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The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids


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Screening of therapeutics relies on representative cancer models. The representation of human glioblastoma by in vitro cell culture models is questionable. We obtained genomic profiles by array comparative genomic hybridization of both short- and long-term primary cell and spheroid cultures, derived from seven glioblastomas and one anaplastic oligodendroglioma. Chromosomal copy numbers were compared between cell cultures and spheroids and related to the parental gliomas using unsupervised hierarchical clustering and correlation coefficient. In seven out of eight short-term cell cultures, the genomic profiles clustered further apart from their parental tumors than spheroid cultures. In four out of eight samples, the genetic changes in cell culture were substantial. The average correlation coefficient between parental tumors and spheroid profiles was 0.89 (range: 0.79-0.97), whereas that between parental tumors and cell cultures was 0.62 (range: 0.10-0.96). In two out of three long-term cell cultures progressive genetic changes had developed, whereas the spheroid cultures were genetically stable. It is concluded that genomic profiles of primary cell cultures from glioblastoma are frequently deviant from parental tumor profiles, whereas spheroids are genetically more representative of the glioblastoma. This implies that glioma cell culture data have to be handled with the highest caution.
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The genomic profile of human malignant glioma is altered early in primary
cell culture and preserved in spheroids
PC De Witt Hamer
, AAG Van Tilborg
, PP Eijk
, P Sminia
, D Troost
, CJF Van Noorden
B Ylstra
and S Leenstra
Department of Neurosurgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Department of
Neuropathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Section Micro Array Facility,
Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands;
Department of Radiation Oncology, VU
University Medical Center, Amsterdam, The Netherlands and
Department of Cell Biology and Histology, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Screening of therapeutics relies on representative cancer
models. The representation of human glioblastoma by
in vitro cell culture models is questionable. We obtained
genomic profiles by array comparative genomic hybridiza-
tion of both short- and long-term primary cell and spheroid
cultures, derived from seven glioblastomas and one anaplas-
tic oligodendroglioma. Chromosomal copy numbers were
compared between cell cultures and spheroids and related to
the parental gliomas using unsupervised hierarchical
clustering and correlation coefficient. In seven out of eight
short-term cell cultures, the genomic profiles clustered
further apart from their parental tumors than spheroid
cultures. In four out of eight samples, the genetic changes in
cell culture were substantial. The average correlation
coefficient between parental tumors and spheroid profiles
was 0.89 (range: 0.79–0.97), whereas that between parental
tumors and cell cultures was 0.62 (range: 0.10–0.96). In two
out of three long-term cell cultures progressive genetic
changes had developed, whereas the spheroid cultures were
genetically stable. It is concluded that genomic profiles of
primary cell cultures from glioblastoma are frequently
deviant from parental tumor profiles, whereas spheroids are
genetically more representative of the glioblastoma. This
implies that glioma cell culture data have to be handled
with the highest caution.
Oncogene (2008) 27, 2091–2096; doi:10.1038/sj.onc.1210850;
published online 15 October 2007
Keywords: glioma; primary cell culture; spheroid; com-
parative genomic hybridization; microarray
Glioblastoma has a poor survival, despite maximal
neurosurgical resection and chemoirradiation (Stupp
et al., 2005). In the development of new treatment
modalities, biologically representative cancer models are
essential. In vivo models appear to retain genetic
hallmarks of human glioblastoma (Pandita et al.,
2004). However, therapeutic screening is limited to a
low-throughput basis, whereas in vitro cancer models are
suited for high-throughput screening of novel therapeu-
tics. In vitro models include established cell line cultures,
short-term primary cell cultures and organotypic spher-
oids. Often, the in vitro sensitivity of cancer cells does not
correlate well with in vivo response. Especially, cell
cultures have been long questioned to represent glioma
biology due to changes under standard culture condi-
tions (Wolff et al., 1999; Voskoglou-Nomikos et al.,
2003; Lee et al., 2006). Therefore, short-term primary cell
cultures (with less than 10 trypsinization passages) and
particularly three-dimensional spheroid models were
considered to reflect the tumor biology better (Suther-
land, 1988; Bjerkvig et al., 1990). We hypothesized that
spheroids more closely mirror the genetics of the primary
tumors than primary cell cultures. Therefore, we
compared the genomic profiles of short-term primary
cell cultures, spheroid cultures and parental tumors of
which cultures were derived. The genetic alterations in
glioblastoma involved in gliomagenesis and progression
are well characterized (Koschny et al., 2002; Kotliarov
et al., 2006). Notably, specific alterations correlate with
response to therapy and prognosis in particular for
oligodendroglioma (Cairncross et al., 1998; Burton et al.,
2002). To compare the genetic stability of primary cell
cultures to those of spheroid cultures, copy number
abnormalities were determined by genome-wide array
comparative genomic hybridization (array CGH) (Snij-
ders et al., 2001). We found that profiles from a
substantial number of short-term primary cell cultures
had changed considerably compared to the parental
tumor and that changes were progressive over weeks,
whereas spheroid profiles were genetically stable.
