Human Cell DOI 10.1007/s13577-013-0086-3
CELL LINE
Establishment and partial characterization of a human tumor cell line, GBM-HSF, from a glioblastoma multiforme Jiagui Qu • Joshua D. Rizak • Yaodong Fan • Xiaoxuan Guo • Jiejing Li • Tanzeel Huma • Yuanye Ma
Received: 22 April 2013 / Accepted: 27 October 2013 Ó Japan Human Cell Society and Springer Japan 2014
Abstract This paper outlines the establishment of a new and stable cell line, designated GBM-HSF, from a malignant glioblastoma multiforme (GBM) removed from a 65-year-old Chinese woman. This cell line has been grown for 1 year without disruption and has been passaged over 50 times. The cells were adherently cultured in RPMI-1640 media with 10 % fetal bovine serum supplementation. Cells displayed spindle and polygonal morphology, and displayed multi-layered growth without evidence of contact inhibition. The cell line had a high growth rate with a doubling time of 51 h. The cells were able to grow without adhering to the culture plates, and 4.5 % of the total cells formed colonies in soft agar. The cell line has also been found to form tumors in nude mice and to be of a highly invasive nature. The cells were also partially characterized with RT-PCR. The RT-PCR revealed that Nestin, b-tubulin
III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed, whereas GFAP, Rex1, and CD133 were negatively transcribed in this cell line. These results suggest that the GBM-HSF cell line will provide a good model to study the properties of cancer stem cells and metastasis. It will also facilitate more detailed molecular and cellular studies of GBM cell division and pathology.
J. Qu University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China e-mail: [email protected]
X. Guo Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China
J. Qu J. D. Rizak T. Huma Y. Ma (&) State Key Laboratory of Brain and Cognitive Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, #32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, People’s Republic of China e-mail: [email protected] J. D. Rizak University of the Chinese Academy of Science, Beijing 100101, People’s Republic of China
Keywords Cell line Glioblastoma multiforme Left prefrontal lobe Gene transcription
Introduction Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans,
J. Li CAS-Max Planck Junior Scientist Group, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, People’s Republic of China T. Huma Reproductive Neuroendocrinology Laboratory, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
Y. Fan Department of Neurosurgery, The Third Affiliated Hospital of Kunming Medical University (Yunnan Province Tumor Hospital), Kunming, Yunnan 650118, People’s Republic of China
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accounting for 52 % of all functional tissue brain tumor cases and 20 % of all intracranial tumors. Despite recent advances in neurosurgery, radiation therapy, and chemotherapy to treat GBM, the prognosis of this cancer is extremely poor, with a median survival time of 12–14 months [1]. Most recent studies have suggested that the presence of cancer stem cells following treatments are the predominant cause of cancer relapse, including GBM relapse [2]. Although cancer stem cells have been studied a great deal, the mechanisms that regulate, conserve, and promote cancer stem cell survival and growth remain largely unknown. Furthermore, cancers are individualistic in nature, each arising from unique cellular lineages. This requires research to be focused on the individual characteristics of each cancer, such as those associated with GBM. One of the most prominent and best tools to study individual cancers is immortalized cell lines, because they can be stably cultured and easily handled. They also provide the means to develop animal models of the cancers through transplantation to immunodeficient animals [3]. Most current research into cancer cell growth, including GBM growth, is still dependent on the use of specific cancer cell lines. There have been a number of cell lines established from GBM, such as U251, U87, and T98G, each with their own biological characteristics. However, there is a need to develop more specific cell lines for therapeutic/anti-cancer research, due to the fact that many cancers are individualistic and the prognosis of GBM remains poor. The establishment of new GBM cell lines will facilitate these studies, and the identification of the biological characteristics of GBM cells will further lead to a better understanding of the variance between GBM growths. This paper describes the establishment of a GBM cell line derived from a malignant GBM removed from a 65-year-old Chinese patient. The cell line was characterized with respect to its morphology, kinetic parameters of cell growth, and its efficiency to form colonies in vitro. Common genes related to tumor origin and malignancy were also characterized by a measure of their transcription levels.
Materials and methods
The surgery was preformed at the First Affiliated Hospital of Kunming Medical University, Kunming, P.R. China. At the time of surgery, the patient did not present with any confounding viral or bacterial infections. Shortly after surgery, the tissue was placed in ice-cold RPMI-1640 media (Gibco, Beijing, China) supplemented with 10 % fetal bovine serum. The tissue was immediately transferred on ice to the State Key Laboratory of Brain and Cognitive Science, Kunming Institute of Zoology, Kunming, P.R. China. The tumor tissue was first washed with ice-cold PBS. Blood vessels were then removed with ophthalmic forceps, and the tissue was finely minced with ophthalmic scissors and treated with 0.25 % trypsin (Sigma, St. Louis, USA) at 37 °C for 15 min to detach individual GBM cells. Afterwards, the cells were dissociated by repeated pipetting through a Pasteur pipette and filtered by a 75-lm cell strainer to remove large clusters. The cells were centrifuged and re-suspended in RPMI-1640 medium (Gibco) supplemented with 10 % fetal bovine serum (Hyclone, Bejing, China) and then plated in a 70-mm culture dish. The cells were cultured at 37.5 °C, 100 % humidity in 95 % air and 5 % CO2. The cells were passaged every 3 days at 80–90 % confluency. The cell line was cultured for more than 50 passages and has been designated GBM-HSF after the donating patient. The cells were frozen in liquid nitrogen and have been successfully recovered several times to test their viability during the freezing process. Growth kinetics GBM-HSF cell cultures were trypsinized and 5,000 cells were plated into each well of a 24-well plate. Twenty-four well plates were cultured as above and the cultured cells were counted daily for 2 weeks with a hemocytometer (Qiujing, Shanghai, China). All measurements were performed in triplicate. The cell doubling time (DT) was determined from the logarithmic growth phase using the following equation: DTðhÞ ¼ 0:693 ðt t0 Þ= ln ðNt N0 Þ Where t0 is the time at which the exponential growth began, t is the time in hours, N0 is cell number at time t0, and Nt is the cell number at t0 [4].
