Isolation and partial characterization of a new human glioblastoma cell line

Introduction
Development of new cell lines derived from human tumors represents an important contribution in the field of human cancer biology. In contrast with the heterogeneous population inside the tumor, the cell line may offer, from genetically point of view, a homogenous material for studying the tumor biology. Moreover, this method may substitute some of the tests performed on animal model (1). However, the homo-genous characteristics of standard cell lines may become a disadvantage at some point in clinical translation process of the results.
Glioblastoma is a primary cerebral tumour with a high malignity degree. In spite of the important progresses achieved in neuroimaging, neurosurgery, radiotherapy and chemotherapy, the average surviving period in operated and irradiated glioblastoma does not usually exceed 12 months. Therefore, it is necessary new experimental therapies to be found for this lethal disease. The chemotherapeutical agents used in clinic have been tested on usual glioblastoma lines like U87, U373 and U251. Even if these glioblastoma lines have the advantages of being standardized and share similar results, their homogenous genotypic and pheno-typic features little resemble the striking heterogeneous diversity of glioblastoma samples obtained from different patients. These conflicting evidences explain the failure of standard chemotherapeutical protocols in extending the survival period in many patients with glioblastoma. In order to safely develop new therapies, researchers have to isolate and characterize new glioblastoma lines with new features regarding the expression of receptors and resistance to chemotherapeutic agents. Moreover, these new drugs should be tested in vitro on as many as possible human glioblastoma lines, to be sure that they will be effective irrespective of genetic and molecular characteristics of the patients.
In this paper the authors present the results obtained regarding the isolation, stabilization and partial characterization of a new glioblastoma cell line (named T11) which has been successfully sub-cultivated for more then 150 passage. This new line has distinct and unique characteristics when compared with standard glioblastoma cell line (e.g. U87) and may become a new and useful in vitro model for glioblastoma.

