Background
Human glioblastoma multiforme (GBM) is one of the most devastating cancers. Extensive tumor cell invasion occurs into normal brain parenchyma, making it virtually impossible to remove the tumor completely by surgery and inevitably causing recurrent disease [
1]. There is therefore a compelling need for more reliable
in vivo preclinical models for studying the disease and for testing new drugs and therapies. For GBM cell lines in common use, comparison of gene expression profiles from cell culture, subcutaneous xenografts, or intracranial xenografts can differ significantly within the same cell line; yet different GBM cell lines from orthotopic models exhibit similar gene profiling patterns [
2]. Recent progress has been made in optimizing experimental models relevant to GBM. For example, glial progenitor cells can form invasive orthotopic glioblastoma tumors when driven by platelet-derived growth factor (PDGF) [
3]. Lee
et al. [
4] established a culture system that allows tumor stem cells to grow in culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) without serum, maintaining both genotype and phenotype similar to that of the primary tumor. Moreover, sorting of CD133-positive tumor stem cells from glioblastoma tumors yields highly angiogenic and aggressive orthotopic tumors in mice [
5].
Significant progress also is being made in developing mouse models that are genetically engineered to develop GBM [
6,
7]. Another approach is to improve the orthotopic human xenograft GBM models. Most commonly used human GBM cell lines grow slowly as orthotopic xenografts or generate poorly invasive tumors in the mouse brain, bearing little resemblance to human GBM. Interestingly, although extracranial GBM metastases rarely happen [
8‐
13], most human GBM tumor cell lines are metastatic from subcutaneous xenografts [
14]. We used experimental lung metastasis (ELM) assays to enrich for metastatic cells. In this model, three of four commonly used GBM lines were highly metastatic, grew more aggressively in the brain and, after two cycles (M2), expressed highly elevated levels of Interleukin-6 (IL6), Interleukin-8 (IL8) and granulocyte macrophage colony-stimulating factor (GM-CSF), thereby resembling GBM in patients [
15‐
18]. We further characterized one line, DBM2, which, when inoculated orthotopically, triggers vascular hyperplasia, and forms areas of central necrosis that are lined by a crowded aggregate of cancer cells. As DBM2 grows orthotopically it creates, in proportion to tumor growth, an opening in the calvarium that allows the use of imaging technologies for non-invasively evaluating and monitoring of therapeutic responses. Here we show that the HSP90 inhibitor 17-(allylamino)-17-demethoxy geldanamycin (17AAG) [
19,
20] significantly inhibits GBM DBM2 orthotopic growth.
Methods
All experiments were performed as approved by the Institutional Animal Care and Use Committee (IACUC) and the Safety Committee of the Van Andel Research Institute.
Cell culture
DBTRG-05MG, U87, and U118 are human glioma cell lines originally purchased from American Type Culture Collection (ATCC, Manassas, VA). DBM2 is a subclone of DBTRG-05MG derived through lung metastases after mouse tail vein injection as described below. U251 cells were provided by Dr. Han-mo Koo of the Van Andel Research Institute. All cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (GibcoTM, Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Corporation) and penicillin and streptomycin (Invitrogen Corporation).
Recovery of invasive GBM cells from lung metastasis
DBTRG-05MG, U251, U87 and U118 cells (106) in 100 μl PBS were injected into nude mice via the tail vein. Individual mice were euthanized when moribund; the pulmonary lesions were collected at necropsy and transplanted subcutaneously into the flank of fresh host mice to propagate the tumors. To generate primary cultures, subcutaneous tumors were harvested at necropsy, washed in PBS, minced, and treated with 0.25% trypsin (Invitrogen Corporation) for 45 min. Released cells were collected at 1500 rpm and resuspended in complete DMEM containing 10% FBS. This procedure was repeated twice to obtain GBM-M2 cell lines. U251-M1 cells were harvested after 1 cycle of selection.
Grading criteria of experimental metastasis
To compare the metastatic potential of GBM cell lines, 106 cells in 100 μl PBS were injected intravenously into nude mice. By time of necropsy, lungs were harvested and a scoring system was established as follows. If no visible lesions were observed in lungs or other organs, mice were scored as (-); if visible and/or hematoxylin and eosin (H&E)-stainable lung lesions were confined to ≤ 50% of the tissue section area, animals were scored as (+); if lesions in the lung exceeded 50% of tissue section area, animals were scored as (++); and if most of the lung was involved and a lesion was present in at least one other organ, animals were scored as (+++).