Tumor tissue and cell culture Colony formation in soft agar A female patient, HSF, at age 65 years was diagnosed with GBM (grade IV) of the left prefrontal lobe. The patient and the patient’s family consented to a minimally invasive surgery to remove the tumor tissue and donated the tissue for use in the development of this immortalized cell line.
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GBM-HSF cells were suspended in RPMI-1640 medium containing 0.4 % agar (Sigma) seeded into a six-well plate pre-coated with a base of 3 ml of 0.5 % agar in RPMI-1640 medium. Cells were plated in triplicate at densities of
Establishment and partial characterization of a human tumor cell line
1 9 104/per well. Colony formation in the soft agar was counted under microscopy after 3 weeks of culture [5]. Sphere formation GBM-HSF cells were trypsinized and suspended in NSC medium (DMEM/F12 supplemented with 1 % ITS-x, 2 lg/ml Heparin, and 2 mM Glutamine) containing 20 ng/ml epidermal growth factor (EGF; Chemicon, CA, USA) or 20 ng/ml basic fibroblast growth factor (bFGF; Chemicon) or both EGF and bFGF. Cells were maintained in a 24-well plate at 37.5 °C, 100 % humidity in 95 % air and 5 % CO2. Flow cytometry-side population assay GBM-HSF cells were trypsinized and suspended at 1 9 106 cells/ml in RPMI-1640 containing 2 % FBS and 10 mM HEPES. For side population (SP) assays, cells were treated either alone with 1 lg/ml Hoechst 33342 or in combination with 100 lM verapamil (both Sigma) for 90 min at 37 °C. After incubation, cells were centrifuged and re-suspended at 2 9 106 in ice-cold HBSS supplemented with 1 lg/ml propidium iodide [6]. The cell suspensions were then analyzed with a flow cytometer (BD influxTM cell sorter, CA, USA). Data are presented as dot plots of Hoechst red (670/30 filter, x-axis) and Hoechst blue (460/50 filter, y-axis).
histological examination. Tumor slices were fixed with 95 % ethanol and stained with hematoxylin and eosin (both Sigma). After being sealed with neutral mounting medium (Jiangyuan, Jiangsu, China), the slices were observed under an upright microscope (Olympus, Tokyo, Japan). RT-PCR The mRNA transcription of 11 target genes (listed in Table 1) was analyzed with RT-PCR. Total RNA was extracted from GBM-HSF cells according to the RNA simple total RNA kit (Tiangen, Beijing, China) protocol. Reverse transcription of the RNA was performed using the M-MLV first strand kit (Invitrogen, Beijing, China). RT products were amplified by PCR, using the HotStart Taq master mix kit (Tiangen). Following a ‘‘hot start’’ at 95 °C for 3 min, samples were cycled at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s. The samples were then given a final 5 min incubation at 72 °C. Primers for RT-PCR are listed in Table 1. Products were run on 2 % agarose gels (Biowest, Spain), stained with ethidium bromide (Sangon Biotech, Shanghai, China) and visualized under UV light. GAPDH was used as a positive control.
