Malignant glioma is the major brain tumor in adults and has a poor prognosis. The failure to control invasive cell subpopulations may be the key reason for local recurrence of glioma after radical tumor resection and may contribute substantially to the failure of adjuvant modalities such as radiotherapy and chemotherapy. Local invasion is the hallmark of malignant glioma. The glioma cells seem to invade following the distinct anatomic structures within the central nervous system. Tumor cell dissemination may occur along structures, such as the basement membranes of blood vessels or the glial limitans externa, which contain extracellular matrix (ECM) proteins. Frequently, invasive glioma cells are also found to migrate along myelinated fiber tracts of white matter.1 Implantation of C6 glioma cells into the rat brain mimics many of the growth and pathological characteristics of human gliomas.2 The invasion study using C6 cells labeled with bromodeoxyuridine (BrdUrd) showed migration of the tumor cells toward the perivascular space that was distant from the primary site of injection after 4 days of the injection.3
Numerous studies indicate that the growth of gliomas is initiated and driven by a subpopulation of cancer cells with stem-like characteristics, including self-renewal capacity and the ability to differentiate. These cells were called glioma stem cells (GSCs), displaying greater tumorigenic potential than matched non-stem tumor cells when xenotransplanted into the brains of immuno-compromised rodents.4 It has been hypothesized that GSCs are more invasive than matched non-stem tumor cells. Do GSCs invade also along the perivascular space or white tracts matter? The direct experimental evidence addressing this issue has been less so far. Here we show experimental data to demonstrate that GSCs display greater invasive potential in vitro than matched non-stem tumor cells derived from C6 cells and the distribution patterns in perivascular niche of tumor-brain interface and along white matter tracts in xenografts.
Reagents and animals
The following reagents and animals were used: rabbit monoclonal to CD133 (Abcam, USA); rabbit polyclonal to nestin (Santa Cruz, USA); epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), 1% luxol fast blue solution (Sigma, USA); B-27 minus vitamin A, DMEM, DMEM/F12 (Gibco, USA); rat glioma line C6 (China Academia Sinica cell repository, Shanghai, China); Sprague-Dawley (SD) rats (Animal Center of PLA General Hospital, Beijing, China); Alexa Fluor-488 F(ab′)2 fragment of goat anti-rabbit IgG, Alexa Fluor-555 F(ab′)2 fragment of goat anti-rabbit IgG (Cell signaling, USA).
C6 cells were cultured in serum-containing medium (SCM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 U/ml streptomycin. C6 cells formed round cell aggregates or spheres when the cells were cultured in serum-free medium (SFM) containing of DMEM-F12 medium, 10 μg/ml bovine insulin, 100 μg/ml bovine serum albumin (BSA), 50 U/ml penicillin, 50 μg/ml streptomycin, as well as 20 ng/ml bFGF, 20 ng/ml EGF, B-27 (1:50). Spheres were centrifuged, triturated with a fire-narrowed pasteur pipette, and resuspended in phosphate buffered saline (PBS) with 0.5% BSA and 2 mmol/L ethylenediaminetetraacetic acid (EDTA). Magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit (Miltenyi Biotec GmbH, Germany). CD133+ and CD133− sorted cell populations were resuspended in SFM. In all experiments, cells were maintained in 100-mm culture dishes (Corning, USA) at 37°C in a humidified 5% CO2/95% air atmosphere.
In vitro matrigel invasion assay
The in vitro cell invasion assay was performed using Matrigel-coated invasion chamber (Millipore, USA) as previously described.5 CD133+ and CD133− cells (1×105) were added to the upper chamber of the Matrigel-coated prehydrate polycarbonate membrane filter. After incubation for 24 hours, the non-invaded cells from the upper side of the filter were scraped using moist cotton swab. The invaded cells in the reverse side of the filter were fixed and stained with crystal violet, and then counted under an inverted microscope.
