Glioma In Nude Mice Research Articles

Autophagic Cell Death of Malignant Glioma Cells Induced by a Conditionally Replicating Adenovirus

Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy. To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad-infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan-Meier method and the Cox-Mantel log-rank analysis. All statistical tests were two-sided. hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad-induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P < .001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P < .001). hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death.

Antisense telomerase RNA inhibits the growth of human glioma cells in vitro and in vivo

Telomerase is implicated in the development of cellular immortality and oncogenesis. It has been shown that telomerase activity is considerably higher in the tissue of many different cancers than in normal tissue, and that the inhibition or downregulation of telomerase activity can prevent the malignant proliferation of tumor cells. Antisense oligonucleotides have been widely used in suppressing the expression of genes and, therefore, in the present research, we evaluated the effect of antisense human telomerase RNA (hTR) on glioma cell growth in vitro and in vivo. We showed that antisense hTR cDNA significantly inhibited TJ905 human glioma cell proliferation in vitro and tumor growth in vivo, as determined by MTT assay and by measuring the volume of glioma in nude mice. Consistent with these results, we found that telomerase activity and the mRNA levels of hTR and hTERT (human telomerase reverse transcriptase) expression were markedly decreased in tumor cells treated with antisense hTR cDNA, as assessed by TRAP (telomeric repeat amplification protocol) assay and RT-PCR (reverse transcription-polymerase chain reaction) analysis. Our study conclusively demonstrates that antisense hTR effectively inhibits the growth of human glioma cells in vitro and in vivo and, thus, may be potentially used for gene therapy of malignant gliomas and other cancers.

Vaccination with MCP-1 cDNA transfectant on human malignant glioma in nude mice induces migration of monocytes and NK cells to the tumor

Recently, studies on vaccination with tumor cells genetically engineered to produce monocyte chemoattractant protein-1 (MCP-1) have provided some encouragement. These studies have shown infiltration of certain types of lymphocytes at the tumor site. However, natural killer (NK) cells have not yet been assessed. We obtained a human malignant glioma cell line producing human MCP-1 constitutively by transfection of MCP-1 cDNA. We then test the effect of vaccination with the MCP-1 transfectant on nude mice. Although vaccination with MCP-1 transfectant did not reduce the tumor in our study, it was associated with the infiltration of large numbers of NK cells and monocytes at the tumor site. The site of vaccination also showed large numbers of monocytes. NK cells were detected with anti-asialo GM1 antibody, and monocytes were detected immunohistochemically with F4/80. We assumed that infiltrating monocytes at the site of vaccination could promote the infiltration of monocytes and NK cells into the tumor site without T-cell mediated transduction because the host lacked T-cell function.

Neural Precursor Cells for Delivery of Replication-Conditional HSV-1 Vectors to Intracerebral Gliomas

Cellular delivery of a replication-conditional herpes simplex virus type 1 (HSV-1) vector provides a means for gene therapy of invasive tumor cells. LacZ-bearing neural precursor cells, which can migrate and differentiate in the brain, were infected with a ribonucleotide reductase-deficient HSV-1 mutant virus (rRp450) that replicates only in dividing cells. Replication of rRp450 in neural precursor cells was blocked prior to implantation into the tumor by growth arrest in late G1 phase through treatment with mimosine. Viral titers in the medium of mimosine-treated, rRp450-infected neural precursor cells were below detection levels 3 days after infection. In culture, after removal of mimosine and passaging, cells resumed growth and replication of rRp450 so that, 7 days later, virus was present in the medium and cell death was evident. Mimosine-treated neural precursor cells injected into established intracerebral CNS-1 gliomas in nude mice migrated extensively throughout the tumor and into the surrounding parenchyma beyond the tumor over 3 days. Mimosine-treated neural precursor cells, infected with rRp450 and injected into intracerebral CNS-1 tumors, also migrated within the tumor with the appearance of foci of HSV-thymidine kinase-positive (TK+) cells, presumably including tumor cells, distributed throughout the tumor and in the surrounding parenchyma over a similar period. This migratory cell delivery method has the potential to expand the range of delivery of HSV-1 vectors to tumor cells in the brain.

The effect of the anti-angiogenic agent TNP-470 on tumor growth and vascularity in low passaged xenografts of human gliomas in nude mice.