Genomic profiles during short-term primary cell and
spheroid culture
As a first approach to determine similarities and
differences in genetics, unsupervised clustering analysis
was performed with 24 genomic profiles collected from
Received 10 April 2007; revised 28 August 2007; accepted 17 September
2007; published online 15 October 2007
Correspondence: Dr P De Witt Hamer, Department of Neurosurgery,
Academic Medical Center, University of Amsterdam, Room H2-238,
PO Box 22660, 1100 DD Amsterdam, The Netherlands.
Oncogene (2008) 27, 2091–2096
2008 Nature Publishing Group
All rights reserved 0950-9232/08 $30.00
eight parental tumor samples, their primary cell and
spheroid cultures (Figure 1a). Profiles derived from the
same tissue material tended to cluster together, specifi-
cally those of samples 33, 32, 153, 60 and 58. In seven
out of eight samples, the spheroid culture profile
clustered closer to the parental tumor profile than the
primary cell culture profile, with the exception of sample
58, where parental tumor, primary cell culture and
spheroid culture had nearly identical profiles. The
primary cell culture profiles of samples 3446, 160, 32
and 55 had altered substantially compared to the
parental tumor profile. Notably, the primary cell
cultures from samples 3446, 160 and 55 had reverted
genome-wide to a near diploid status. This diploidy
suggests contamination of primary cultures with normal
cells, but the glial origin of cultured cells was confirmed
by their GFAP positivity (primary antibody from Dako,
Haverlee, Belgium; data not shown), indicating that
glioma cells and not normal cells were cultured.
The copy numbers that changed in the primary cell
culture profiles included genomic regions that are
associated with therapeutic response and prognosis
(Cairncross et al., 1998; Burton et al., 2002). For instance,
1p loss as observed in the parental tumor of samples 3446
and 55 was reconstituted in the corresponding primary
cell culture profiles. Furthermore, loss of chromosome 10
as observed in the parental tumor of samples 3446, 160
and 55 was reconstituted in the primary cell culture
profiles. Amplification on 7p, the EGFR gene locus, as
observed in the parental tumor samples 33 and 160, was
lost in the short-term primary cell cultures.
Genomic profiles were in general well-preserved in
short-term spheroid cultures (Figure 1a), which is in
contrast with profiles of short-term primary cell
cultures. The average correlation coefficient between
the whole-genomic primary cell culture and parental
tumor profiles was 0.62 (range: 0.11–0.96) and between
the whole-genomic spheroid culture and parental tumor
profiles was 0.89 (range: 0.79–0.97) (Figure 1b).
Genomic changes occur progressively in primary cell
cultures, whereas spheroid cultures are genetically stable
To study the genetic stability of cell and spheroid cultures
during an expanded period of time, we compared array
CGH profiles from three patients after 2, 6 and 12 weeks
in culture with the parental tumor samples as a reference
(Figures 2a–c). Primary cell culture profiles were preserved
initially in samples 58 and 60. However, progressive
changes occurred, most markedly at 12 weeks of culture
(Figure 2d). For instance, an initial 9p loss in the parental
tumor of sample 55 became a 9p gain in primary cell
culture at 12 weeks (Figure 2a). A normal copy number of
2p and 11p in the parental tumor of sample 58 became a
2p and 11p gain in primary cell culture at 6 weeks and loss
of the entire chromosome 2 and 11p occurred at 12 weeks
(Figure 2b). A gain in chromosome 3 in the parental tumor
of sample 60 was initially preserved in primary cell culture
at 2 weeks and was lost at 12 weeks. Finally, an initial loss
of 19q in the parental tumor of sample 60 was
reconstituted at 12 weeks of primary cell culture
(Figure 2c).
The genomic profiles of spheroid cultures were stable
up to 12 weeks of culture, resembling those of their
parental tumors (Figure 2).
