Gliomas are the most common primary brain tumors, and one of the most insidious biological features of glioma is the potential of single cells to invade normal brain tissues and establishing numerous micro-tumors at a distance from the primary tumor, which can make surgical resection palliative but not curative.1 More details about the relationship of the immunity of the central nervous system and the genesis and development of gliomas need to be developed. Animal models are the primary method for studying the characteristics of malignant gliomas and their response in vivo.2 Although the C6/Wistar rat model has been used extensively, the origin for C6 cells is still unclear. And the phenomenon of spontaneous regression of intracranial C6 rat glioma presages the limited use of this model.3–5 In the present work, G422 glioma was implanted in the brain of BALB/c mice (immuno-competent mice), nude mice (T cell related immuno-deficient) and complement C3 knock-out mice (complement C3 related immuno-deficient, C3-KO). The survival time of the host, growth and histopathology of the tumor, and concentrations of the inflammatory factors, tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ), in the tumor tissue were assessed.
Cell lines and mouse tumor models
The G422 mouse glioma cell line was contributed by Xijing Hospital, the Fourth Military Medical University, Chongqing, China, and cultured in RPMI media 1640 containing 10% fetal calf serum (Hyclone, USA). All animal experiments were approved by the Subcommittee on Research Animal Care of the Third Military Medical University. Twelve male BALB/c mice (immunocompetent mice), nude mice (T cells related immunodeficient) and complement C3 knock-out mice (complement C3 related immuno-deficient, C3-KO mice) weighed (20–25) g, were designed to observe the survival time (n=6 in each kind of animals) and to measure the cytokine concentrations (n=6 in each kind of animals). They were depilated on the head, and the next day, skull holes were pierced 1.0 mm to the right of the sagittal suture and 2.0 mm anterior to the fontanel. They were injected with 106 G422 cells suspended in 5 μl of phosphate-buffered saline (PBS) by the direction of a stereotaxis system. All procedures were performed aseptically.
Tumor volume and survival time assessment
Orthogonal diameters of tumors were measured twice every week with Magnatic Resonance Imaging (MRI, BioSpec In-vivo MR Spectroscopy/Imaging System. Bruker BioSpin International AG, Switzerland). The maximal diameters of the tumor were measured in three trans-planes:6 coronal (d1), sagittal (d2), and transverses (d3), and the tumor volume were calculated with the formula: d1×d2×d3×π/6. Survival time was recorded after G422 tumor implantation and mice were sacrificed when the primary tumor reached 5.0 mm in diameter or the body weight dropped≥15%.
For histopathological examination, the tumor tissues were taken for electron microscopy and light microscopy inspection. Immunohistochemistry (IHC) was performed on 3 μm thick sections from 10% formalin-fixed, paraffin-embedded tissues. Sections were deparaffinized in xylene, rehydrated, washed in PBS and blocked with normal serum for 20 minutes and then incubated overnight with anti-glial fibrillary acidic protein (GFAP) antibody (dilution 1:50, Santa Cruz Biotechnology, USA). Sections were then incubated with streptavidin using the BIOS kit (Beijing Boisynthesis Biotechnology, China), washed in carbonate buffer, stained with 3,3′-diaminobenzidine (DAB) and nuclei counterstained with hematoxylin.
Determination of cytokine concentrations
TNF-α and INF-γ concentrations in tumor tissue homogenates were determined by commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the instructions of the manufacturer. Assays were conducted 35 days after G422 glioma implantation in BALB/c mice, 21 days in nude mice and 14 days in C3-KO mice; when the tumor sizes were similar.
Infiltrated CD68+ Cells
The proportion of CD68+ cells in the tumor tissue was determined in BALB/c, nude and C3-KO mice by flow cytometry. Briefly, tumors were disaggregated in 25 ml of Hank's blanced salt solution (HBSS) containing 10% bovine serum albumin (BSA) at 37°C for 30 minutes. After washing a minimum of three times in PAB buffer (HBSS w/o Ca2+ Mg2+ containing 5 mg/ml BSA and 1 mg/ml sodium azide), cells were stained with CD68 monoclonal antibodies directly conjugated with FITC (Santa Cruz Biotechnology). Flow cytometric analysis was performed on a two-laser FACStar Plus flow cytometer (Becton-Dickinson, San Jose, CA), and data were acquired from a minimum of 20 000 cells.
All measured values were presented as means ± standard error of the mean (SEM). The two-tailed Student's t-test and analysis of variance (ANOVA) were used for comparison. P values of 0.05 or less was regarded as statistically significant.
After G422 glioma implantation in the brain of the BALB/c mice, nude mice and C3-KO mice, survival was observed. The longest median survival time of (44.3±6.0) days was found in BALB/c mice, followed by (24.8±5.2) days in nude mice and the shortest was (18.6±5.8) days in the C3-KO mice.
The tumor spheres can be shown by hematoxylina and eosin stained histology in all of the mice after injection. Figure 1A shows the tumor spheres 49 days after G422 glioma was transplanted in the brains of BALB/c mice. Figure 1B shows the G422 glioma 24 days after transplantation into the brains of nude mice and Figure 1C shows the G422 glioma 21 days after it was transplanted into the C3-KO mice.