Expression of cytokines and growth factors
To prepare GBM-conditioned media, 5 × 105 cells were seeded into 10-cm dishes and grown to 80% confluency. Cells were washed with PBS twice, and complete medium was replaced with DMEM lacking serum. After culture for an additional 24 hrs medium was collected and spun at 13,000 × rpm for 5 min (Sorvall RT7 Plus) and the supernatant fraction was collected and stored at -80C for Multi-Analyte Profile (MAP) testing (Rules-Based Medicine, Austin, TX). To do the data analysis, the concentration levels of cytokines and growth factors from each cell line was normalized based on cell numbers. The fold change in expression of 89 cytokines and proteins are determined by comparing expression levels of GBM-M2 sub-lines to their parental DBTRG-05MG, U87 and U251 cell lines. R version 2.6.1 was used to generate the heat-map of the expression level fold change.
Intracranial injection
Immunocompromised [athymic nude (nu/nu)] mice at about six weeks of age were used for intracerebral injections. Mice were anesthetized using isoflurane gas anesthesia (~2%) and placed into the ear bars of a stereotaxic frame. A burr hole was created through the skull 2 mm posterior to the bregma, and 5 × 105 cells in 5 μl PBS were injected into the brain at 3 mm depth.
Immunohistochemistry staining of GBM orthotopic tumors
Tumor tissues were harvested, fixed with formalin, and embedded in paraffin. Paraffin blocks were sectioned to perform H&E and immunohistochemistry (IHC) staining for microscopic evaluation. IHC was performed using the Discovery XT Staining Module (Ventana Medical Systems, Inc., Tucson, Arizona). Briefly, deparaffinized sections were incubated in Tris/Borate/EDTA, pH 8 at 95°C for 8 minutes and at 100°C for 36 minutes for antigen retrieval. For Met staining, slides were then incubated with primary antibodies MET4, a mouse monoclonal antibody (mAb) against the extracellular domain of human MET [
21] at 1:250 dilution (8 μg/ml), anti-uPAR (R&D, Minneapolis, MN) at 1:200, and anti-CD31 (Neomarkers, Fremont, CA) at 1:200 for 60 minutes. The slides were then incubated with a universal secondary antibody, which is an anti-mouse and rabbit cocktail (Ventana Medical Systems, Inc.) for 30 minutes followed by diaminobenzidine (DAB) staining (Ventana Medical Systems, Inc.).
Treatment of DBM2 mouse tumor models with 17AAG
17AAG was purchased from LC Laboratory (Woburn, MA). 17AAG was first dissolved in 100% DMSO and stored at -80°C and then freshly diluted with vehicle PBST (PBS with 0.05% Tween 80) just prior to injection [
22]. For all tumor models, host mice (6-week old female nude mice) were given vehicle alone (control), 17AAG in vehicle at a daily dose of 20 mg/kg (single injection daily), or 60 mg/kg body weight (administered as two divided doses 6 hrs apart), all administered by intraperitoneal injection [
22]. For drug testing in the GBM subcutaneous xenograft model, tumor volume (V
t) was measured with manual calipers twice a week (V
t = length × width × depth). Results are expressed as mean ± SE.
With the orthotopic GBM xenograft model, DBM2 cells were inoculated intracranially and tumor growth was monitored by serial high-resolution ultrasound as described in the supplementary figures [Additional Files
1 and
2]. Weekly measured tumor volume was normalized with the initial tumor size upon group to achieve the fold change of tumor volume. Result is expressed as mean ± SE. With lung metastasis model, 28 nude mice were divided into control (n = 8), 20 mg/kg (n = 10) and 60 mg/kg (n = 10) groups. Each mouse received a single intravenous tail vein injection of 10
6 DBM2 cells in 100 μl PBS. Treatment started the second day after the cells were injected and continued for 8 weeks, by which time most of the control mice were moribund. At necropsy, lungs were harvested and scored as described above; body weight and lung weight of each mouse were also recorded.