Table 1 Primers for the RT-PCR Nestin
Forward: 50 -TTGCCTGCTACCCTTGAGAC-30 Reverse: 50 -GGGCTCTGATCTCTGCATCTAC-30
In vitro cell invasion assay GFAP 5
GBM-HSF glioma cells (1 9 10 ) were plated in the upper chamber of a Transwell (Millipore, Billerica, USA) with a membrane pre-coated with matrigel (Sigma) in 200 ll RPMI-1640 medium supplemented with 2 % N2. The transwells were then placed into the wells of a 24-well plate filled with 1.5 ml RPMI-1640 containing 10 % FBS. Cells were cultured at 37.5 °C, 100 % humidity in 95 % air and 5 % CO2 [7]. After that, the cells remaining on the top layers were removed with a 200-ll tip and the invaded cells on the underside of the membrane were fixed with 4 % paraformaldehyde and stained with hematoxylin (Sigma). Heterotransplantation into nude mice GBM-HSF cells (2 9 106) were injected subcutaneously into the flank of 6- to 8-week-old Bulb/c nude mice (n = 6, male). Growth of the tumor was monitored every 2 days by measuring the size of tumors with a ruler [5]. The mice were then sacrificed by cervical dislocation and the tumors were excised, cropped, and cut into slices (7 lm thick) with a cryostat microtome (Leica CM1850, Germany) for
Forward: 50 -CTGCGGCTCGATCAACTCA-30 Reverse: 50 -TCCAGCGACTCAATCTTCCTC-30
b-tubulin III
Forward: 50 -TCTTCTCACAAGTACGTGCCT-30 Reverse: 50 -CCCCACTCTGACCAAAGATGAA-30
Map2
Forward: 50 -GAAGGACTTGTCCGAAGCG-30
Klf4
Forward: 50 -GACCACCTCGCCTTACAC-30
Reverse: 50 -TCTCCGTTGATCCCATTCTCTT-30 Reverse: 50 -CTCAGTTGGGAACTTGACC-30 Oct4
Forward: 50 -CTTGAATCCCGAATGGAAAGGG-30 Reverse: 50 -CCTTCCCAAATAGAACCCCCA-30
Nanog
Forward: 50 -AAGGTCCCGGTCAAGAAACAG-30 Reverse: 50 -CTTCTGCGTCACACCATTGC-30
Sox2
Forward: 50 -TGGACAGTTACGCGCACAT-30 Reverse: 50 -CGAGTAGGACATGCTGTAGGT-30
Rex1
Forward: 50 -CATACGCCTGTGTTCCC-30 Reverse: 50 -GCTCTTGCTGTTATCCAGTC-30
CD133
Forward: 50 -GCGGTAAAACTGGCTAAGTA-30 Reverse: 50 -TGTGATGGGCTTGTCATAAC-30
CD26
Forward: 50 -CTACACAGCAGCGTGAATG-30 Reverse: 50 -GGCAAGATCATCTGATACCA-30
GAPDH
Forward: 50 -CAACTACATGGTTTACATGTTC-30 Reverse: 50 -GCCAGTGGACTCCACGAC-30
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Results
Colony formation
Morphology and growth kinetics
HSF cells were plated in soft agar in triplicate at 10,000 cells per well in a six-well plate. The cells formed 450 ± 12 colonies per well 21 days after being plated and incubated in the soft agar. The average ratio of colony formation contained 4.5 % of the total cells after 21 days incubation. Figure 3 displays the morphology of GBMHSF colonies on day 21.
GBM-HSF cells displayed a heterogeneous morphology at 60–70 % confluency. Most of the cells were multipolar cells with a spindle or oval morphology. A small subset of cells presented with long dendrites (Fig. 1a). When the GBM-HSF cells were grown to a very high density, the majority of the cells displayed long spindle morphologies (Fig. 1b). The high density and compacted cell shape suggested that the GBM-HSF cells did not exhibit contact inhibition during proliferation. The cell line was found to have a high growth rate with a doubling time of about 51 h (Fig. 2).
Identification of cancer stem cells GBM-HSF cells were able to form tumor spheres in NSC medium with or without growth factors, such as EGF or bFGF, in the media (Fig. 4). EGF and bFGF were found
Fig. 1 Morphology of GBM-HSF cells at a 60–70 % confluency and b high density, multi-layered growth. Bar 100 lm
Fig. 2 Growth curve of GBM-HSF cells (n = 3)
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Fig. 3 Morphology of GBM-HSF colony formed after 21 days of culture. Bar 100 lm
Establishment and partial characterization of a human tumor cell line
Fig. 4 Sphere formation of GBM-HSF cells in conditioned medium. a NSC medium; b NSC medium supplemented with EGF; c NSC medium supplemented with bFGF; d NSC medium supplemented with EGF and bFGF. Bar 100 lm
to increase the rate of growth of the cell line and the formation rate of tumor spheres (Fig. 4b–d). A side population (SP) assay was used to determine if the cell line contains the cells with stem cell potential which can efflux Hoechst 33342 dye [8]. SP cells were found to be present in the cell line, and the number of SP cells decreased dramatically in the presence of verapamil (Fig. 5). These results suggested that the cell line might contain cancer stem cells.
1-week latency period, all the mice (n = 6) transplanted with the cells developed round-shaped nodules with a diameter around 5 mm (Fig. 7a), which was indicative that the cell line had formed tumors in the nude mice. Histological evaluation of the tumors with hematoxylin and eosin found the heterotransplanted cells to be highly clustered together and to have similar morphology to astrocytic neoplasm (Fig. 7b). RT-PCR
Invasion assay and heteroplantation tumorgencity The GBM-HSF cell line was found to have a highly invasive nature in an in vitro transwell assay. Investigation of the bottom of the Matrigel-coated porous membrane found a large number of cells, which suggested that the cells have migrated through the membrane (Fig. 6). To evaluate the cell line in vivo, a small subcutaneous injection of cells was given to BALB/c nude mice. After a
RT-PCR was employed to study the transcription of genes related to tumor origin, malignancy, and metastasis. Nestin, b-tubulin III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed in the cell line, whereas no transcriptions of GFAP, Rex1, or CD133 were found HSF cells. Among the positively transcribed genes, b-tubulin III, Klf4, Oct4, Sox2, and CD26 had higher mRNA levels than Nestin, Map2, and Nanog (Fig. 8).
