Experimental Intracranial Glioma Research Articles

Abstract 3255: The role of Hdj2 protein in metastatic progression of experimental intracranial glioma

Abstract Glioblastoma is one of the most malignant cancer types with median survival of 15 months regardless of the application of combined radio- and chemotherapy. Expression of Hdj2 negatively correlates with the increase of tumor aggressiveness. For assessment of Hdj2 role in tumor progression, invasiveness and metastasis formation in gliomas we developed three C6-based cell lines with Hdj2 and related proteins knock-down: C6 shHsp70, C6 shHdj1 and C6 shHdj2 itself. Following intracranial injection of the modified tumor cells the animals’ survival was estimated. As compared to control C6 (25.4±3.9 days) and C6 shHdj1 (25.5±3.8) we observed a nearly 1.5-fold decrease in survival in C6 shHdj2 (16.8±3.5 days) (P<0.05). On the contrary, in C6 shHsp70 group the survival increased up to 42.5±12.0 days (the increase is completely explainable by the slower growth rate of the culture). Subsequent MR imaging and histological analysis of the tumors demonstrated elevated invasiveness and metastatic activity in C6 shHdj2 group in comparison to C6 shHsp70, C6 shHdj1 and control C6. High migration activity and the ability of detached C6 shHdj2 cells to settle on the substrate was proved in wound-healing assay, colony forming assay and calcium-dependent cell aggregation assay. Immunofluorescence analyses showed loss of membrane-expressed N-cadherin and loss of intercellular contacts mediated by N-cadherin in C6 shHdj2 cells in comparison to other considered cell lines. Actin staining with rhodamine-falloidin revealed highly abundant leading edges in C6 shHdj2 culture. Decrease of Hdj2 expression in gliomas has proved to be of high importance in tumor progression and formation of metastasis. Citation Format: Darya A. Meshalkina, Maxim A. Shevtsov, Boris A. Margulis, Irina V. Guzhova. The role of Hdj2 protein in metastatic progression of experimental intracranial glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3255. doi:10.1158/1538-7445.AM2015-3255

FasL gene knock-down therapy enhances the antiglioma immune response

Malignant glioma is a lethal form of brain cancer that is very difficult to treat. The aggressive behavior of these neoplasms and their limited responsiveness to therapy has been attributed in part to the ability of these tumors to evade the immune system. Gliomas, like many other solid tumors, express components of numerous immune escape mechanisms, including immunosuppressive proteins such as TGF-beta, IL-10, and FasL. Here, we show that FasL expression can support the growth of experimental intracranial glioma. We show that FasL is readily detected in human glioblastoma multiforme clinical specimens. FasL was found to be expressed by three well-characterized rat glioma cell lines (9L, F98, and C6) and glioma cell-derived FasL mediated the death of phytohemagglutinin-stimulated Jurkat T-lymphocytes when cocultured with glioma cells in vitro. We asked if inhibiting 9L-derived FasL altered the growth of experimental glioma. FasL expression knockdown using shRNA reduced the growth of subcutaneous and intracranial 9L gliomas by approximately 50% in immune competent Fisher 344 rats. In contrast, FasL expression knockdown had no affect on the growth of intracranial 9L glioma in T-cell deficient athymic rats. Intracranial tumors derived from FasL knockdown 9L glioma cells contained up to 3-fold more tumor infiltrating T-cells than tumors derived from control 9L cells. These results demonstrate that down-regulating FasL expression and/or function in glial malignancies can enhance T-cell tumor infiltration and inhibit tumor growth. The findings suggest that targeting endogenous FasL in glial malignancies could enhance the efficacy of emerging immune-based treatment strategies.

Inhibition of Akt inhibits growth of glioblastoma and glioblastoma stem-like cells

A commonly activated signaling cascade in many human malignancies, including glioblastoma multiforme, is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor, PTEN deletion, or PIK3CA mutations. In this study, we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule, A-443654, showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings, this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654, methylated at a region that blocks Akt binding, was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion, respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma, with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions.