Statistical analysis
Statistical analysis of 17AAG-treated DBM2 intracranial tumor growth was performed with a student's "t" test. Log-rank test was used to analyze survival time. Chi-square test was used for comparison of 17AAG treatments against DBM2 pulmonary metastases.
Discussion
The limited number of preclinical models that recapitulate the invasive GBM tumor growth is a major hurdle to drug development. Subjecting human melanoma cells to ELM yielded highly metastatic cells with higher proliferative and invasive potential [
23,
32]. We applied this method to GBM cell lines for the purpose of improving their invasiveness in orthotopic models. The ELM assay has been used to select for metastatic cancer cells in a number of other cancer types [
33‐
35], but has not been tested previously with GBM, most likely because of the notion that extracranial metastases of human GBM are clinically rare.
Here we show that GBM cell lines can be highly invasive after ELM selection, but they still are not metastatic when implanted in the brain. The lack of extracranial metastasis of the derivative GBM-M2 cell lines strongly suggests that rapid tumor growth or the unique CNS environment curtails the escape of tumor cells [
14]. A previous study confirms the intrinsic metastatic nature of GBM tumor cells: GBM tumor cells were metastatic in spontaneous metastasis assays and no different than other types of cancer cells when tested in these assays [
14]. Although stem cells isolated from primary tumor tissues [
4,
36] have not yet been tested for metastatic potential, the stem-cell like sub-populations from rat C6 glioma cells form neurospheres and like our GBM-M2 cells, are metastatic to lungs, as well as to other organs in nude mice upon intraperitoneal (i.p.) injection [
37], again supporting that GBM tumor cells have intrinsic metastatic potential. Consistent with these reports we show that three of four commonly used GBM lines are highly metastatic in ELM assays (Table
1) and form metastasis in lungs and lymph nodes, similar to the destinations of some of the rare clinical GBM metastases in patients [
8,
9,
11‐
13]. It is quite remarkable that GBM tumor cell lines, which came from primary tumors that have never grown as metastases and are selected to grow
in vitro in tissue culture, have the capacity to be highly metastatic. This indicates that some aspect of GBM malignancy also satisfies the requirements for the metastatic process, or that the metastatic genotype is acquired early in tumor progression as has been proposed [
38,
39]. We have proposed that once cells acquire an invasive phenotype, they have the ability to acquire a proliferative phenotype again to become a metastatic colony [
40].
The changes in cytokine and growth factor expression that occur after ELM GBM-M2 selection are similar to those that predict aggressive disease and poor patient outcome, demonstrating the similarity of cell lines to the scenario in patients. Interestingly, after ELM selection, all three GBM-M2 lines show highly elevated GM-CSF, IL-6, IL-8 and Brain-derived neurotrophic factor (BDNF) compared with parental cell lines (Figure
2, [Additional File
4]). Both GM-CSF and its receptor are absent in normal brain but expressed at high levels in glioma tissues [
17].
In vitro, GM-CSF stimulates glioma cells to both proliferate and migrate [
17]. IL-6 gene amplification in patients distinguishes GBM from low-level astrocytoma and is associated with poor prognosis [
18]. In addition, IL-8 expression is highly associated with gliomagenesis and tumoral angiogenesis. Taken together, the co-elevation of these 3 cytokines appears to be an important indicator for GBM or poor prognosis. BDNF, a member of the neurotrophin family, plays an important role in neuronal development and survival [
41]. Although a role for BDNF in GBM is not elucidated, its downstream signaling through Ras, ERK as well as PI3K pathways [
42], would suggest it could play a role in GBM disease. Furthermore all of the GBM lines express high levels of MCP-1, also a marker of poor prognosis in patient gliomas [
26]. All of these markers are consistent with the GBM nature of the GBM-M2 cells.