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Fig. 5 Detection of SP cells in GBM-HSF cell line. Cells were incubated with Hoechst 33342 alone (a) or in combination with verapamil (b). SP cells which decreased dramatically by the treatment of verapamil were outlined and shown as percentage of total 20,000 cells
Fig. 6 In vitro invasion assay of GBM-HSF cells. GBM-HSF cells were able to migrate through the matrigel coated porous membrane. The invaded cells were stained with hematoxylin and observed under a microscope. Bar 100 lm
Discussion This study reports the establishment of a novel human cell line, GBM-HSF, from the biopsy of a GBM from a 65-year-old woman as well as the characterization of the cell morphology, cell growth kinetics, colony formation efficiency, sphere formations, in vitro invasion abilities, and tumorigenicity of the cell-line. The transcription of genes related to tumor origin, malignancy, and metastasis were also investigated. The GBM-HSF cells adhered to culture plates and presented with multiple morphologies and a relatively high growth rate (DT = 51 h). The cell
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Fig. 7 Tumorigenicity of GBM-HSF cells in nude mice. a Representative photo of the growth of tumor nodule growth 1 week after heterotransplantation; b histological staining of a tumor nodule slice (7 lm) with hematoxylin and eosin. Bar 100 lm
line also had a relatively high efficiency of colony formation, where 4.5 % of the total cells formed colonies in soft agar. Furthermore, the cell line was found to have a highly invasive nature and to potentially contain cancer stem cells.
Establishment and partial characterization of a human tumor cell line
Fig. 8 Detection of embryonic and neuronal cell growth, cancer cell growth and metastasis biological markers by RT-PCR in GBM-HSF cells. Lane 1 GAPDH (181 bp), 2 Nestin (145 bp), 3 GFAP (209 bp) 4 b-tubulin III (127 bp), 5 Map2 (119 bp), 6 Klf4 (167 bp), 7 Oct4 (206 bp), 8 Nanog (237 bp), 9 Sox2 (215 bp), 10 Rex1 (123 bp), 11 CD133 (158 bp), 12 CD26 (149 bp), M marker
This GBM-HSF cell-line was also found to be capable of developing tumors in BALB/c nude mice. RT-PCR revealed that the neural stem cell-related gene Nestin [9], neuronal markers b-tubulin III and Map2 [10], embryonic stem cell-related genes Klf4, Oct4, Sox2, and Nanog [11, 12], and cancer metastasis-related gene CD26 [13] were positively transcribed in the GBM-HSF cell line. Two additional stem cell-related genes, Rex1 [14, 15] and CD133 [16–18], were found to be negatively transcribed in the cell line. Nonetheless, the cumulative results presented here suggest that the GBM-HSF cell line contains cancer stem cells and that this cell line will provide a good model to study the properties of GBM cancer stem cells and metastasis. The transcription of Nestin, b-tubulin III, and Map2 has been used as an indicator of neuroepithelial origin [19–21], which suggests that GBM-HSF cell line has a similar origin. Nestin has also been related to the malignancy of tumors, where cancers that present with Nestin positive tumors have a poor prognosis [22–26]. Klf4, Oct4, Sox2, and Nanog are common markers of stem cells [11, 12]. Positive transcription of these four genes suggested that the GBM-HSF cell line has similar properties to stem cells. These four genes play a crucial role in embryo development and construct the core transcription regulatory circuitry in embryonic stem cells [11, 12]. Recent studies also suggested that activation of these four genes correlated with malignancy in human gliomas [27]. Transcription of CD26 suggested that the GBM-HSF cells may have properties associated with metastasis, as CD26 has been found to be closely related to cancer cell metastasis [13]. In addition to these transcriptional changes that indicate cancerous properties of the GBM-HSF cell line, a battery of in vitro tests and heteroplantation experiments identified the cell line to have cancerous properties, as well as to be likely to contain a sub-population of cancer stem cells. All told, this supports the notion that the GBM-HSF cell line established here carries properties associated with the development of glioblastoma multiforme, which will allow it to be used in further investigations into the growth and treatment of these cells in vitro and in vivo.
It is also noteworthy that the astrocyte marker GFAP was negatively transcribed in the GBM-HSF cells. A previous study had suggested that GFAP expression was negatively related to the proliferation of glioma cells [28], which may suggest that there is a genetic transition sequelae from normal glia cells in brain to glioma cells which is differentially represented in the GBM-HSF cell line. This may also have coincided with the high growth rate and colony formation of the GBM-HSF cells. Although the low transcription levels of certain genes in the GBM-HSF cell line, like Rex1 and CD133, may not be fully representative of their expression in cancer stem cells due to the presence of non-stem cells in the culture, it is worth discussing the potential relationships between these stem cell markers as their interactions may provide fruitful insights in the future. Rex1 is a stem cell marker that is closely related to Nanog, Sox2, and Oct4 [14, 15]. The promotor of Rex1 is thought to be both repressed and activated by Oct4 [15]. This suggests that the high level of Oct4 transcription in the GBM-HSF cell line may have led to the negative transcription of Rex1. However, further work is required to confirm this relationship. Similarly, the glycoprotein CD133 has been used as a marker for the isolation of cancer stem cells in brain tumors, despite it not having a known function [16–18]. Nonetheless, the null transcription levels of CD133 found in the GBM-HSF cells may not directly reflect the cancer status of the GBM-HSF cell line. CD133 negative cells have been found to cause tumor growth in nude rats as well as to give rise to CD133 positive cells in subsequent studies [29]. Taken together, these findings suggest that the GBMHSF cell line will provide a new and useful tool to facilitate molecular and cellular investigations into the development of GBM and in the search for effective new therapies.