Array CGH confirms genetic alterations characteristic for
To validate the robustness of the array CGH technique,
known genetic alterations were identified in the parental
Figure 1 Comparison of genomic profiles of short-term primary cell and spheroid cultures derived from seven glioblastomas and one
anaplastic oligodendroglioma. The clinical, histological and culture characteristics of the samples are shown in Supplementary Table
S1. (a) Genomic profiles from the parental tumor (T, white), 2-week primary cell culture (C, yellow) and 2-week spheroid culture
(S, blue) are displayed in cluster tree order. Data were obtained using an array comparative genomic hybridization (aCGH) with
custom arrays containing 6.4K BAC clones, prepared as described (Smeets et al., 2006). Therefore, BACs were spotted in triplicate
onto CodeLink slides (Amersham Biosciences, Buckinghamshire, UK), using the OmniGrid 100 Microarrayer (Genomic Solutions,
Ann Arbor, MI, USA) equipped with SMP3 pins (TeleChem, Sunnyvale, CA, USA). DNA was extracted from samples and peripheral
blood from 18 male blood donors as reference using standard SDS-proteinase K methods and collected by ethanol precipitation.
Labeling of extracted DNA with Cy3/Cy5 and hybridization to the arrays were performed as described (Smeets et al., 2006). The array
slides were scanned with Microarray Scanner G2505B (Agilent Technologies, Palo Alto, CA, USA), and the spot intensities were
measured with BlueFuse (release 3.2; BlueGnome, Cambridge, UK; The data set was deposited at
GEO (accession GSE6042) (Barrett et al., 2005). For further analysis, spots were excluded with quality flag o1 or confidence value
o0.85. Triplicates of spot ratios (Cy3/Cy5 intensity signals) were fused within BlueFuse in a confidence weighted fashion. The resulting
transformed ratios were normalized to their mode value and shown as black dots. The gray reference lines represent 1 and þ 1
ratio values. Data are truncated at 1.4 and þ 1.4 (B0.1% of data points). The chromosomal positions of the BAC clones were
retrieved from the UCSC genome browser (h16 freeze, 07/2003, NCBI built 34; Chromosomal numbering is
printed above the data. After ordering the clones by chromosomal position, gains and losses for genomic regions were called with CGH
plotter using default settings (release 7.1.2005; moving average filtering of 5 and a default constant value of 6) (Autio et al., 2003).
Outlined in red and green are estimated copy number gains and losses, respectively, of genomic regions according to the CGH-plotter
method. The ordered whole-genome profiles with data points that retain the amplitude of genomic changes as called by CGH-plotter
were hierarchically clustered using Pearson’s uncentered correlation coefficient as metric with average linking in Cluster (release 3.0),
based on an algorithm as described (Eisen et al., 1998) and plotted in TreeView (release 1.0.12). Qualitatively similar results were
obtained when the correlation coefficients were calculated by Kendall’s t or Spearman’s r statistics (data not shown). The
chromosomes X and Y were omitted from the cluster analysis, because the intensity ratios were considered noninformative regarding
cancer-related copy number abnormalities. Nodes in the cluster tree to the right increase in similarity of genomic profiles from right to
left. (b) Bar chart comparing similarity of genomic profiles for primary cell culture and parental tumor (yellow), and spheroid culture
and parental tumor (blue) of each of the eight samples, expressed as Pearson’s correlation coefficients using the R package (release
Genomic profiles of glioblastoma in culture
PC De Witt Hamer et al
Genomic profiles of glioblastoma in culture
PC De Witt Hamer et al
glioma samples. Several specific genetic alterations have
been observed in human glioblastoma, including losses
of 9p21 (CDKN2A), 10q23 (PTEN) and 13q14 (Rb), and
gains of 1q32 (MDM4), 7p12 (EGFR), 8q24 (c-MYC)
and 12q13–15 (CDK4); and in oligodendroglioma, loss
of 1p and 19q (Koschny et al., 2002). The frequency of
involvement of copy number changes in the present
analysis with relatively few samples was consistent with
this. In our study, loss of 9p21 was observed in five out
of eight samples, loss of chromosome 10 in all samples,
four of which included apparent homozygote losses
ratioo0.8) of PTEN at 10q23 and loss of 13q14
in three out of eight samples. Furthermore, gain of 1q32
was observed in two samples, gain of chromosome 7 in
four samples, two of which were apparent EGFR
amplifications (log
ratio>3) at 7p12, gain of 8q24 in
one sample and gain of 12q13–15 in one sample.