Material and Methods
Cell line establishment
The tumor sample was obtained from a 58 years-old female patient, with the preoperative written consent of the patient. The histological exam showed a grad IV glioma (glioblastoma) according with World Health Organization’s (WHO) classification. The sample was prepared by manual fragmentation into pieces up to few millimeters each, followed by enzymatic digestions using 0.125% trypsin and 20 mM EDTA. Then, the cells have been cultivated for more then 150 passages, using Dulbecco Modified Eagle’s medium (Sigma-Aldrich, USA) supplemented with 10% foetal bovine serum, 100 IU/ml penicillin, 100µg/ml streptomycin and 50 µg/ml neomycin.
Morphological studies
Cells at different passages were cultivated on glass slides inside the Petri dishes. At 48-72 hours the glass slides were washed with phosphate buffered saline (PBS), cells were treated with Bouin’s solution for 10 minutes, and colored with haematoxylin for 7 minutes and eosin 1 % for 5 minutes. The images were taken on an inverted microscope.
Growth curve
1x105 T11 cells were plated in 35 mm dishes. At every 24 hours, for 7 consecutive days, cells were tripsynized and counted. Total number of viable cells was determined using trypan blue exclusion test. The experiment was done in triplicate and the results were graphically represented as average and standard deviation (SD).
Plating efficiency (PE)
1x102 T11 cells were plated in a 6-well dish with medium change at every 4 days. U87 cells were used as positive controls. Colonies were assessed at 4 weeks after paraformaldehyde fixation and cresyl violet staining. PE (the proportion of cells that attach and grow to the number of cells originally plated, expressed as a percentage) was determined by the following formulae: PE (%) = (Colonies Counted / Cells Inoculated) x 100.
Immunocytochemistry
Cells were seeded on glass slides inside the Petri dishes. At a 70-80% confluence, slides were washed with PBS and treated with acetone and methanol for 10 minutes. In order to block the unspecific interactions, hydrogen peroxide and albumin 2 % were added. Further, cells were incubated with the primary antibody over night (mouse monoclonal antibody anti-GFP and anti-vimentin) and with he secondary antibody (rabbit antibody coupled with peroxidase) for 2 hours at room temperature. The peroxidase reaction was obtained with 3, 3’-diaminobenzidine (DAB).
For nestin and NSE analysis, T11 cells were treated, when reached 70% confluences, with PFA 4% for 10 minutes. For blocking the unspecific interactions, hydrogen peroxide and albumin 2 % were added then the cells were incubated with primary antibodies over night at 4ºC (NSE mouse monoclonal – Abcam, nestin mouse monoclonal – Abcam). Further, cells were washed with PBS and incubated with secondary antibody (rabbit antibody coupled with Alexia red - alexa 555) for 2-3 hours at room temperature. The cells were contra stained with DAPI (1 µg/ml) for 10 minutes at room temperature then they were mounted in Mowiol and examined with a confocal microscope Leica SP2.
RT PCR
Isolation of RNA was performed using TRIzol kit (Invitrogen, USA) according to the manufacturer recommendations. The quality of the extracted RNA samples was assessed by the 260/280 nm ratios. Revers transcription (RT) started from 2 mg RNA, using the kit Access Quick RT-PCR System (Promega, USA). For RT-PCR the amplification cycles and the primers are specified in the Table 1. For the semi- quantitative analysis of RT-PCR products, GAPDH was used as an internal control. Total PCR products were detected by electrophoresis on a 2% agarose gel. Additionally, cDNA was used as a template in TaqMan gene expression assays for GFAP, Nestin, hTERT (Applied Biosystems, USA) according with manufacturer protocol.
Flow cytometry
Acquisition of labeled cells was performed using a Beckman Coulter EPICS XL flow cytometer. Cells were collected, washed twice in PBS, 0.1% BSA, 0.1% sodium azide at 4°C and finally fixed in 1% paraformaldehyde. Analysis of T11 and U87 was performed using monoclonal antibodies against the following markers: CD133/2 (Miltenyi Biotec, Germany), CD45 (Coulter Immunotech, France), NCAML1 (R&D Systems, UK), anti-oligodendrocyte marker O4 (R&D Systems, UK), anti-A2B5 (R&D Systems, UK); b3-tubulin (Chemicon International, USA), Anti-human Vascular Endothelial Growth Factor (Sigma-Aldrich, USA), GFAP (Abcam, USA). 5 X 105 cells were incubated for 1 h with primary antibodies (5:100). Cells were then washed, incubated with the secondary antibody and analyzed on a Beckman Coulter flow cytometer. Ten thousand events were acquired and data were analyzed with WinMDI. Positive cells were determined as percentages of gated cells.
Western Blot
Cells lysate were performed in T-PER Tissue Protein Extractor (Thermo Scientific, USA). 10-20 µg of total protein was run on SDS-PAGE-12%. Proteins were transferred on PVDF membrane using dry transfer system: iBlot TM Gel Transfer Stacks PVDF (Invitrogen, USA). Non-specific sites were blocked with Western Breeze solution (Invitrogen, USA). Then the membrane was incubated with monoclonal antibody. The incubation with secondary antibody (IgG conjugated with alkaline phosphates) and the staining were made using Western Breeze kit (Invitrogen, USA) according with manufacturer protocol. The following markers were followed: Bax antibody (BD Pharmingen), PDGF (Abcam, USA) Nestin antibody (Abcam, USA), CD133 (Abcam, USA).
In vivo experiments
A total of 20 nude mice, 8–10-weeks-old (Crl: CD-1-Foxn1nu; Charles River Breeding Laboratories, Germany) were used in experiments. Animals were anesthetized by intraperitoneal injection of xylazine 10 mg/kg and ketamine 80 mg/kg. 10 animals received stereotactically guided injections over 3 min into forebrain (2 mm lateral and 1 mm anterior from bregma; depth 3.5 mm from dura) of 5 x 105 U87 cells in a volume of 3 µl PBS. The other 10 animals received 5 x105 T11 cells using the same parameters. All the surgical and experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee, in accordance with Romanian governmental guidelines for ethics in animal experiments.