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CELL LINE
Establishment and partial characterization of a human tumor cell line, GBM-HSF, from a glioblastoma multiforme Jiagui Qu • Joshua D. Rizak • Yaodong Fan • Xiaoxuan Guo • Jiejing Li • Tanzeel Huma • Yuanye Ma
Received: 22 April 2013 / Accepted: 27 October 2013 Ó Japan Human Cell Society and Springer Japan 2014
Abstract This paper outlines the establishment of a new and stable cell line, designated GBM-HSF, from a malignant glioblastoma multiforme (GBM) removed from a 65-year-old Chinese woman. This cell line has been grown for 1 year without disruption and has been passaged over 50 times. The cells were adherently cultured in RPMI-1640 media with 10 % fetal bovine serum supplementation. Cells displayed spindle and polygonal morphology, and displayed multi-layered growth without evidence of contact inhibition. The cell line had a high growth rate with a doubling time of 51 h. The cells were able to grow without adhering to the culture plates, and 4.5 % of the total cells formed colonies in soft agar. The cell line has also been found to form tumors in nude mice and to be of a highly invasive nature. The cells were also partially characterized with RT-PCR. The RT-PCR revealed that Nestin, b-tubulin
III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed, whereas GFAP, Rex1, and CD133 were negatively transcribed in this cell line. These results suggest that the GBM-HSF cell line will provide a good model to study the properties of cancer stem cells and metastasis. It will also facilitate more detailed molecular and cellular studies of GBM cell division and pathology.
J. Qu University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China e-mail: [email protected]
X. Guo Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China
J. Qu J. D. Rizak T. Huma Y. Ma (&) State Key Laboratory of Brain and Cognitive Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, #32 Jiao Chang Dong Lu, Kunming, Yunnan 650223, People’s Republic of China e-mail: [email protected] J. D. Rizak University of the Chinese Academy of Science, Beijing 100101, People’s Republic of China
Keywords Cell line Glioblastoma multiforme Left prefrontal lobe Gene transcription
Introduction Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans,
J. Li CAS-Max Planck Junior Scientist Group, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, People’s Republic of China T. Huma Reproductive Neuroendocrinology Laboratory, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan
Y. Fan Department of Neurosurgery, The Third Affiliated Hospital of Kunming Medical University (Yunnan Province Tumor Hospital), Kunming, Yunnan 650118, People’s Republic of China
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accounting for 52 % of all functional tissue brain tumor cases and 20 % of all intracranial tumors. Despite recent advances in neurosurgery, radiation therapy, and chemotherapy to treat GBM, the prognosis of this cancer is extremely poor, with a median survival time of 12–14 months [1]. Most recent studies have suggested that the presence of cancer stem cells following treatments are the predominant cause of cancer relapse, including GBM relapse [2]. Although cancer stem cells have been studied a great deal, the mechanisms that regulate, conserve, and promote cancer stem cell survival and growth remain largely unknown. Furthermore, cancers are individualistic in nature, each arising from unique cellular lineages. This requires research to be focused on the individual characteristics of each cancer, such as those associated with GBM. One of the most prominent and best tools to study individual cancers is immortalized cell lines, because they can be stably cultured and easily handled. They also provide the means to develop animal models of the cancers through transplantation to immunodeficient animals [3]. Most current research into cancer cell growth, including GBM growth, is still dependent on the use of specific cancer cell lines. There have been a number of cell lines established from GBM, such as U251, U87, and T98G, each with their own biological characteristics. However, there is a need to develop more specific cell lines for therapeutic/anti-cancer research, due to the fact that many cancers are individualistic and the prognosis of GBM remains poor. The establishment of new GBM cell lines will facilitate these studies, and the identification of the biological characteristics of GBM cells will further lead to a better understanding of the variance between GBM growths. This paper describes the establishment of a GBM cell line derived from a malignant GBM removed from a 65-year-old Chinese patient. The cell line was characterized with respect to its morphology, kinetic parameters of cell growth, and its efficiency to form colonies in vitro. Common genes related to tumor origin and malignancy were also characterized by a measure of their transcription levels.
Materials and methods
The surgery was preformed at the First Affiliated Hospital of Kunming Medical University, Kunming, P.R. China. At the time of surgery, the patient did not present with any confounding viral or bacterial infections. Shortly after surgery, the tissue was placed in ice-cold RPMI-1640 media (Gibco, Beijing, China) supplemented with 10 % fetal bovine serum. The tissue was immediately transferred on ice to the State Key Laboratory of Brain and Cognitive Science, Kunming Institute of Zoology, Kunming, P.R. China. The tumor tissue was first washed with ice-cold PBS. Blood vessels were then removed with ophthalmic forceps, and the tissue was finely minced with ophthalmic scissors and treated with 0.25 % trypsin (Sigma, St. Louis, USA) at 37 °C for 15 min to detach individual GBM cells. Afterwards, the cells were dissociated by repeated pipetting through a Pasteur pipette and filtered by a 75-lm cell strainer to remove large clusters. The cells were centrifuged and re-suspended in RPMI-1640 medium (Gibco) supplemented with 10 % fetal bovine serum (Hyclone, Bejing, China) and then plated in a 70-mm culture dish. The cells were cultured at 37.5 °C, 100 % humidity in 95 % air and 5 % CO2. The cells were passaged every 3 days at 80–90 % confluency. The cell line was cultured for more than 50 passages and has been designated GBM-HSF after the donating patient. The cells were frozen in liquid nitrogen and have been successfully recovered several times to test their viability during the freezing process. Growth kinetics GBM-HSF cell cultures were trypsinized and 5,000 cells were plated into each well of a 24-well plate. Twenty-four well plates were cultured as above and the cultured cells were counted daily for 2 weeks with a hemocytometer (Qiujing, Shanghai, China). All measurements were performed in triplicate. The cell doubling time (DT) was determined from the logarithmic growth phase using the following equation: DTðhÞ ¼ 0:693 ðt t0 Þ= ln ðNt N0 Þ Where t0 is the time at which the exponential growth began, t is the time in hours, N0 is cell number at time t0, and Nt is the cell number at t0 [4].