Experimental modeling of glioblastoma
Evident advantages of two-dimensional monolayer cell
systems for experimental studies are ease of culture, low
Figure 2 Comparison of genomic profiles in time of primary cell and spheroid cultures for three glioblastoma samples. (ac) Genomic
profiles of the parental tumor, primary cell and spheroid culture ordered by culture time in weeks (w). The short-term culture profiles at
2 weeks are similar to those shown in Figure 1a, and same scaling, numbering and color coding were used. (d) Bar chart comparing the
Pearson’s correlation coefficients of genomic profiles of primary cell and spheroid cultures with the parental tumor. Numbers 2, 6 and
12 below the bars refer to the culture time in weeks.
Genomic profiles of glioblastoma in culture
PC De Witt Hamer et al
expense and prompt and reproducible results on a large
scale. Crucial, however, for studies involving therapeutic
screening is whether tumor biology is adequately
portrayed by this model system. Recently, significant
genomic rearrangements in early passages (up to p10) of
primary cell culture were reported based on single
nucleotide polymorphism analysis in one glioblastoma
sample (Lee et al., 2006). In the present study, we
demonstrate genetic discordance between the parental
tumor and derived primary cell cultures to be frequent
and to appear early, already after 2 weeks of culture.
Therefore, primary cell cultures inappropriately reflect
glioblastoma genetically. Although we provide no direct
evidence that the genetic discordance results in altered
therapeutic responses, it emphasizes the invalid repre-
sentation of tumor biology by primary cell cultures. This
was also shown by alterations in ploidy status of glioma
cell cultures at later passages (Bigner et al., 1987). In
contrast, several studies report concordant cytogenetics
in cell culture and parental tissue (Izumoto et al., 1995;
Hartmann et al., 1999). We suspect that the discrepancy
with our current study is a result of either the low
resolution of techniques such as ploidy analysis and
karyotyping or the limited number of target genes. The
organotypic spheroid model appears to be more suitable
for therapeutic screening on a large scale, since genetic
alterations are better preserved.
There are several potential explanations for the
preservation of genomic profiles in spheroids and the
progressive genetic changes in primary cell cultures.
First, subpopulations may have been selected during
primary cell culture, due to culture conditions and
technical procedures, such as trypsinization, the serum
type or the affinity for plastic. The existence of cell
subpopulations is supported by considerable genetic
heterogeneity observed within glioblastomas and glioma
cell cultures (Hartmann et al., 1999; Steilen-Gimbel
et al., 1999; Loeper et al., 2001). Second, the divergent
clonal evolution may have been accelerated during
primary cell culture, because cell proliferation is more
pronounced in monolayer cell cultures than in spheroid
cultures. Third, the clonal evolution may be more
divergent in primary cell culture, because the progenitor
cells or tumor stem cells that presumably drive the cancer
cell population were possibly lost early in primary cell
culture and maintained in spheroid culture. This
assumption is supported by preliminary data showing
stem cell marker (CD133)-positive immunostaining in
frozen sections of spheroids after long-term culture (data
not shown). However, the continuing debate on cancer
stem cell detection in glioma precludes convincing proof
of this assumption. Selection of subpopulations at
initiation of culture due to microheterogeneity in the
original tumor sample is not a very likely explanation,
because fragments of tumors used for primary cell and
spheroid cultures were carefully homogenized. The exact
nature of the genetic instability of cells in culture is not
known, but it casts doubt on the representation of
glioblastoma biology by cultured glioma cells.
In summary, the present study demonstrates genomic
profiles of primary cell cultures that are deviant from
their parental tumors within the limitations of array
CGH analysis. Characteristic genetic abnormalities are
preserved in organotypic spheroid cultures until at least
12 weeks. Therefore, spheroids are genetically a more
representative model for human glioblastoma. These
observations imply that the relevance of primary cell
cultures for therapeutic screening studies, even after
short-term culture, should be seriously questioned.
