Brains Of Syngeneic Rats Research Articles

Pericytes promote abnormal tumor angiogenesis in a rat RG2 glioma model.

In glioma angiogenesis, tumor vessels cause morphological and functional abnormalities associated with malignancy and tumor progression. We hypothesized that certain structural changes or scantiness of functional pericytes may be involved in the formation of dysfunctional blood vessels in gliomas. In this study, we performed morphological examinations to elucidate the possible involvement of pericytes in brain tumor vessel abnormalities using a rat RG2 glioma model. After implantation of RG2 glioma cells in the syngeneic rat brain, gliomas were formed as early as day 7. In immunohistochemical examinations, desmin-positive pericytes, characterized by morphological abnormalities, were abundantly found on leaky vessels, as assessed by extravasation of lectin and high-molecular-weight dextrans. Interestingly, desmin-positive pericytes seemed to be characteristic of gliomas in rats. These pericytes were also found to express heat-shock protein 47, which plays an important role in the formation of the basement membrane, suggesting that RG2 pericytes promoted angiogenesis by producing basement membrane as a scaffold for newly forming blood vessels and caused functional abnormalities. We concluded that RG2 pericytes may be responsible for abnormal tumor angiogenesis lacking the functional ability to maintain the blood-brain barrier.

Antiphosphatidylserine Antibody Combined with Irradiation Damages Tumor Blood Vessels and Induces Tumor Immunity in a Rat Model of Glioblastoma

The vascular targeting antibody bavituximab is being combined with chemotherapy in clinical trials in cancer patients. Bavituximab targets the membrane phospholipid, phosphatidylserine, complexed with beta2-glycoprotein I. Phosphatidylserine is normally intracellular but becomes exposed on the luminal surface of vascular endothelium in tumors. Phosphatidylserine exposure on tumor vessels is increased by chemotherapy and irradiation. Here, we determined whether treatment with the murine equivalent of bavituximab, 2aG4, combined with irradiation can suppress tumor growth in a rat model of glioblastoma. F98 glioma cells were injected into the brains of syngeneic rats where they grow initially as a solid tumor and then infiltrate throughout the brain. Rats with established tumors were treated with 10 Gy whole brain irradiation and 2aG4. Combination treatment doubled the median survival time of the rats, and 13% of animals were rendered disease free. Neither treatment given individually was as effective. We identified two mechanisms. First, irradiation induced phosphatidylserine exposure on tumor blood vessels and enhanced antibody-mediated destruction of tumor vasculature by monocytes/macrophages. Second, the antibody treatment induced immunity to F98 tumor cells, which are normally weakly immunogenic. Surviving rats were immune to rechallenge with F98 tumor cells. In vitro, 2aG4 enhanced the ability of dendritic cells (DCs) to generate F98-specific cytotoxic T cells. Phosphatidylserine exposure, which is induced on tumor cells by irradiation, likely suppresses tumor antigen presentation, and 2aG4 blocks this tolerogenic effect. Bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma.

Spheres Isolated from 9L Gliosarcoma Rat Cell Line Possess Chemoresistant and Aggressive Cancer Stem‐Like Cells

The rat 9L gliosarcoma is a widely used syngeneic rat brain tumor model that closely simulates glioblastoma multiforme when implanted in vivo. In this study, we sought to isolate and characterize a subgroup of cancer stem-like cells (CSLCs) from the 9L gliosarcoma cell line, which may represent the tumor-initiating subpopulation of cells. We demonstrate that these CSLCs form clonal-derived spheres in media devoid of serum supplemented with the mitogens epidermal growth factor and basic fibroblast growth factor, express the NSC markers Nestin and Sox2, self-renew, and differentiate into neuron-like and glial cells in vitro. More importantly, these cells can propagate and recapitulate tumors when implanted into the brain of syngeneic Fisher rats, and they display a more aggressive course compared with 9L gliosarcoma cells grown in monolayer cultures devoid of mitogens. Furthermore, we compare the chemosensitivity and proliferation rate of 9L gliosarcoma cells grown as a monolayer to those of cells grown as floating spheres and show that the sphere-generated cells have a lower proliferation rate, are more chemoresistant, and express several antiapoptosis and drug-related genes, which may prove to have important clinical implications. Disclosure of potential conflicts of interest is found at the end of this article.

Utility of the F98 Rat Glioma Model for Photodynamic Therapy

A syngeneic rat brain tumor model consisting of F98 glioma cells in Fischer rats was investigated for its utility in PDT studies. Results of in vitro studies demonstrated that the F98 cell line was sensitive to ALA-PDT, especially at low light irradiances. Histological examination revealed that F98 tumors share many fundamental characteristics with human GBMs, including rapid growth and infiltrative behavior. ALA-PDT in normal brain showed that high light fluences (26 J) delivered at relatively low powers (10 mW) are capable of causing significant edema. These findings suggest that light irradiation parameters should be chosen carefully when treating tumor-bearing animals. Rats inoculated with F98 cells preincubated in ALA showed a significant survival advantage following light exposure. Taken together, the results suggest that the F98 rat glioma model is appropriate for PDT studies of malignant gliomas.

Effects of lipid association on lomustine (CCNU) administered intracerebrally to syngeneic 36B-10 rat brain tumors

A syngeneic, intracerebral rat brain tumor model was developed and characterized and then used to evaluate the therapeutic enhancement of lipid-associated 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The Fisher rat glioma cell 36B-10 (100,000–500,000 cells) was implanted intracranially to Fisher F-344 rats into the caudate nucleus. Animals treated with lipid-associated CCNU showed a 2- to 10-fold decrease in tumor size compared with animals treated with free CCNU, indicating that lipid association increases the therapeutic index of intracerebral CCNU treatment. Moreover, the syngeneic rat brain tumor model may be useful for evaluation of other therapeutic modalities.