Tumor tissue and cell culture Colony formation in soft agar A female patient, HSF, at age 65 years was diagnosed with GBM (grade IV) of the left prefrontal lobe. The patient and the patient’s family consented to a minimally invasive surgery to remove the tumor tissue and donated the tissue for use in the development of this immortalized cell line.
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GBM-HSF cells were suspended in RPMI-1640 medium containing 0.4 % agar (Sigma) seeded into a six-well plate pre-coated with a base of 3 ml of 0.5 % agar in RPMI-1640 medium. Cells were plated in triplicate at densities of
Establishment and partial characterization of a human tumor cell line
1 9 104/per well. Colony formation in the soft agar was counted under microscopy after 3 weeks of culture [5]. Sphere formation GBM-HSF cells were trypsinized and suspended in NSC medium (DMEM/F12 supplemented with 1 % ITS-x, 2 lg/ml Heparin, and 2 mM Glutamine) containing 20 ng/ml epidermal growth factor (EGF; Chemicon, CA, USA) or 20 ng/ml basic fibroblast growth factor (bFGF; Chemicon) or both EGF and bFGF. Cells were maintained in a 24-well plate at 37.5 °C, 100 % humidity in 95 % air and 5 % CO2. Flow cytometry-side population assay GBM-HSF cells were trypsinized and suspended at 1 9 106 cells/ml in RPMI-1640 containing 2 % FBS and 10 mM HEPES. For side population (SP) assays, cells were treated either alone with 1 lg/ml Hoechst 33342 or in combination with 100 lM verapamil (both Sigma) for 90 min at 37 °C. After incubation, cells were centrifuged and re-suspended at 2 9 106 in ice-cold HBSS supplemented with 1 lg/ml propidium iodide [6]. The cell suspensions were then analyzed with a flow cytometer (BD influxTM cell sorter, CA, USA). Data are presented as dot plots of Hoechst red (670/30 filter, x-axis) and Hoechst blue (460/50 filter, y-axis).
histological examination. Tumor slices were fixed with 95 % ethanol and stained with hematoxylin and eosin (both Sigma). After being sealed with neutral mounting medium (Jiangyuan, Jiangsu, China), the slices were observed under an upright microscope (Olympus, Tokyo, Japan). RT-PCR The mRNA transcription of 11 target genes (listed in Table 1) was analyzed with RT-PCR. Total RNA was extracted from GBM-HSF cells according to the RNA simple total RNA kit (Tiangen, Beijing, China) protocol. Reverse transcription of the RNA was performed using the M-MLV first strand kit (Invitrogen, Beijing, China). RT products were amplified by PCR, using the HotStart Taq master mix kit (Tiangen). Following a ‘‘hot start’’ at 95 °C for 3 min, samples were cycled at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s. The samples were then given a final 5 min incubation at 72 °C. Primers for RT-PCR are listed in Table 1. Products were run on 2 % agarose gels (Biowest, Spain), stained with ethidium bromide (Sangon Biotech, Shanghai, China) and visualized under UV light. GAPDH was used as a positive control.