Mr RH Wessel, Mr T Krugers, Mr A Mrs
ic¸, Mrs M Tijssen
and Mrs W Tigchelaar are kindly acknowledged for their
skillful technical assistance, and the Departments of Neuro-
surgery of the Academic Medical Center from the University
of Amsterdam and of the VU University Medical Center,
for providing tumor material. We thank Mrs TMS Pierik for
her excellent assistance with preparation of the manuscript
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Supplementary Information accompanies the paper on the Oncogene website (
Genomic profiles of glioblastoma in culture
PC De Witt Hamer et al
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Glioblastoma (GBM) is the most common and dismal primary brain tumor. Unfortunately, despite multidisciplinary treatment, most patients will perish approximately 15 months after diagnosis. For this reason, there is an urgent need to improve our understanding of GBM tumor biology and develop novel therapies that can achieve better clinical outcomes. In this setting, three-dimensional tumor models have risen as more appropriate preclinical tools when compared to traditional cell cultures, given that two-dimensional (2D) cultures have failed to accurately recapitulate tumor biology and translate preclinical findings into patient benefits. Three-dimensional cultures using neurospheres, organoids, and organotypic better resemble original tumor genetic and epigenetic profiles, maintaining tumor microenvironment characteristics and mimicking cell–cell and cell–matrix interactions. This chapter summarizes our methods to generate well-characterized glioblastoma neurospheres, organoids, and organotypics.Key wordsOrganoidsOrganotypicsNeurosphereGlioblastomaStem cell
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... in vitro, the establishment of patient-derived GSC cultures in a serum-free medium promotes enrichment in stem-like cells, and has long been demonstrated as the most reliable culture procedure for retaining initial patient tumor features [96,97]. In that sense, GSC cultures from pHGG and DMG H3 K27 altered tumors were shown to be proliferative, positive for several stem cell markers, able to differentiate, and endowed with a tumorigenic potential. ...
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In children, high-grade gliomas (HGG) and diffuse midline gliomas (DMG) account for a high proportion of death due to cancer. Glioma stem cells (GSCs) are tumor cells in a specific state defined by a tumor-initiating capacity following serial transplantation, self-renewal, and an ability to recapitulate tumor heterogeneity. Their presence was demonstrated several decades ago in adult glioblastoma (GBM), and more recently in pediatric HGG and DMG. In adults, we and others have previously suggested that GSCs nest into the subventricular zone (SVZ), a neurogenic niche, where, among others, they find shelter from therapy. Both bench and bedside evidence strongly indicate a role for the GSCs and the SVZ in GBM progression, fostering the development of innovative targeting treatments. Such new therapeutic approaches are of particular interest in infants, in whom standard therapies are often limited due to the risk of late effects. The aim of this review is to describe current knowledge about GSCs in pediatric HGG and DMG, i.e., their characterization, the models that apply to their development and maintenance, the specific signaling pathways that may underlie their activity, and their specific interactions with neurogenic niches. Finally, we will discuss the clinical relevance of these observations and the therapeutic advantages of targeting the SVZ and/or the GSCs in infants.
... These 3D tumor models are found to recapitulate 3D cell-cell and cell-matrix interactions and transport properties [19][20][21], thus promotingin vivo-like tumor behavior. For example, glioma spheroids more closely recapitulate the molecular makeup of the parental tumor and present more stable molecular profiles over time compared to 2D cultures [22,23]. 3D tumor spheroids can develop a hypoxic inner core region mimicking in vivo solid tumors that contribute to more realistic drug sensitivity compared to 2D monolayers [24][25][26]. ...
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Brain tumors are the leading cause of cancer-related deaths in children. Tailored therapies need preclinical brain tumor models representing a wide range of molecular subtypes. Here, we adapted a previously established brain tissue-model to fresh patient tumor cells with the goal of establishing3D in vitro culture conditions for each tumor type.Wereported our findings from 11 pediatric tumor cases, consisting of three medulloblastoma (MB) patients, three ependymoma (EPN) patients, one glioblastoma (GBM) patient, and four juvenile pilocytic astrocytoma (Ast) patients. Chemically defined media consisting of a mixture of pro-neural and pro-endothelial cell culture medium was found to support better growth than serum-containing medium for all the tumor cases we tested. 3D scaffold alone was found to support cell heterogeneity and tumor type-dependent spheroid-forming ability; both properties were lost in 2D or gel-only control cultures. Limited in vitro models showed that the number of differentially expressed genes between in vitro vs. primary tissues, are 104 (0.6%) of medulloblastoma, 3,392 (20.2%) of ependymoma, and 576 (3.4%) of astrocytoma, out of total 16,795 protein-coding genes and lincRNAs. Two models derived from a same medulloblastoma patient clustered together with the patient-matched primary tumor tissue; both models were 3D scaffold-only in Neurobasal and EGM 1:1 (v/v) mixture and differed by a 1-mo gap in culture (i.e., 6wk versus 10wk). The genes underlying the in vitrovs. in vivo tissue differences may provide mechanistic insights into the tumor microenvironment. This study is the first step towards establishing a pipeline from patient cells to models to personalized drug testing for brain cancer.