The effect of the anti-angiogenic agent TNP-470 on tumor growth, vascular area, vascular density and tumor perfusion of two different subcutaneously implanted human glioma xenografts (E98 and E106) in nude mice was evaluated. Vascular parameters were investigated with an image analysis system. For both tumor lines a small but significant tumor growth suppression was observed. However, no differences in vascular parameters between TNP-470 treated tumors and controls could be found after 6 weeks of treatment. It is concluded that although TNP-470 is a promising anti-angiogenic agent in many tumor types, at least 2 glioma lines seem to be partly resistant to its anti-angiogenic effects. Further evaluation of the effects of combination of TNP-470 and cytostatic agents or radiotherapy in human glioma xenografts are required to determine the place of anti-angiogenic therapy in general and treatment with the anti-angiogenic agent TNP-470 more specifically in the treatment of human gliomas.

Growth inhibition of intracerebral rat glioma by transfection-induced human interferon-beta.

In our previous study on liposome-mediated transfection of the human interferon-beta (HuIFN-beta) gene into subcutaneously implanted human gliomas in nude mice, we found that HuIFN-beta was produced and secreted by the tumor cells and that the growth of solid tumors was completely inhibited. The present study investigated the growth-inhibitory effect of liposomes containing the HuIFN-beta gene inserted into a vector (pSV2IFN-beta) on T9 rat glioma implanted into the brains of rats. Tumor cells and liposomes containing pSV2IFN-beta or other additives were simultaneously injected into the brains of rats. HuIFN-beta was detected in solid gliomas growing in the brains of rats injected with liposomes and magnetic resonance imaging (MRI) showed that tumor growth was inhibited. In addition, the latent period until the appearance of neurological symptoms was significantly prolonged in rats treated with liposomes containing pSV2IFN-beta. However, the survival time of the treated rats was not significantly increased.

Efficacy of direct intratumoral therapy with targeted protein toxins for solid human gliomas in nude mice

Targeted protein toxins are a new class of reagents with the potential for great tumor selectivity and cytotoxic potency. Two such compounds were studied: 1) Tf-CRM107, a conjugate of human transferrin (Tf) and diphtheria toxin with a point mutation (CRM107); and 2) 454A12-rRA, a conjugate of a monoclonal antibody (454A12) to the human Tf receptor and recombinant ricin A chain (rRA). Both compounds are potent and specific in killing human glioblastoma cell lines in vitro. The authors investigated the activity of these reagents administered intratumorally against solid U251 MG human gliomas in vivo. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were randomly assigned to be treated with 100-microliters intratumoral injections of Tf-CRM107 (10 micrograms) or 454A12-rRA (10 micrograms), equimolar doses of CRM107 (4.3 micrograms), 454A12 antibody (7.5 micrograms), or rRA (1.5 micrograms), or phosphate-buffered saline (PBS) every 2 days for a total of four doses. Tumor volume and animal weight were assessed by a blinded observer before each treatment and biweekly for 30 days after initiating therapy. With Tf-CRM107 administration, tumor regression of greater than 95% occurred by Day 14 (p < 0.01) and tumors did not recur by Day 30. Treatment with 454A12-rRA caused a 30% decrease in tumor volume by Day 14 (p < 0.01). Treatment with equimolar doses of the unconjugated targeted protein toxin components CRM107, 454A12, or rRA caused significant U251 MG tumor growth inhibition, but the effects were less potent than the antitumor effects of the conjugates. This study also characterized the dose-response effect of Tf-CRM107 on tumor growth and tumor weight on Day 30. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were treated with 100-microliters intratumoral injections of 10, 1.0, or 0.1 microgram of Tf-CRM107 or PBS every 2 days for a total of four doses. All three doses of Tf-CRM107 significantly inhibited tumor growth by Day 14 (p < 0.01) and at Day 30 (p < 0.05), with a significant dose-response relationship. This study demonstrated in vivo efficacy of the targeted toxins Tf-CRM107 and 454A12-rRA against a human glioma. With intratumoral administration, the effect of Tf-CRM107 was tumor-specific and in some animals curative. Regional therapy with these potent tumor-specific agents using direct intratumoral infusion should limit systemic toxicity and may be efficacious against brain tumors.

Growth inhibition of experimental glioma grafts by monoclonal antibody treatment.