Results
Morphological studies
The features observed during the morphological studies were: the cellular polymorphism, the characteristics of nuclear and cytoplasm coloration, the size, shape and number of the nuclei inside of T11 cells. The Figure 1 represents the morphological aspect of the glioblastoma cell line at the passages: 40, 64 and 80, observed by optic microscope. The culture exhibit a polymorphic aspect with star like cells and big-round nuclei, or fusiform cells, dendritic-like cells and even giant cells with multiple nuclei.
Growth curve
The growth curve of the glioblastoma population at the passage 50 is represented in figure 2. The doubling time of cell population was estimated at 74 hours at 37ºC. After this time the cells reached the confluence, became older and died progressively. The greatest number of viable cells was obtained between the fourth and fifth day (Fig. 2). These results suggest that T11 cell culture must be split at every 4 day (plus/minus one day).
Comparing plating efficiency between T11 and U-87
Plating efficiency is a measure of the number of colonies originating from single cells (the number of cells that grow into colonies per 100 cells inoculated), considering that cell growth in culture generally undergoes a decline after plating, and graphically, plating efficiency is the global minima (lowest point) of the growth curve at day one, after which growth rises again. So, the decrease in viable cells after plating is due to "anchorage-dependence" - cells must attach to the bottom of the culture dish. Because plating efficiency is one of the parameters typically used to define growth properties of cells in culture, we compared the number, size and morphology of T11 with U-87 (Fig. 3).

Phenotype characterization of tumor cells
T11 characteristics were assessed and compared with those of U-87 cells. We found markers specific for glial progenitors, astrocytes (glial fibrillary acidic protein - GFAP), oligo- dendrocites (A2B5; O4), microglia (CD45, CD11b) (Table 2). U-87 cells were found to be positive for A2B5 (Han J., 1994); the expression was higher (20.24%) comparing with T11 (13.97%). O4 was expressed at low level in both T11 (2.09%) and U87 cells (5.51%). Some markers tested by us, specific for neuronal lineage (b3-tubulin and NCAM) were negative.
In order to test the hypothesis which claims that neural stem cells are the source of initiation for brain cancer, we tested the presence of markers for this type of cells. Using determination by flow cytometry, we observed that T11 cells express CD133, but at low level comparing with U87 (19.56% vs. 22.56%, Table 2 and fig. 4). According with this result, mRNA CD133 had a similarly expression (data not shown). The GFAP quantification was done using flow cytometry (fig. 4) and real time PCR (fig. 5). Neural stem cells frequently express GFAP, but this marker is heavily and specifically expressed in astrocytes and certain other astroglia in the central nervous system, in satellite cells, in peripheral ganglia and in non-myelinating Schwann cells in peripheral nerves. It was surprising to see that T11 line expressed GFAP at higher levels comparing with U87 (Table 2 and fig. 5).
Nestin has been widely used as a high-specificity marker for stem/progenitor cells, glioma cells, and tumor endothelial cells in the mammalian CNS. The authors identified nestin in the cytoplasm of T11 using immunofluorescence (fig. 6A). Nestin mRNA expression was increased when compared with U-87 by relative quantification PCR. The result was confirmed by Western Blot technique (fig. 7). Neuron-specific enolase (NSE) was detected in T11 using immunofluorescence technique (fig. 6 B).

Identifying genetic prognostic markers
The level of VEGF, well known as an important angiogenic factor and as a prognosticator marker of glioma progression, was high in both T11 and U-87 (97.4 vs 87.8) (fig. 8). EFGR, another important factor in tumor progression, has been detected at low levels in RT-PCR (fig. 9). Bcl-2, a protein involved in apoptosis inhibition and protection of normal and neoplastic cells from toxicity, may cause resistance against adjuvant treatment. The Bcl-2 expression in T11 was lower in comparison with U-87 (fig. 9). Bax protein expression, a promoter of cell death, could not be detected by Western Blot. Another prognostic marker tested was MDR1, a protein involved in multidrug resistance that act as a drug efflux pump and is responsible for decreased drug accumulation in multidrug-resistant cells and often mediates the development of resistance to anticancer drugs. MDR1 expression was not detected in T11 (fig. 9). These effects are probably due to the fact that our line was initiated from a patient who has not undergone radio-/chemotherapy.
In vivo experiments
All mice injected with U87 cells developed glioblastoma xenograft. The size of the xenografts at 4 weeks (28 days) was in accordance with literature data (data not shown). No animal which received T11 cells developed glioblatoma xenografts at 2, 4, 6 or 8 weeks after injection.