... Organotypic multicellular spheroids (OMSs) and tissue-derived tumorspheres were both obtained from patient tumor fragments, but in the first case, they were nondissociated, and in the second case, they were partially dissociated and remodeled and compacted later (Fig. 3B). Thus, OMSs are able to maintain elements of the stroma, such as immune cells and extracellular matrix, for up to 70 days after their establishment, while tissue-derived tumorspheres are exclusively composed of tumoral cells [162,163]. ...
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In nature, cells reside in tissues subject to complex cell–cell interactions, signals from extracellular molecules and niche soluble and mechanical signaling. These microenvironment interactions are responsible for cellular phenotypes and functions, especially in normal settings. However, in 2D cultures, where interactions are limited to the horizontal plane, cells are exposed uniformly to factors or drugs; therefore, this model does not reconstitute the interactions of a natural microenvironment. 3D culture systems more closely resemble the architectural and functional properties of in vivo tissues. In these 3D cultures, the cells are exposed to different concentrations of nutrients, growth factors, oxygen or cytotoxic agents depending on their localization and communication. The 3D architecture also differentially alters the physiological, biochemical, and biomechanical properties that can affect cell growth, cell survival, differentiation and morphogenesis, cell migration and EMT properties, mechanical responses and therapy resistance. This latter point may, in part, explain the failure of current therapies and affect drug discovery research. Organoids are a promising 3D culture system between 2D cultures and in vivo models that allow the manipulation of signaling pathways and genome editing of cells in a body-like environment but lack the many disadvantages of a living system. In this review, we will focus on the role of stem cells in the establishment of organoids and the possible therapeutic applications of this model, especially in the field of cancer research.
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Glioblastoma (GBM) tumor microenvironment (TME) is a highly heterogeneous and complex system, which in addition to cancer cells, consists of various resident brain and immune cells as well as cells in transit through the tumor such as marrow derived immune cells. The TME is a dynamic environment which is heavily influenced by alterations in cellular composition, cell-to-cell contact and cellular metabolic products as well as other chemical factors, such as pH and oxygen levels. . Emerging evidence suggests that GBM cells appear to reprogram their the TME, and hijack microenvironmental elements to facilitate rapid proliferation, invasion, migration, and survival thus generating treatment resistance. GBM cells interact with their microenvironment directly through cell-to-cell by interaction mediated by cell-surface molecules, or indirectly through apocrine or paracrine signaling via cytokines, growth factors and extracellular vehicles. The recent discovery of neuron-glioma interfaces and neurotransmitter-based interactions has uncovered novel mechanisms that favor tumor cell survival and growth. Here, we review the known and emerging evidence related to the communication between GBM cells and various components of its TME, discuss models for studying the TME and outline current studies targeting components of the TME for therapeutic purposes.
Glioblastoma (GBM) remains a fatal diagnosis despite the current standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) therapy. One aspect that impedes drug development is the lack of an appropriate model representative of the complexity of patient tumors. Brain organoids derived from cell culture techniques provide a robust, easily manipulatable, and high-throughput model for GBM. In this review, we highlight recent progress in developing GBM organoids (GBOs) with a focus on generating the GBM microenvironment (i.e., stem cells, vasculature, and immune cells) recapitulating human disease. Finally, we also discuss the use of organoids as a screening tool in drug development for GBM.