Photodynamic therapy of newly implanted glioma cells in the rat brain

A syngeneic rat brain tumor model is used to investigate the effects of aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on small clusters of tumor cells sequestered in normal brain. Biodistribution studies on tumor-bearing animals were undertaken in order to determine the occurrence of photosensitizer in tumor cells invading normal brain. ALA-PDT toxicity in normal brain and gross tumor were evaluated from histopathology. Effects of PDT on isolated glioma cells in normal brain were investigated by treating animals 48 hours after tumor cell implantation. Fluorescence microscopy of frozen tissue sections showed that photosensitizer content was limited and variable in tumor tissue invading normal brain. ALA-PDT with high light doses resulted in significant damage to both gross tumor and normal brain, however, the treatment failed to prolong survival of animals with newly implanted glioma cells. In contrast, animals inoculated with tumor cells pre-incubated in vitro with ALA showed a significant survival advantage in response to PDT. The results show that ALA-PDT could not prevent tumors from forming if treatment was performed shortly after tumor initiation. This was likely due to inadequate levels of ALA/PpIX in the glioma cells.

Development of a Syngeneic Rat Brain Tumor Model Expressing EGFRvIII and Its Use for Molecular Targeting Studies with Monoclonal Antibody L8A4

The goals of the present study were 2-fold: (a) to develop and characterize a rat brain tumor model that could be used for studies of molecular targeting of EGFRvIII and (b) to study the tumor localizing properties of radiolabeled monoclonal antibody (mAb) L8A4, specifically directed against EGFRvIII, following systemic, i.t., and convection enhanced delivery to brain tumor-bearing rats. F98 wild-type (F98WT) rat glioma cells were transfected with a gene encoding human EGFRvIII, and following selection and cloning, a cell line, designated F98(npEGFRvIII), was identified, which expressed a nonconstitutively phosphorylated form of the receptor. As determined by a radioligand binding assay, there were 1.2 x 10(5) EGFRvIII sites per cell compared with an undetectable number on F98WT cells. The tumorigenicity of the F98(npEGFRvIII) glioma was studied following i.c. implantation of 10(3), 10(4), or 10(5) cells into CD-Fischer rats. Mean survival times were 23, 17, and 13 days, respectively, which were equivalent to those obtained with F98EGFR and F98WT cells. As determined by magnetic resonance imaging, the mean doubling times for the F98WT and F98(npEGFRvIII) gliomas were similar (59.8 +/- 4.8 versus 52 +/- 3.3 hours). Following i.v. administration to glioma-bearing rats, mAb L8A4 specifically targeted the F98(npEGFRvIII) glioma, and at 24 hours, 7.7% of the injected dose per gram (ID/g) localized in the tumor. This increased 5-fold to 39.5% ID/g following i.t. injection and 7-fold to 59.8% ID/g at 24 hours following convection enhanced delivery. Based on these data, we have concluded that the F98(npEGFRvIII) glioma should be a valuable animal model for therapy studies focusing on molecular targeting of EGFRvIII by receptor specific mAbs.

Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN–VEGF (sense) and pLEN–FGEV (antisense) expression vectors were used to transfect the packaging cell line GP+E86 and to establish ecotropic virus producer cell lines. GP+E86:LEN–FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+E86:LEN–VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP+E86:LEN–FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.

Effect of rat tumor necrosis factor on syngeneic rat brain tumor.

The effect of partially purified rat tumor necrosis factor (TNF) was tested against 9L rat brain tumor both in vivo and in vitro. The TNF-containing serum (TNS) was produced by intravenous injection of OK432 and lipopolysaccharide (LPS). Injection of TNS significantly (p <0.05) prolonged the survival time of brain tumor-bearing rats (29.9±12.6 days after tumor cell inoculation, as compared to 20.8±4.4 days in the untreated group). In the in vitro assay, medium containing 50% TNS significantly decreased the viability of 9L brain tumor cells, by 57.6%, 50.0%, and 57.0% at 3, 5, and 7 days after the beginning of culture, respectively. TNS also displayed significant inhibition of cell growth, indicating a cytostatic effect. To verify TNF activity, TNS was partially purified by means of the DEAE-Sephadex A 50 batch ion exchange method and Sephadex G 200 column chromatography. Four fractions were tested in TNF-sensitive L (S) cells, TNF-resistant L (R) cells, and 9L brain tumor cells. Fraction 4 of TNS demonstrated 37.5%, 88.1 %, and 43.2% cell viability against L (S), L (R), and 9L cells, respectively. On the other hand, fraction 4 of normal rat serum showed 87.5%, 87.8%, and 82.2% cell viability, respectively. These results strongly suggest the presence of TNF in the TNS produced by OK432 and LPS.

Autoimmune encephalomyelitis: activation of thymus lymphocytes against syngeneic brain antigens in vitro.

Thymus lymphocytes of normal adult rats were autosensitized in vitro against soluble antigens extracted from the brains of syngeneic rats. Injection of the autosensitized lymphocytes into syngeneic rats led to the development of brain lesions suggestive of autoimmune encephalomyelitis. Injection of control lymphocytes or the antigen extract alone did not cause lesions. Since sensitization in vitro requires the presence of lymphocytes programmed with specific receptors, the results indicate that normal rats have lymphocytes capable of recognizing central nervous system self-antigens. Hence, regulatory mechanisms, inoperative in vitro, probably function in vivo to prevent immune activation of self-recognizing lymphocytes and autoimmunity. This concept suggests a new approach to exploring the pathogenesis of autoimmune diseases.