Intracranial transplantation into SD rats
Intracranial transplantation of C6 cells into SD rats was performed as described.6 Briefly, C6 cells were suspended in PBS (1×105/μl). Twenty-six-week-old SD rats were anesthetized with 10% chloral hydrate (3 ml/kg) and placed in the stereotactic frame using ear bars. A hole was made in the skull 3.0 mm lateral to the bregma, 1 mm anterior to the coronal suture. Cell suspension (10 μl) was transplanted into the right caudate 5.0 mm below the surface of the brain using a Hamilton syringe. When rats developed neurological deficits, they were sacrificed, brains of bearing tumor were quickly dissected out and fixed with 4% paraformaldehyde (PFA). After cutting 5-μm-thick sections from coronal dissected brain and placing them on lysine-coated slides, mirror sections, comprising a pair of consecutive slices, were prepared to examine whether GSCs invasion along with white fiber tracts. The sections were placed on the slides so that their adjacent surfaces faced upwards, and appeared as reversed images of each other, the same as an object and its image in a mirror.7 One of mirror sections was prepared for immunohistochemistry (IHC) staining, another for luxol fast blue (LFB) staining. Brain sections were then subjected to hematoxylin and eosin (H&E) staining. All animal experiments were accordance with the guidelines of the Tianjin Medical University Committee for Ethics of Animal Experimentation.
Immunofluorescent and IHC staining
Immunofluorescence was performed on spheres and serum-cultured cells as described.8 Previously, for immunostaining of undifferentiated tumor spheres, cells were plated onto poly-L-lysine-coated glass coverslips in SCM for 4 hours. Cells were then fixed with 4% PFA and incubated with the following antibodies: CD133 (1:1000), nestin (1:200). Secondary antibodies (anti-rabbit IgG conjugated with Alexa 488 555) were used. For immunostaining of serum-cultured cells, serum-cultured cells C6 were plated onto glass coverslips for 24 hours. Cells were counterstained with anti-fade sealant containing 4′6-diamidino-2-phenylindole (DAPI) (Vectashield, USA). IHC staining of mirror sections and paraffin slips were performed as described7 using following primary antibodies: CD133 (1:100), nestin (1:100). Images were examined under fluorescence microscope DP70 (Olympus, Japan). Pictures were captured with DP70 CCD digital camera (Olympus).
LFB staining was performed as described.9 Mirror sections were immersed in 1% LFB solution overnight at 57°C in tightly sealed staining jars. Removal of excess LFB with 95% ethanol rinses was followed by distilled H2O rinses and an initial differentiation for a few seconds in 0.05% LiCO3. Differentiation of the sections was continued in several changes of 70% ethanol until the grey and white matter were clearly distinguished. Thereafter, the sections were washed thoroughly by distilled H2O and stained for three minutes at 57°C in a preheated 1% solution of cresyl violet acetate (Sigma) containing 0.1% acetic acid (pH 3.7) that had been made up 16 to 20 hours prior to use. After differentiation in 95% ethanol, the sections were dehydrated, cleared in xylene and mounted.
The rat brains of bearing tumor were quickly removed after anesthesia with 10% chloral hydrate (3 ml/kg) and placed on ice. Tissues were taken from tumor core, the junctional zone between the tumor and the normal brain tissue, the surrounding area of the tumor margin (2-3 mm), and the corpus callosum of contralateral hemisphere with microinstrument. They were kept at -80°C. A big piece of frozen tissue was broken into small pieces and transferred into a 1.5-ml microcentrifuge tube; 500-μl cell lysis buffer was added to the tube. The tissue was homogenized with 10 to 15 strokes (3-4 s/stroke) using a mini-homogenizer and plastic pestle on ice. The protein was concentrifuged at 12 000 × g for 15 minutes at 4°C. The supernatant was transferred to a fresh tube; 50-μg protein and an equal volume of 2× sample buffer were heated at 94°C for 5 minutes. Proteins were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel and transblotted onto a polyvinylidene difluoride (PVDF) transfer membrane (Millipore). The blot was blocked in PBS-T and 5% skim milk at 37°C for 1 hour. The membrane was then incubated in primary antibody (CD133, 1:500) at 4°C overnight, followed by treatment with secondary antibody conjugated with horseradish peroxidase (1:1000). Blots were developed using the enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia, UK) and visualized using the GeneGenius Imaging System (Frederick, USA).
Statistical evaluations were carried out using SPSS 15.0 software (SPSS Inc., USA). Values were expressed as mean ± standard deviation (SD). Student's t test and one-way analysis of variance (ANOVA) test were used. Statistical significance was set at P <0.05.