Saponins represent a category of diverse, natural glycoside molecules that belong to the triterpenoid or the steroid class. They vary in terms of their solubility and permeability characteristics and are classifiable based on the biopharmaceutics classification system. They have drug delivery potential as surfactants that can solubilize cholesterol in the plasma membrane of tumorigenic cells. Glioblastoma is an important malignancy that can aggressively afflict the brain of humans with a poor prognosis. Glioblastoma Stem Cells (GSCs), are an important subset of cancer cells and are major determinants for drug resistance and tumour relapse. These cells are quiescent and have been known to survive current therapeutic strategies. Certain saponins have shown potential to eliminate glioblastoma cells in a variety of model systems and hence provide a sound scientific basis for their development as a “stand-alone” drug or as part of a drug combination (from the existing arsenal of drugs) developed for the treatment of glioblastoma. However, due to their reactogenicity towards the immune system and hemolytic potential, selective delivery to the tumorigenic site is essential. Hence, nano-formulations (liposome/emulsion-based delivery systems/nano-structured lipid and calix[n]arenes-based carriers) and variants that are resistant to saponin may serve as delivery tools that can be functionalized to improve the selectivity. It is necessary to develop/validate/refine in vitro higher order models that replicate the features of the glioma microenvironment (BBB/BTB). Reproducible validation of the model as well as the drug/delivery system will help in the development of formulations that can augment cell death in this recalcitrant brain tumour.
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Background/methods: Gliomas are common malignant neoplasms of the central nervous system. Among the major subtypes of gliomas, oligodendrogliomas are distinguished by their remarkable sensitivity to chemotherapy, with approximately two thirds of anaplastic (malignant) oligodendrogliomas responding dramatically to combination treatment with procarbazine, lomustine, and vincristine (termed PCV). Unfortunately, no clinical or pathologic feature of these tumors allows accurate prediction of their response to chemotherapy. Anaplastic oligodendrogliomas also are distinguished by a unique constellation of molecular genetic alterations, including coincident loss of chromosomal arms 1p and 19q in 50%-70% of tumors. We have hypothesized that these or other specific genetic changes might predict the response to chemotherapy and prognosis in patients with anaplastic oligodendrogliomas. Therefore, we have analyzed molecular genetic alterations involving chromosomes 1p, 10q, and 19q and the TP53 (on chromosome 17p) and CDKN2A (on chromosome 9p) genes, in addition to clinicopathologic features in 39 patients with anaplastic oligodendrogliomas for whom chemotherapeutic response and survival could be assessed. Results/conclusions: Allelic loss (or loss of heterozygosity) of chromosome 1p is a statistically significant predictor of chemosensitivity, and combined loss involving chromosomes 1p and 19q is statistically significantly associated with both chemosensitivity and longer recurrence-free survival after chemotherapy. Moreover, in both univariate and multivariate analyses, losses involving both chromosomes 1p and 19q were strongly associated with longer overall survival, whereas CDKN2A gene deletions and ring enhancement (i.e., contrast enhancement forming a rim around the tumor) on neuroimaging were associated with a significantly worse prognosis. The inverse relationship between CDKN2A gene deletions and losses of chromosomes 1p and 19q further implies that these differential clinical behaviors reflect two independent genetic subtypes of anaplastic oligodendroglioma. These results suggest that molecular genetic analysis may aid therapeutic decisions and predict outcome in patients with anaplastic oligodendrogliomas.
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The disappointing results of chemotherapy in glioblastoma might be caused by the choices of agents, which mostly include nitrosurea. We compared the in vitro efficacy of chemotherapeutic agents, developing a method to summarize published data. Between 1966 and 1995 chemotherapy in glioma cells was reported in 1643 articles. Efficacy was mostly described by the drug concentration that killed 50% of the cells (LC50). It was measured with various cell-culture techniques, of which a colorimetric test [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltrazolium bromide] was mostly used. We calculated factors from these data to transform results to LC50 values as if the colorimetric test was used in all of them. This allowed data from all publications to be summarized in a new type of meta analysis. The most important agents and the average LC50 values (mg/l) were actinomycin-D 0.042, vincristine 0.075, mitoxantrone 0.12, vinblastine 0.21, doxorubicin 0.29, diaziquone 0.76, cisplatin 1.1, methotrexate 1.1, cytasine arabinoside 1.59, 5-flurouracil 2.33, bleomycin 18.6, carboplatin 29.8, carmustine 37.0, nimustine 48.9, and lomustine 76.7. The most resistant cell was SNB56, followed with increasing sensitivity by SF128 and A172, and primary cultures P497, SF210, U87MG, SF126, 9L, P540, U251MG, HU62, C6. The complete list of original data is available upon request. The efficacy of nitrosourea in vitro is low.
