%, respectively. In grafts with moderately invasive properties, most of the cells (76.8±6.8%) invading the surrounding brain tissue returned to the tumor bulk or stopped centrifugal migration.
Our data suggest that the invasion of individual GBM tumors can be conditioned by the prevalence of a cell fraction with particular invasive morphology and by signaling between the tumor core and invasive cells. These findings can be important for the development of new therapeutic strategies that target the invasive GBM cells.
Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres
2014, Cancer Letters
Citation Excerpt :
A weakening of these signals in monolayer cultures may have rendered their CSCs arrhythmic. Tumorspheres derived from glioma cell cultures have a gradient of increasing differentiation from the sphere’s core outward to the surface [74], and this pattern was observed in the C6 tumorspheres grown in SM. This distribution of H33− cells agrees with reports of a gradient of increasing hypoxia towards the core, and that hypoxia promotes the stem-like state or induces de-differentiation [75].
Increased cancer risk is linked to disruption of circadian rhythms. Cancer stem cells (CSCs) are a known cause of cancer aggressiveness, but their circadian properties have not been described. We discovered circadian rhythms in gene expression within C6 glioma tumorspheres enriched in CSCs and found that the circadian clock is particularly robust in medium lacking any growth factors. A method is introduced for identifying individual CSCs in culture for single-cell analysis. CSCs in monolayer cell culture failed to show a circadian rhythm in nuclear localization of mPER2 protein, suggesting that cell interactions or the tumor-like microenvironment within tumorspheres enable circadian timing.
Gliomas: a reflection of temporal gliogenic principles
2024, Communications Biology

View all citing articles on Scopus

View full text

Research ArticleA comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies

Abstract

Introduction

Section snippets

Biopsies and cell culturing

Primary cultures of brain tumor stem cells had high intersample variation of tumor stem cell marker positive populations

Discussion

Acknowledgments

Lancet Oncol.

Neuron

Cancer Cell

J. Biol. Chem.

Exp. Neurol.

Brain Res.

J. Biol. Chem.

Brain Res.

Brain Res.

J. Biosci. Bioeng.

J. Neurosci. Meth.

Exp. Eye Res.

Cell Stem Cell

Dev. Biol.

A Classification of the Tumors of the Glioma Group on a Histogenetic Basis With a Correlated Study of Prognosis

A comparison between stem cells from the adult human brain and from brain tumors

Neurosurgery

Brain tumor stem cells maintain overall phenotype and tumorigenicity after in vitro culturing in serum free conditions

Neuro Oncol.

Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma

Cancer Res.

Cancerous stem cells can arise from pediatric brain tumors

Proc. Natl Acad. Sci. USA

Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro

Glia

Isolation of cancer stem cells from adult glioblastoma multiforme

Oncogene

Neurosphere formation is an independent predictor of clinical outcome in malignant glioma

Stem Cells

Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme

Clin. Cancer Res.

CD133(+) and CD133(−) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles

Cancer Res.

Identification of A2B5+CD133− tumor-initiating cells in adult human gliomas

Neurosurgery

CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells

Int. J. Cancer

Efficient tumour formation by single human melanoma cells

Nature

Research Article
A comparative study of the structural organization of spheres derived from the adult human subventricular zone and glioblastoma biopsies