Characterization of GSCs
The C6 glioma cell line contained a small subpopulation of cancer stem-like cells.10 GSCs of C6 cells grew into neurosphere-like glioma spheres when cultured in SFM (Figure 1B); but in SCM, C6 cells showed adherent growing (Figure 1A). CD133+ GSCs could also be found in an adhere monolayer (Figure 1C). Immunofluorescence staining showed highly expressed CD133 marker in gliomasphere (Figure 1D).
In vitro matrigel invasion assay
CD133+ GSCs and CD133− cells were assessed for their invasive potential by examining cell migration through a matrigel invasion assay. GSCs populations showed more cells migrated through the matrigel than matched non-stem tumor cell populations (Figure 2A and 2B). A total of 85.3±4.0 GSCs passed the matrigel, meanwhile the number of the passed non-stem cells was 25.9±3.1. Quantified data confirmed that GSCs had more (3-4 folds) cells migrated through the matrigel than matched non-stem tumor cells (Figure 2C). These data suggested that GSCs were significantly more invasive in vitro than matched non-stem tumor cells (t=14.5, P <0.05).
Distribution of GSCs in the brain tumor
Aggressive invasion of cancer cells into brain tissue is one of the most significant characteristics of malignant glioma. To further compare the invasive potential of GSCs and non-tem tumor cells in vivo, the distribution of GSCs in the brain tumor was studied. GSCs invaded into the brain diffusely and the border line was unclear. In the tumor, CD133+ GSCs located around a blood vessel as a single cell and as cell clusters as well. The junction of tumor and brain tissues maintained numerous CD133+ GSCs which distributed around vascular even the long axis of blood vessels. The sites distant from tumor also had this phenomenon of perivascular satellitosis (Figure 3).
GSCs and white fiber tracts
The infiltrative path of gliomas into the normal brain presents as a non-random process, often following white matter tracts.11 These preferred anatomical routes for invasion suggest the importance of interactions between migrating cells and their microenvironment. Mirror sections staining showed that GSCs around vascular located at the edge of white matter tracts. The polarity of glioma cells in fiber was parallel to the white matter tracts. GSCs were observed migrating along vessels and reaching the white matter tracts, and then GSCs migrating into other sites transited through the white matter tracts (Figure 4).
The expressions of CD133 in different regions of brain tumor were identified by Western blotting. Data were presented as the mean of triplicate experiments, differences between the mean of each group were tested using the one-way ANOVA test (F=2488, P <0.05) (Figure 5). Significantly higher amounts of protein were observed in the center (0.0378±0.0007) and edge of brain tumor (0.0464±0.0010) than in the surrounding of the tumor (0.0295±0.0004). CD133 protein expressed also in corpus callosum between hemispheres but the level of expression was very low (0.0115±0.0002), which might imply that GSCs was migrating into the contralateral hemisphere along corpus callosum.
Treatment of adult glioma, in particular glioblastoma, remains a significant clinical challenge, despite modest advances in surgical technique, radiation, and chemotherapeutics. Two major aspects of glioma biology that contributes to this recalcitrance are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells through white matter tracts, which are hallmarks of glioblastoma. Recent insight into the relationship between GSCs with invasion provide a renewed hope for development of novel strategies aimed at reducing the morbidity of this uniformly fatal disease. Singh and colleagues12 successfully isolated cancer stem cells (CSCs) from different types of brain tumors, GSCs make up a small fraction of the glioma that have been shown to be capable of giving rise to the entire tumor and which are believed to represent a source of treatment resistance. Although malignant glioma cells rarely spread outside the central nervous system, glioma cells often infiltrate into normal brain tissue preventing curative surgical resection.