Table 1 Primers for the RT-PCR Nestin
Forward: 50 -TTGCCTGCTACCCTTGAGAC-30 Reverse: 50 -GGGCTCTGATCTCTGCATCTAC-30
In vitro cell invasion assay GFAP 5
GBM-HSF glioma cells (1 9 10 ) were plated in the upper chamber of a Transwell (Millipore, Billerica, USA) with a membrane pre-coated with matrigel (Sigma) in 200 ll RPMI-1640 medium supplemented with 2 % N2. The transwells were then placed into the wells of a 24-well plate filled with 1.5 ml RPMI-1640 containing 10 % FBS. Cells were cultured at 37.5 °C, 100 % humidity in 95 % air and 5 % CO2 [7]. After that, the cells remaining on the top layers were removed with a 200-ll tip and the invaded cells on the underside of the membrane were fixed with 4 % paraformaldehyde and stained with hematoxylin (Sigma). Heterotransplantation into nude mice GBM-HSF cells (2 9 106) were injected subcutaneously into the flank of 6- to 8-week-old Bulb/c nude mice (n = 6, male). Growth of the tumor was monitored every 2 days by measuring the size of tumors with a ruler [5]. The mice were then sacrificed by cervical dislocation and the tumors were excised, cropped, and cut into slices (7 lm thick) with a cryostat microtome (Leica CM1850, Germany) for
Forward: 50 -CTGCGGCTCGATCAACTCA-30 Reverse: 50 -TCCAGCGACTCAATCTTCCTC-30
b-tubulin III
Forward: 50 -TCTTCTCACAAGTACGTGCCT-30 Reverse: 50 -CCCCACTCTGACCAAAGATGAA-30
Map2
Forward: 50 -GAAGGACTTGTCCGAAGCG-30
Klf4
Forward: 50 -GACCACCTCGCCTTACAC-30
Reverse: 50 -TCTCCGTTGATCCCATTCTCTT-30 Reverse: 50 -CTCAGTTGGGAACTTGACC-30 Oct4
Forward: 50 -CTTGAATCCCGAATGGAAAGGG-30 Reverse: 50 -CCTTCCCAAATAGAACCCCCA-30
Nanog
Forward: 50 -AAGGTCCCGGTCAAGAAACAG-30 Reverse: 50 -CTTCTGCGTCACACCATTGC-30
Sox2
Forward: 50 -TGGACAGTTACGCGCACAT-30 Reverse: 50 -CGAGTAGGACATGCTGTAGGT-30
Rex1
Forward: 50 -CATACGCCTGTGTTCCC-30 Reverse: 50 -GCTCTTGCTGTTATCCAGTC-30
CD133
Forward: 50 -GCGGTAAAACTGGCTAAGTA-30 Reverse: 50 -TGTGATGGGCTTGTCATAAC-30
CD26
Forward: 50 -CTACACAGCAGCGTGAATG-30 Reverse: 50 -GGCAAGATCATCTGATACCA-30
GAPDH
Forward: 50 -CAACTACATGGTTTACATGTTC-30 Reverse: 50 -GCCAGTGGACTCCACGAC-30
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Results
Colony formation
Morphology and growth kinetics
HSF cells were plated in soft agar in triplicate at 10,000 cells per well in a six-well plate. The cells formed 450 ± 12 colonies per well 21 days after being plated and incubated in the soft agar. The average ratio of colony formation contained 4.5 % of the total cells after 21 days incubation. Figure 3 displays the morphology of GBMHSF colonies on day 21.
GBM-HSF cells displayed a heterogeneous morphology at 60–70 % confluency. Most of the cells were multipolar cells with a spindle or oval morphology. A small subset of cells presented with long dendrites (Fig. 1a). When the GBM-HSF cells were grown to a very high density, the majority of the cells displayed long spindle morphologies (Fig. 1b). The high density and compacted cell shape suggested that the GBM-HSF cells did not exhibit contact inhibition during proliferation. The cell line was found to have a high growth rate with a doubling time of about 51 h (Fig. 2).
Identification of cancer stem cells GBM-HSF cells were able to form tumor spheres in NSC medium with or without growth factors, such as EGF or bFGF, in the media (Fig. 4). EGF and bFGF were found
Fig. 1 Morphology of GBM-HSF cells at a 60–70 % confluency and b high density, multi-layered growth. Bar 100 lm
Fig. 2 Growth curve of GBM-HSF cells (n = 3)
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Fig. 3 Morphology of GBM-HSF colony formed after 21 days of culture. Bar 100 lm
Establishment and partial characterization of a human tumor cell line
Fig. 4 Sphere formation of GBM-HSF cells in conditioned medium. a NSC medium; b NSC medium supplemented with EGF; c NSC medium supplemented with bFGF; d NSC medium supplemented with EGF and bFGF. Bar 100 lm
to increase the rate of growth of the cell line and the formation rate of tumor spheres (Fig. 4b–d). A side population (SP) assay was used to determine if the cell line contains the cells with stem cell potential which can efflux Hoechst 33342 dye [8]. SP cells were found to be present in the cell line, and the number of SP cells decreased dramatically in the presence of verapamil (Fig. 5). These results suggested that the cell line might contain cancer stem cells.
1-week latency period, all the mice (n = 6) transplanted with the cells developed round-shaped nodules with a diameter around 5 mm (Fig. 7a), which was indicative that the cell line had formed tumors in the nude mice. Histological evaluation of the tumors with hematoxylin and eosin found the heterotransplanted cells to be highly clustered together and to have similar morphology to astrocytic neoplasm (Fig. 7b). RT-PCR
Invasion assay and heteroplantation tumorgencity The GBM-HSF cell line was found to have a highly invasive nature in an in vitro transwell assay. Investigation of the bottom of the Matrigel-coated porous membrane found a large number of cells, which suggested that the cells have migrated through the membrane (Fig. 6). To evaluate the cell line in vivo, a small subcutaneous injection of cells was given to BALB/c nude mice. After a
RT-PCR was employed to study the transcription of genes related to tumor origin, malignancy, and metastasis. Nestin, b-tubulin III, Map2, Klf4, Oct4, Sox2, Nanog, and CD26 were positively transcribed in the cell line, whereas no transcriptions of GFAP, Rex1, or CD133 were found HSF cells. Among the positively transcribed genes, b-tubulin III, Klf4, Oct4, Sox2, and CD26 had higher mRNA levels than Nestin, Map2, and Nanog (Fig. 8).