Tumor cell nests were organized around blood vessels both within the tumor and at the margins of the tumor, and they infiltrated into the brain tissue around the tumors (Figure 2A). Undifferentiated cells and large polygonal cells with amphophilic cytoplasm, and vesicular and pleomorphic nuclei with prominent nucleoli and mitotic figures were frequently found (Figure 2B). The GFAP positive staining of the cells declared their glioma origin (Figure 2C). The G422 glioma demonstrated similar histological manifestations when grown in three kinds of mice with different immune backgrounds.
The growth of the tumors in different strains of mice was not the same. Tumors grew the fastest in the C3-KO mice, followed by the nude mice and grew the most slowly in BALB/c mice (Figure 3). However, the size of tumors, (37.43±1.67), (37.46±2.31) and (39.10±2.89) mm3 in BALB/c, nude and C3-KO mice respectively, were similar before the hosts died. We detected similar tumor volumes at 35 days in BALB/c mice ((34.87±1.35) mm3), 21 days in nude mice ((34.31±1.02) mm3) and 14 days in C3-KO mice ((33.68±1.98) mm3).
Inflammatory factors in the tumor tissue
TNF-α and INF-γ levels in the tumors were examined 35 days after G422 glioma implantation in BALB/c mice, 21 days in nude mice and 14 days in C3-KO mice (Figure 4); when there tumor sizes were similar. In nude and C3-KO mice, there were significantly lower levels of TNF-α than in the BALB/c mice; (28.11±4.86) μmol/L in nude mice and (22.87±6.36) μmol/L in C3-KO mice vs. (230.21±39.17) μmol/L in BALB/c mice (P <0.01). The concentrations of INF-γ were different in the three kinds of mice. The highest level was seen in the BALB/c mice, (180.76±29.19) μmol/L, followed by the nude mice, (113.46±23.76) μmol/L and C3-KO mice, (16.84±4.45) μmol/L (Significance was determined by ANOVA, P <0.01). However, the proportions of infiltrated CD68 + lymphocytes in tumor tissues were similar; 24.6%±3.5% in BALB/c mice, 26.7%±4.2% in nude mice and 25.0%±6.7% in C3-KO mice.
With the development of tumor immunology, it is recognized that the central nervous system is not a completely immune privileged organ.7 The relationship of the immunity of the central nervous system and the genesis and development of glioma remains unclear. Establish reliable animal brain glioma models of different backgrounds would provide appropriate subjects for various studies. In our earlier experiments, we found that photodynamic therapy can significantly kill the rat brain C6 glioma, but it only showed inhibitory effects in the nude mouse C6 model.8 This may be related to the brain micro-environment and immune background. But as a result of the phenomenon of spontaneous regression of intracranial C6 rat glioma, the evaluation of the effect of treatment should be treated carefully. The G422 glioma cell line, which can be transplanted successfully without spontaneous regression, has recently been implanted in the brains of BALB/c mice, nude mice and C3-KO mice.9–11
In this study, microinjection by a stereotactic instrument approach was used to plant G422 glioma cells into the brains of mice with different immune backgrounds. The high rate of tumorigenesis, rapid growth, and significant mass effect of clinical cases were imitated without spontaneous tumor regression. HE staining and electron microscopy showed the characteristics of tumor and the positive GFAP immunohistochemical staining identified their glioma origin.12 Although the growth of tumors in three strains of mice was significantly different, there was no significant difference in tumor volume just before death. This suggests that the three strains of mice have similar tolerance to the tumor mass. Similar tumor volumes were found at 35 days in BALB/c mice, 21 days in nude mice and 14 days in the C3-KO mice. Therefore, the opportunity for intervention should not based on the time of tumor implanted but on the tumor volume.
The significant differences in survival time, growth rate of G422 gliomas and concentrations of TNF-α and INF-γ in different mice suggest that both acquired immunity and innate immunity may influence the growth of glioma. With acquired immunity, the host would identify tumor antigen, release cytotoxic cytokines such as TNF-α and INF-γ, activate lymphocytes and produce specific antibodies.13 The complement system is an important component of innate immunity. The activation of complement promotes the formation of the complement membrane attack complex (MAC), which will not only kill the target cells by complement dependent cytotoxicity (CDC), and it can also induce changes of cell metabolism and activate specific immune responses.14 Because all of the three pathways of complement activation depend on the existence of complement C3, the formation of MAC can not be activated in C3-KO mice. The deficiency of innate immunity might lead to the significantly shorter survival time in the C3-KO mice. Nude mice, with acquired immune deficiency, still have intact natural immunity. They showed weaker inhibition of tumor growth than immune competent BALB/c mice, but slower tumor growth than C3-KO mice.
Ardavin et al15 found that complement activation was important in an anti-tumor immune response in liver cancer. There are a series of soluble and membrane bound factors that serve as a bridge between innate immunity and specific immunity. So we assumed that the normal activation of the complement system would promote the formation of intermediate products in the anti-tumor immune response, improve the tumor-specific response, and inhibit glioma formation and growth. Some studies have shown that caveolin-1, locating in the membrane of vascular endothelial cells, can clear the MAC depositing on the surface of cells and is related to the invasion and migration of glioma.16,17 The possibility that glioma cells can clear MAC with a caveolin-1 dependent mechanism to escape immune surveillance is worthy of further study. In short, our study suggest that not only acquired immunity18 but also innate immunity can affect the growth of tumors transplanted into the brain.
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