The effects of 14AC1 monoclonal antibody (McAb) on 79FR-G-41 rat glioma cells in vitro, on the formation of metastases in lung by antibody coated glioma cells, and on the growth of glioma grafts in BALB/c-nu/nu mice were investigated. The 14AC1 antibodies - isotyped as IgG2a - were obtained from a hybridoma clone established after fusion of X63-Ag8.653 myeloma cells and spleen cells of BALB/c mice hyperimmunized with 79FR-G-41 glioma cells. Antibody treatment of glioma cells in vitro caused evident cell surface alterations and pronounced growth depression of most cells. However, a few tumor cells remained unchanged in morphology and continued to proliferate. Moreover, 14AC1 antibodies drastically reduced lung metastasis by pretreated and i.v. delivered glioma cells. Additionally, 14AC1 antibodies suppressed the growth of transplanted rat gliomas in nude mice as evidenced by a longer latency period and a smaller volume of glioma grafts in treated than in control tumor bearers. Nevertheless, glioma grafts showed accelerated growth after termination of antibody treatment. Further experimental investigation is required in order to identify the precise mechanisms of the effects of McAbs on tumor cells in vitro and in vivo.

Interactions of human fibroblast interferon with chemotherapeutic agents and radiation against human gliomas in nude mice.

The growth inhibitory effects of the combination of beta-interferon (beta-IFN) and conventional anticancer drugs such as adriamycin (ADM) and ACNU were evaluated in nude mice receiving subcutaneous transplants of human glioblastoma. Next, the anticancer and radiation sensitizing effects of beta-IFN on human glioblastoma was also evaluated in nude mice model. After three weeks of combined treatment, tumour reduction rates (treated/control values) were evaluated by the Battelle's method. In conclusion, the growth inhibitory effect of IFN was most enhanced when IFN was administered following radiotherapy. Furthermore, the combined effect was enhanced in proportion to the dose of radiation.

Interaction of Human Fibroblast Interferon with Chemotherapeutic Agents and Radiation against Human Gliomas in Nude Mice

Growth inhibitory effects of the combination of β-interferon (β-IFN) and conventional anticancer drugs such as adriamycin (ADM) and 1-(4-amino-2-methyl pyrimidine-5-yl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were evaluated in nude mice receiving subcutaneous transplants of human glioblastoma. Next, the anticancer and radiation sensitizing effects of β-IFN on human glioblastoma were also evaluated in the nude mouse model. Tumor reduction rates (treated/control values of the tumor volume) were evaluated by Battelle's method after 3 weeks of the combination treatment. The tumor reduction rates of each treatment group were as follows: β-IFN alone, 63%; ADM (8 mg/kg) alone, 48%; β-IFN plus ADM, 36%; ACNU (20 mg/kg) alone, 44%; β-IFN plus ACNU, 36%; radiation (390 rad) alone, 61%; β-IFN plus radiation (390 rad), 35%; radiation (650 rad) alone, 51%; and β-IFN plus radiation (650 rad), 26%. The growth inhibitory effect of β-IFN was most enhanced when β-IFN was administered following radiotherapy. Furthermore, the combination effect was enhanced in proportion to the dose of radiation.

Quantification of glucose utilization in an experimental brain tumor model by the deoxyglucose method.

Reevaluation of lumped and rate constants is necessary when Sokoloff's 2-deoxyglucose (DG) method is used to measure glucose utilization in pathological tissue. We describe here a modification of Sokoloff's lumped constant measurement that permits simultaneous estimation of both lumped and rate constants from a single animal experiment. A subcutaneous tumor model (AA ascites tumor) was used for measurement of these constants with a procedure similar to Sokoloff's that kept the plasma tracer concentration constant. Measured constants were as follows: lumped constant, 0.654 +/- 0.081; k1, 0.196 +/- 0.038 min-1; k2, 0.262 +/- 0.067 min-1; k3, 0.117 +/- 0.044 min-1. These constants were used to quantify glucose utilization in the implanted brain tumor. To test the validity of this method, we compared a fraction of the free DG pool calculated using the tumor constants with a fraction measured directly by chromatographic analysis of tissue samples from both subcutaneous tumor and implanted brain tumor. The values derived by chemical analysis agreed well with those predicted by the calculations. The value of k4 varied from 0.0031 +/- 0.0018 min-1 for the tumor tissue to 0.0214 +/- 0.0024 min-1 for tumors with a large necrotic center. This method would be especially useful when applied to xenograft human gliomas in nude mice for quantification of glucose utilization in human gliomas by means of positron emission tomography.