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Fig. 5 Detection of SP cells in GBM-HSF cell line. Cells were incubated with Hoechst 33342 alone (a) or in combination with verapamil (b). SP cells which decreased dramatically by the treatment of verapamil were outlined and shown as percentage of total 20,000 cells
Fig. 6 In vitro invasion assay of GBM-HSF cells. GBM-HSF cells were able to migrate through the matrigel coated porous membrane. The invaded cells were stained with hematoxylin and observed under a microscope. Bar 100 lm
Discussion This study reports the establishment of a novel human cell line, GBM-HSF, from the biopsy of a GBM from a 65-year-old woman as well as the characterization of the cell morphology, cell growth kinetics, colony formation efficiency, sphere formations, in vitro invasion abilities, and tumorigenicity of the cell-line. The transcription of genes related to tumor origin, malignancy, and metastasis were also investigated. The GBM-HSF cells adhered to culture plates and presented with multiple morphologies and a relatively high growth rate (DT = 51 h). The cell
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Fig. 7 Tumorigenicity of GBM-HSF cells in nude mice. a Representative photo of the growth of tumor nodule growth 1 week after heterotransplantation; b histological staining of a tumor nodule slice (7 lm) with hematoxylin and eosin. Bar 100 lm
line also had a relatively high efficiency of colony formation, where 4.5 % of the total cells formed colonies in soft agar. Furthermore, the cell line was found to have a highly invasive nature and to potentially contain cancer stem cells.
Establishment and partial characterization of a human tumor cell line
Fig. 8 Detection of embryonic and neuronal cell growth, cancer cell growth and metastasis biological markers by RT-PCR in GBM-HSF cells. Lane 1 GAPDH (181 bp), 2 Nestin (145 bp), 3 GFAP (209 bp) 4 b-tubulin III (127 bp), 5 Map2 (119 bp), 6 Klf4 (167 bp), 7 Oct4 (206 bp), 8 Nanog (237 bp), 9 Sox2 (215 bp), 10 Rex1 (123 bp), 11 CD133 (158 bp), 12 CD26 (149 bp), M marker
This GBM-HSF cell-line was also found to be capable of developing tumors in BALB/c nude mice. RT-PCR revealed that the neural stem cell-related gene Nestin [9], neuronal markers b-tubulin III and Map2 [10], embryonic stem cell-related genes Klf4, Oct4, Sox2, and Nanog [11, 12], and cancer metastasis-related gene CD26 [13] were positively transcribed in the GBM-HSF cell line. Two additional stem cell-related genes, Rex1 [14, 15] and CD133 [16–18], were found to be negatively transcribed in the cell line. Nonetheless, the cumulative results presented here suggest that the GBM-HSF cell line contains cancer stem cells and that this cell line will provide a good model to study the properties of GBM cancer stem cells and metastasis. The transcription of Nestin, b-tubulin III, and Map2 has been used as an indicator of neuroepithelial origin [19–21], which suggests that GBM-HSF cell line has a similar origin. Nestin has also been related to the malignancy of tumors, where cancers that present with Nestin positive tumors have a poor prognosis [22–26]. Klf4, Oct4, Sox2, and Nanog are common markers of stem cells [11, 12]. Positive transcription of these four genes suggested that the GBM-HSF cell line has similar properties to stem cells. These four genes play a crucial role in embryo development and construct the core transcription regulatory circuitry in embryonic stem cells [11, 12]. Recent studies also suggested that activation of these four genes correlated with malignancy in human gliomas [27]. Transcription of CD26 suggested that the GBM-HSF cells may have properties associated with metastasis, as CD26 has been found to be closely related to cancer cell metastasis [13]. In addition to these transcriptional changes that indicate cancerous properties of the GBM-HSF cell line, a battery of in vitro tests and heteroplantation experiments identified the cell line to have cancerous properties, as well as to be likely to contain a sub-population of cancer stem cells. All told, this supports the notion that the GBM-HSF cell line established here carries properties associated with the development of glioblastoma multiforme, which will allow it to be used in further investigations into the growth and treatment of these cells in vitro and in vivo.
It is also noteworthy that the astrocyte marker GFAP was negatively transcribed in the GBM-HSF cells. A previous study had suggested that GFAP expression was negatively related to the proliferation of glioma cells [28], which may suggest that there is a genetic transition sequelae from normal glia cells in brain to glioma cells which is differentially represented in the GBM-HSF cell line. This may also have coincided with the high growth rate and colony formation of the GBM-HSF cells. Although the low transcription levels of certain genes in the GBM-HSF cell line, like Rex1 and CD133, may not be fully representative of their expression in cancer stem cells due to the presence of non-stem cells in the culture, it is worth discussing the potential relationships between these stem cell markers as their interactions may provide fruitful insights in the future. Rex1 is a stem cell marker that is closely related to Nanog, Sox2, and Oct4 [14, 15]. The promotor of Rex1 is thought to be both repressed and activated by Oct4 [15]. This suggests that the high level of Oct4 transcription in the GBM-HSF cell line may have led to the negative transcription of Rex1. However, further work is required to confirm this relationship. Similarly, the glycoprotein CD133 has been used as a marker for the isolation of cancer stem cells in brain tumors, despite it not having a known function [16–18]. Nonetheless, the null transcription levels of CD133 found in the GBM-HSF cells may not directly reflect the cancer status of the GBM-HSF cell line. CD133 negative cells have been found to cause tumor growth in nude rats as well as to give rise to CD133 positive cells in subsequent studies [29]. Taken together, these findings suggest that the GBMHSF cell line will provide a new and useful tool to facilitate molecular and cellular investigations into the development of GBM and in the search for effective new therapies.
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