Smear preparations of 23 fresh astrocytoma biopsies were analyzed by two-color fluorescence in situ hybridization with cosmids specific for the P16 and the TP53 genes. Additionally, tissue sections of the same tumors were immunostained with the use of a monoclonal antibody that recognizes both wild-type and mutant TP53 protein. In 21 astrocytomas, loss of P16 was observed in a significant proportion of cells. Cells with homozygous P16 loss were present in 13 astrocytomas; 14 astrocytomas showed cells with heterozygous loss of P16. Remarkably, 5 astrocytomas showed a scattered mosaic pattern of cells with homozygous and, respectively, heterozygous p16 loss. Homozygous deletion of TP53 was not observed. Cells with heterozygous TP53 loss were detected in 12 tumors, in 7 of them in association with P16 loss. One tumor showed aberrant cells for neither TP53 nor P16 but strong immunostaining for TP53. Positive TP53 immunostaining was found in 16 astrocytomas. Heterozygous loss of TP53 was significantly correlated with TP53 protein expression. We conclude that, unlike typical tumor suppressor genes, P16 might enhance cellular proliferation after heterozygous loss through a dosage effect and that the distribution of cells with homozygous loss of P16 speaks in favor of a polyclonal loss of the second copy of this gene.
Karyotypes of four malignant human gliomas were followed from direct preparation and/or short-term culture through their establishment in vitro to determine whether the cultured cells maintained the original karyotypes, or were the products of selection, progression, or alteration in vitro. The karyotypes of these four human glioma-derived cell lines showed the same evolutionary pattern consisting of a doubling of the stem line or a closely related population; one line changed ploidy again to near-pentaploid. Marker types seen originally were generally retained, but new markers were acquired in the later passages. We concluded that the eventual chromosomal compositions of these four human glioma-derived cell lines were the products of karyotypic evolution, rather than simple selection of a minor population of polyploid cells.
Tumor tissue from seven human gliomas was maintained in long-term agar overlay culture as multicellular organotypic spheroids. Light microscopic and ultrastructural observation of the spheroids displayed morphological features similar to those of the original tumor tissue in vivo ; in this respect they were different from spheroids obtained from permanent cell lines. The spheroids contained preserved vessels, connective tissue, and macrophages, revealing a close resemblance to the conditions in the original tumor. Flow cytometric deoxyribonucleic acid measurements of cells from the tumor spheroids and from biopsy material obtained directly from the operation revealed the same ploidy and the same amount of proliferating cells in the spheroids as in the original tumor. Fluorescence microscopy using bromodeoxyuridine (BUdR) incorporation and anti-BUdR monoclonal antibody confirmed the proliferative potential of tumor cells in the spheroids. Diameter measurements showed that the size of the spheroids from two of the tumors increased over time while in three other cases it decreased. Spheroids from the remaining two tumors showed no change in size, even after 80 days in culture. These growth data and the relatively high number of proliferating cells, as measured by flow cytometry, indicate that the degree of cell proliferation and cell loss from the spheroids are closely linked, as is the case for tumors in vivo . The culture system presented provides a valuable alternative to propagation of human tumors in animals.
Abnormal vascularization of malignant tumors is associated with the development of microregions of heterogeneous cells and environments. Experimental models such as multicell spheroids and a variety of new techniques are being used to determine the characteristics of these microregions and to study the interactions of the cells and microenvironments. The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.
The p16INK4A/MTS1 (p16) and p15INK4B/MTS2 (p15) genes map to 9p21 where genetic alterations have been frequently reported in various human tumors. Using the polymerase chain reaction (PCR), we investigated the loss of these genes on primary glioma samples and cultured glioma cells. All or any of three exons of the p16 gene were homozygously delted in 11 (35.5%) of 31 glioblastomas, none of 9 anaplastic astrocytomas and 5 astrocytomas, and in all 6 human glioma cell lines. Exon 2 of the p15 gene was homozygously deleted in 4 (12.9%) of 31 glioblastomas, but not in lower grade gliomas. It was homozygously deleted in 5 (83.3%) of 6 glioma cell lines. In 12 short-term cultures of cells derived from primary glioma samples, 5 (41.7%) and 2 (16.7%) glioblastoma-derived cells had homozygous deletion of all or any of the three exons of the p16 gene and exon 2 of the p15 gene, respectively. The deletion pattern of these genes in cultured cells was completely consistent with that seen in the primary tumors. Furthermore, two long-term cultures retained both genes that were identical to those in the original tumor tissues. Our results indicate that loss of the p16 and p15 genes may be involved in tumor progression in human gliomas, especially in the development of glioblastoma, that this loss may give growth advantage to the cells in culture, and that it is not the result of culture artifacts.
A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.