We chose to further develop DBM2 cells as an orthotopic model. DBM2 cells, when inoculated orthotopically, not only show significant invasive growth, but also central necrosis, extensive vascular hyperplasia, and glomeruloid body-like vasculature formation. Brat et al. (2004, 2005) have reported the pathological features associated with poor diagnosis in GBM patients as well as the possible mechanisms. Necrosis is a hallmark of glioblastoma occurring in 60% of GBM patients while intravascular tumor-thrombus formation is found in over 90% of GBM cases. In addition, vascular hyperplasia is a characteristic of GBM and associated with poor prognosis [
15,
30,
43]. As an explanation for their highly invasive nature, we show that DBM2 tumors not only express both c-Met and uPAR, the receptor of urokinase signaling pathway, but also strongly respond to HGF (data not shown) indicating that the c-Met signaling pathway may play an important role in the invasion of DBM2 orthotopic tumors into the brain parenchyma [
24,
27,
40,
44]. Brain tumors seldom invade the skull, but there are reports of GBM with skull-erosion phenotypes and metastases to other organs [
45,
46]. The exact mechanism of the osteolytic phenotype of DBM2 is unknown. It is possibly mediated through activation of bone-resorbing osteoclasts and may be facilitated by elevated IL-6 and IL-8 levels [
47,
48].
Real-time noninvasive imaging technologies permit longitudinal monitoring of tumor progression. Magnetic resonance imaging (MRI) is commonly used for human brain tumor imaging and is being refined in preclinical models [
7]. Bioluminescence-based
in vivo imaging systems are also used to rapidly measure tumor volume and evaluate drug efficacy in animal models [
49]. Cranial window models have been developed in which part of the mouse skull is replaced with a cover glass so that the blood vessels can be observed microscopically [
50]. Here, taking advantage of the osteolytic phenotype, we show high-resolution ultrasound can be used to monitor real-time, non-invasive imaging of brain tumor growth and vascularization. In addition, with Doppler and contrast injection ultrasound, directional blood flow can easily be visualized in the tumor.
We show that our xenograft model is versatile in that it can be used with SQ implantation for measuring tumor growth potential [
25], with systemic injection for measuring invasive and metastatic growth potential in EML assays [
51], or with orthotopic administration of tumor cells for measuring tumor growth in a macro- and micro-environment that recapitulates GBM in patients. Thus this model is particularly suitable for testing therapeutics. We chose here to test the drug, 17AAG, because of its diversity in targeting the destabilization of numerous oncoproteins [
52]. 17AAG, a derivative of geldanamycin, an HSP90 inhibitor that has been in clinical trials in patients with advanced cancer [
19,
20]. It has not been considered for GBM treatment largely, we suspect, because of anticipated blood brain barrier interference with drug delivery. We show here that in all three tumor settings, 17AAG at 60 mg/kg, significantly inhibits tumor growth (Table
2, Figure
4). Thus 17AAG prevents SQ xenograft formation, the formation of metastatic lesions in ELM assays and importantly, at the same dose, inhibits DBM2 orthotopic tumor growth and prolongs animal survival time. It is certainly possible that the highly invasive GBM tumors compromise the BBB in our DBM2 orthotopic model leading to significant 17AAG anti-tumor activity. Studies with orthotopic GBM mouse models have shown that imaging reagents can leak from the intracranial tumors, indicating that the BBB is compromised [
7] and anti-HGF mAbs, despite their large molecular size can inhibit orthotopic tumor growth in the brain [
53,
54]. Our results indicate that 17AAG may be used clinically to treat malignant GBM patients providing there is limited BBB interference with drug penetration.
In conclusion, we report that commonly used GBM cells have metastatic potential which can easily be selected in ELM assays. When implanted in the brain, the metastatic potential of GBM cells can be converted to a highly invasive phenotype. Importantly we show that 17AAG is an effective inhibitor of orthotopic tumor growth and that the response to treatment can be measured in real-time by ultrasound. We anticipate that this orthotopic model with high-resolution ultrasound technology will serve as a valuable tool in preclinical screening for drugs effective in targeting GBM.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
QX designed study, isolated and characterized cell lines, performed ultrasound imaging, performed data analysis and interpretation and prepared manuscript. RT performed animal experimentation. KH served as sonographer. LD performed ultrasound imaging and assisted with animal studies. BB performed immunohistochemistry, staining procedures and evaluation. RS reviewed pathological slides and provided interpretation. BK served as pathologist and assisted with preparation of manuscript. SC served as pathologist. PZ prepared Met4 antibody. KD performed statistical analysis. BC prepared Met4 antibody. JR performed histology and immunohistochemistry. RH performed ultrasound imaging. GVW developed the concept and designed study, interpreted data, prepared manuscript, and supervised study.