Discussions
Harrison (1907) defined for the first time a method of studying the living cells isolated from the metabolic interaction of the organism; he named it cell culture (2). Starting with 1950, the researchers initiated the first cell line obtained by the dispersion of the tissue fragments (3). Depending of their life-time, the cell lines could be stabilized cell lines with an unlimited number of passages, and limited cell lines, which, after a specific number of passages, become older and die progressively. Although many cell lines are constantly initiated, their critical evaluation is very important. These cell lines are useful model systems for studying different modifications appeared in tumors, and also for assessing variable effects of new therapeutic drugs.
One part of the characterisation of tumour cell cultures involves the observation of culture morphology starting with the primary culture and continuing until the successfully establishment of the cell line. These observations are performed on live cells, in culture, using the inverted microscope and on coloured cells using different coloured agents (hematoxylin-eozin, Giemsa, May-Grunwald). In the primary culture, the cells have polymorphic aspects like: dendritic cells with abundant cytoplasm and big-round nuclei, astrocytic cells with star-like shape and round cells with hypercromic nucleus. The typical shape of the cells obtained from human glioblastoma is a star-like shape with thin filaments which expand form the body of the cell (4). The solid tumours are not homogenous from the point of cellular composition, but rather are composed of different cells. However by sub-cultivation, the process of selection isolates those cells with the most malignant features and the culture acquires a monomorphic aspect (Perzelova et al., 1998) (5). T11 showed a cellular polymorphism in the characteristics of nuclear and cytoplasm coloration, the size, shape and number of the nuclei inside the cells, which remained unchanged even at higher passage number. The culture exhibit a polymorphic aspect with star-like cells and big-round nuclei, or fusiform cells, dendritic-like cells and even giant cells with multiple nuclei.
In order to demonstrate the glial origin of the cells, we identified the presence of markers specific for glial progenitors, astrocytes (glial fibrillary acidic protein -GFAP), oligodendrocites (A2B5; O4) and microglia (CD45, CD11b). The presence of the GFAP has been considered to be an important marker which attests the glial origin (Bocchini et al., 1991; Osborne & Weber 1983) (6, 7). However a large number of human glioblastoma lines do not express this glial marker (Bigner et al., 1981) (8). It has been demonstrated that for some glioblastoma lines, the positive staining for GFAP decrease rapidly along with the prolonged in vitro cultivation (Bocchini et al., 1991) (6). Other line maintained the GFAP expression during sub-cultivation (Bigner et al., 1981; Jones et al., 1981; Di Tomaso et al., 2000) (8, 9, and 1).
Additionally, T11 cells were negative for neuronal specific lineage markers like b3-tubulin and NCAM, but positive for neuron-specific enolase by immunofluorescence technique. In normal cells from central nervous system, neuron-specific enolase (NSE) is demonstrable by immunohistochemistry in neurons and their processes. However, it has been shown that NSE may also be expressed in reactive astrocytes and in various neoplastic cells of non-neuronal origin, including those of astrocytomas and glioblastomas. In glial tumours, many of the neoplastic cells examined were positive either for GFAP protein or NSE, but usually not for both. However, it was observed that GFAP protein and NSE could be expressed simultaneously by the same cell in gliomas, but only occasional (Vinores S.A., Rubinstein L.J., 1985) (10). So, it is not surprising that we detected both proteins in T11 cells, a line initiated from a glioblastoma. On the other hand, the presence of NSE among neurofilament (NF), sustain the neuroectodermal origin of the tumour cells.