Interstitial Docetaxel (Taxotere), Carmustine and Combined Interstitial Therapy: a Novel Treatment for Experimental Malignant Glioma

Docetaxel (Taxotere) is a hemisynthetic, anti-cancer compound with good preclinical and clinical activity in a variety of systemic neoplasms. We tested its activity against malignant gliomas using local delivery methods. Antitumor activity was assessed in vitro against human (U87 and U80 glioma) and rat brain-tumor (9L gliosarcoma and F98 glioma) cell lines. For in vivo evaluation, we incorporated docetaxel into a biodegradable polymer matrix, determined associated toxicity in the rat brain, and measured efficacy at extending survival in a rat model of malignant glioma. Also, we examined the combined local delivery of docetaxel with carmustine (BCNU) against the experimental intracranial glioma. Rats bearing intracranial 9L gliosarcomas were treated 5 days after tumor implantation with various polymers (placebo, 5% docetaxel, 3.8% BCNU, or 5% docetaxel and 3.8% BCNU combination). Animals receiving docetaxel polymers (n=15, median survival 39.1 days) had significantly improved survival over control animals (n=12, median survival 22.5 days, P=0.01). Similarly, animals receiving BCNU polymers (n=15, median survival 39.3 days, 13.3% long-term survivors) demonstrated an increase in survival compared to the controls (P=0.04). Animals receiving the combination polymers demonstrated a modest increase in survival compared to either chemotherapeutic agent alone (n=14, median survival 54.9 days, 28.6% long-term survivors) with markedly improved survival over controls (P=0.003). We conclude that locally delivered docetaxel shows promise as a novel anti-glioma therapy and that the combination of drug regimens via biodegradable polymers may be a great therapeutic benefit to patients with malignant glioma.

Local Delivery of Interleukin-2 and Adriamycin is Synergistic in the Treatment of Experimental Malignant Glioma

Local delivery of adriamycin (ADR) via biodegradable polymers has been shown to improve survival in rats challenged intracranially with 9L gliosarcoma. Likewise, local delivery of interleukin-2 (IL-2) has been shown to extend survival in experimental brain tumor models. In the current study, we hypothesized that local delivery of ADR and IL-2 might act synergistically against experimental intracranial glioma. Polyanhydride polymers (PCPP-SA) containing 5% ADR by weight were prepared using the mix-melt method. IL-2 polymer microspheres (IL-2 MS) were produced via the complex coacervation of gelatin and chondroitin sulfate in the presence of IL-2. Sixty male Fisher 344 rats received an intracranial challenge with a lethal dose of 9L gliosarcoma cells. In addition, a group of rats were injected with either IL-2 MS or empty microspheres. Five days later they received ADR or blank polymer. There were a total of four treatment groups: (1) empty microspheres, blank polymer; (2) empty microspheres, ADR polymer; (3) IL-2 MS, blank polymer; and (4) IL-2 MS, ADR polymer. Compared to control animals treated with empty microspheres and blank polymer, animals receiving empty microspheres and ADR polymer (P < 0.0004), IL-2 MS and blank polymer (P < 0.0005), and IL-2 MS combined with ADR polymer (P < 0.0000002) all showed statistically significant improvement in survival. In addition, animals receiving the IL-2/ADR combination had significantly extended survival compared to either ADR or IL-2 alone (P < 0.000003 and P < 0.0004, respectively). Both ADR and IL-2, when delivered locally, are effective monotherapeutic agents against experimental intracranial gliosarcoma. The combination ADR and IL-2 therapy is more effective than either agent alone.

Intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and non-neural origin.

The remarkable migratory and tumor-tropic capacities of neural stem cells (NSCs and/or neuroprogenitor cells) represent a potentially powerful approach to the treatment of invasive brain tumors, such as malignant gliomas. We have previously shown that whether implanted directly into or at distant sites from an experimental intracranial glioma, NSCs distributed efficiently throughout the main tumor mass and also tracked advancing tumor cells, while stably expressing a reporter transgene. As therapeutic proof-of-concept, NSCs genetically modified to produce the prodrug activating enzyme cytosine deaminase (CD), effected an 80% reduction in the resultant tumor mass, when tumor animals were treated with the systemic prodrug, 5-fluorocytosine. We now extend our findings of the tumor-tropic properties of NSCs (using a well-characterized, clonal NSC line C17.2), by investigating their capacity to target both intracranial and extracranial tumors, when administered into the peripheral vasculature. We furthermore demonstrate their capacity to target extracranial non-neural tumors such as prostate cancer and malignant melanoma. Well-characterized NSC lines (lacZ and/or CD-positive) were injected into the tail vein of adult nude mice with established experimental intracranial and/or subcutaneous flank tumors of neural and non-neural origin. The time course and distribution of NSCs within the tumor and internal organs was assessed in various models. Resulting data suggest that NSCs can localize to various tumor sites when injected via the peripheral vasculature, with little accumulation in normal tissues. Our findings suggest the novel use of intravascularly administered NSCs as an effective delivery vehicle to target and disseminate therapeutic agents to invasive tumors of neural and nonneural origin, both within and outside of the brain.