Cell surface molecules differentially expressed in GSCs and functionally associated with the maintenance of GSCs may be ideal markers for sorting or identifying GSC population. Several molecules, including CD133, CD15, A2B5, L1CAM have been identified on cell surface of GSCs. CD15 (SSEA-1) originally identified as a surface marker of mouse embryonic stem cells has been recently used as an alternative marker to enrich GSCs from some glioblastoma multiforme (GBM) tumors in which CD133 is not an informative maker for GSCs population.13 CD15 in normal stem cells and CSCs remains poorly understood. A2B5 have been used for the enrichment of GSC population.14 L1CAM is a differentially expressed surface glycoprotein that plays critical roles in the maintenance, survival and cellular functions of GSCs.15 Some important stem cell transcription factors (SCTFs) involved in regulating normal stem cells are also required for the maintenance of a GSC phenotype. These stem cell transcription factors such as Sox2, Oct4, Nanog, c-Myc, Olig2 and Bmil are critical for maintaining the self-renewal, proliferation, survival, and multi-lineage differentiation potential of GSCs.16 Some of above glioma stem cell markers could only identify the particular stage of a certain type of CSCs ascribed to a some degree of GSCs' heterogeneity. Among these stem cell markers, CD133 and Nestin are currently the most accredited markers for the identification of GSCs. The C6 rat glioma cell line is known to contain a subpopulation of GSCs. In this study, we isolated and characterized CSCs from the C6 cells, and assessed their invasive potential. GSCs deprived from C6 cells had greater invasive potential than matched non-stem tumor cells in vitro cell invasion assay. On the basis of this, we speculate that GSCs are likely to be primary tumor cells invading into brain tissue.
Descriptions in the 1940s of tumor cells migrating into the normal brain around blood vessels (perivascular satellitosis), first suggested that malignant glioma cells might have a special relationship with the surrounding vasculature.17 Nestin+/CD133+ cancer cells locate next to capillaries in brain tumors, the brain tumor microvasculature forms a niche that is critical for the maintenance of GSCs.18,19 IHC study using C6 orthotopic tumor models show that CD133+ glioma cells locate and distribute in the tumor-brain interface and the space being distant from the site of tumor, meanwhile these GSCs reside perivascular niche, even around the long vessel axis. Perivascular niche exhibit restricted oxygen availability and distinct ECM profiles. In parallel, GSCs are enriched in perivascular niches, and possibly also within regions of hypoxia and at the invasive edge of the tumor.20 Why is GSCs performing “vasoinvasion”? In malignant glioma, there exists an imbalance between the supply and consumption of oxygen as a result of limited O2 delivery to the cancer cells.21 Hypoxic can be caused by abnormal vascular structure, increased diffusion distance, a temporary disruption in blood flow and/or low haemoglobin level. Hypoxic stress is a key important factor of niche.22 Unlike normal cells, tumor cells are much better equipped to cope with hypoxia, in hypoxic conditions, hypoxia-tolerant tumor cell clones are selected, while tumor stem cells in hypoxic niches escape anti-angiogenic treatment.23 The core relationship between a tumor and its host niche is the nutritional and signal interactions between CSCs and endothelial cells, the nutritional interaction between the cancer and the host triggers the invasion and metastasis of cancers.24 For more nutrition and O2, GSCs may reach “fertile soil” alongside “oxygen pipes”.25
The infiltrating GSCs probably reached white matter tracts by through perivascular space, white matter tracts under certain circumstances, acted as barriers to invasion of gliomas cells. The polarity of glioma cells which show intrafascicular, perifascicular, and interfibrillary migration is parallel to the white matter tracts. Herein, cells migrating follow the path of white matter tracts maybe a forced process, it is an easy way for glioma cells to invade along white matter tracts, because of minimal resistance. Most of tumor cells are polarized in the tumor-brain interface and white matter tracts, but in the tumor center, there are only a few. Soluble motility factors (such as CXCL12/CXCR4) function as autocrine or paracrine signaling, leading to changes in cell morphology: the cell becomes polarized and membrane protrusions including pseudopodia, lamellipodia, filopodia and invadopodia are extended from the leading edge of the cells.26,27 The polarized cells are easy to invasion and migration. Corpus callosum of contralateral hemisphere express CD133 protein show that GSCs probably migrated into contralateral hemisphere across the corpus callosum.
In short, the GSCs under the influence of hypoxic pressure, prone to migrate to more fertile areas along blood vessels and white matter tracts, guided by chemokine factor. Targeting GSCs through anti-invasive therapies in combination with traditional GBM therapeutic paradigms may inhibit cancer invasion, overcome therapeutic resistance and reduce tumor recurrence, which may lead to a significant improvement of GBM treatment.
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