In order to assess if neural stem cells are the source of initiation for brain cancer, we tested the presence of markers for this type of cell: CD133 and nestin. T11 cell line maintained expression of nestin and CD133, which are both specific markers for glioblastoma-derived stem-like cells (11), even at high number passage (passage number 80). A variety of malignant cancers have been found to contain a subpopulation of stem cell-like tumor cells, or cancer stem cells. Recently it was demonstrated that U87 cell line contains a fraction of tumor cells that could form tumor spheres and were enriched by progressively increasing the concentration of serum-free neural stem cell medium (Yu S.C., et al., 2008) (12). This capacity was also observed in T11 cell line (data not shown).
Another marker identified in T11 cells population was CD45. This is a marker usually found in cancer stem-like cells isolated from glioblastoma samples (13) but is also characteristic for microglia cells which are the smallest of the glial cells and acts as the first and main form of active immune defence in the central nervous system.
An important role in the development of the tumour plays the growth factors like: epidermal growth factor (EGF), the receptor of epidermal growth factor (EGFR), transforming growth factor (TGF), insuline-like growth factor (IGF-I), platelet-derived growth factor (PDGF). The EGFR pathway may represent an important driver for tumor growth and tumor invasion. On the other hand, the autocrine loop of PDGF (by simultaneous expression of both PDGF and PDFR), which has been demonstrated in secondary glioblastoma, may play an important role in the development of the tumor (14). All these data underline the importance of EGFR and PDGFR expression in glioblastoma development.
Surprisingly, our results showed a lack expression for PDGFR and a very weak expression of EGFR on T11 cells. However, in the literature, conflicting data on the prognostic relevance of EGFR amplification and/or overexpression in glioblastomas have been reported. While EGFR amplification and/or overexpression has been associated with a poor prognosis in some studies (Hiesiger et al., 1993; Zhu et al., 1996; Korshunov et al., 1999) (15, 16, 17) other authors could not substantiate a prognostic significance (Quan et al., 2005) (18), or even reported an association with better prognosis (Houillier et al., 2006; Smith et al., 2001) (19, 20). It was observed that only 26% from long-term survivors revealed EGFR amplification comparing with 44% from the control group of unselected consecutive primary glioblastomas (Krex D., et al., 2007) (21). On the other hand, it was noted that glioblastoma multiforme cell lines fail to preserve epidermal growth factor receptor (EGFR) gene amplification (Pandita et al., 2004) (22) and in spite of a reproducible EGFR amplification incidence of 30% to 40% in patient with glioblastoma, there is only a single glioblastoma cell line with stable EGFR amplification and nontumorigenic effects (Filmus et al., 1985; Thomas et al., 2001) (23, 24). Alternative methods, the direct transplantation of patient surgical material into the brains of nude mice (Horten et al., 1981; Shapiro et al., 1979), (25, 26), have been more successful in maintaining the invasive features of these CNS tumors. In order to preserve tumor EGFR amplification status as well as tumor invasiveness in the orthotopic setting, a short-term cell culture step that facilitates the injection of a constant number of tumor cells and transplantation in several mice (Giannini C., et al., 2005) were combined (27). The absence of both EGFR and PDGFR in T11 cell line may explain the failure of this line to develop xenograft in nude mice. However, in order to draw a final conclusion regarding the tumorigenity of this new line, SCID mice should be used for in vivo experiments, as there are other glioblastoma lines (like U1242) which develop glioblastoma xenografts only when injected in SCID mice.
Another important finding was the virtually absent expression of MDR1 gene and PDGFR, as it has been shown at RT-PCR respectively Western Blot experiments. These results recommend T11 cell line as a good in vitro model for testing a large number of chemotherapeutical agents in order to study the mechanism of inducing of drug resistance at continuous exposure of different drugs.

Conclusion
Our new line has distinct and unique characteristics when compared with standard glioblastoma cell line (e.g. U87) and may become a new and useful in vitro model for glioblastoma.

Acknowledgements
This study was supported by national research project VIASAN-CEEX, grant agreement no.176/2006, and PNII 41_035/2007.

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