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Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts.

Liu Y, Lang F, Xie X, Prabhu S, Xu J, Sampath D, Aldape K, Fuller G, Puduvalli VK - Cell Death Dis (2011)

Bottom Line: It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays.Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity.Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77035, USA.

ABSTRACT
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

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Effect of AdsTRAIL on human glioma organotypic slice cultures. Organotypic slice cultures were generated from freshly resected human glioma specimens and the slices treated with PBS, AdEGFP, or AdsTRAIL (100 MOI) for 48 h. (a) Paraffin-embedded slices were stained with H&E or immunostained with a cleaved caspase 3 antibody. Arrows indicate pyknotic and fragmented apoptotic bodies in the H&E sections and positive staining for cleaved caspase 3. (b) In situ TUNEL staining of glioma slices with arrows showing positively stained cells in the lower panel and apoptotic cells in the upper panel. (c) A single-cell suspension was generated from slice cultures treated with PBS, AdEGFP, or AdsTRAIL and subjected to flow-cytometric analysis. The time course of appearance of the sub-G1 fraction indicating apoptotic cells was determined for the periods indicated
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fig6: Effect of AdsTRAIL on human glioma organotypic slice cultures. Organotypic slice cultures were generated from freshly resected human glioma specimens and the slices treated with PBS, AdEGFP, or AdsTRAIL (100 MOI) for 48 h. (a) Paraffin-embedded slices were stained with H&E or immunostained with a cleaved caspase 3 antibody. Arrows indicate pyknotic and fragmented apoptotic bodies in the H&E sections and positive staining for cleaved caspase 3. (b) In situ TUNEL staining of glioma slices with arrows showing positively stained cells in the lower panel and apoptotic cells in the upper panel. (c) A single-cell suspension was generated from slice cultures treated with PBS, AdEGFP, or AdsTRAIL and subjected to flow-cytometric analysis. The time course of appearance of the sub-G1 fraction indicating apoptotic cells was determined for the periods indicated

Mentions: Serially passaged cell lines are subject to genetic drift that often limits their utility in determining biological behavior and treatment response of human gliomas. Preclinical studies that rely solely on testing the effects of novel agents against such established cell lines or in vivo tumors derived from them retain the limitation of the cells. Although these limitations can be partly overcome by the direct use of human tumor tissue in preclinical studies, there are no models that currently allow such ex vivo studies in cancer. To address this, we developed a novel human glioma organotypic slice culture model based on similar cultures of normal rodent brain slices used in neurophysiological studies,17 which maintains genetic identity with the patient's tumor; this model was utilized to test the effect of AdsTRAIL in viable human glioma tissue. Freshly resected gliomas were processed to obtain organotypic slices, which were then treated with PBS, AdEGFP, or AdsTRAIL for up to 72 h. The slices were harvested, fixed in formalin, and embedded in paraffin. Slides were generated and used for immunohistochemical studies. Hematoxylin and eosin (H&E)-stained sections of the untreated control slices showed the characteristic features of malignant glioma. Slices treated with AdsTRAIL showed the presence of cells with condensed and pyknotic nuclei consistent with apoptotic bodies; these cells showed positivity for adenoviral hexon expression, indicating generation of viral protein in the transduced cells. The slices were also positive for cleaved caspase 3 (Figure 6a) and in situ TUNEL staining (Figure 6b), indicating activation of caspases and DNA fragmentation. In contrast, AdEGFP-treated cells, which also showed hexon expression, did not show positivity for either cleaved caspase 3 or TUNEL staining. To confirm these findings, the slices were disaggregated into single-cell suspension after treatment, fixed and stained with propidium iodide, and analyzed by flow cytometry for evidence of apoptosis. AdsTRAIL-treated cells showed a time-dependent increase in the sub-G1 fraction (Figure 6c). These results provide direct evidence of the ability of AdsTRAIL to induce apoptosis in human glioma tissue and strongly suggest that a similar effect could be expected in vivo if the agent is delivered to the tumor.


Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts.

Liu Y, Lang F, Xie X, Prabhu S, Xu J, Sampath D, Aldape K, Fuller G, Puduvalli VK - Cell Death Dis (2011)

Effect of AdsTRAIL on human glioma organotypic slice cultures. Organotypic slice cultures were generated from freshly resected human glioma specimens and the slices treated with PBS, AdEGFP, or AdsTRAIL (100 MOI) for 48 h. (a) Paraffin-embedded slices were stained with H&E or immunostained with a cleaved caspase 3 antibody. Arrows indicate pyknotic and fragmented apoptotic bodies in the H&E sections and positive staining for cleaved caspase 3. (b) In situ TUNEL staining of glioma slices with arrows showing positively stained cells in the lower panel and apoptotic cells in the upper panel. (c) A single-cell suspension was generated from slice cultures treated with PBS, AdEGFP, or AdsTRAIL and subjected to flow-cytometric analysis. The time course of appearance of the sub-G1 fraction indicating apoptotic cells was determined for the periods indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101700&req=5

fig6: Effect of AdsTRAIL on human glioma organotypic slice cultures. Organotypic slice cultures were generated from freshly resected human glioma specimens and the slices treated with PBS, AdEGFP, or AdsTRAIL (100 MOI) for 48 h. (a) Paraffin-embedded slices were stained with H&E or immunostained with a cleaved caspase 3 antibody. Arrows indicate pyknotic and fragmented apoptotic bodies in the H&E sections and positive staining for cleaved caspase 3. (b) In situ TUNEL staining of glioma slices with arrows showing positively stained cells in the lower panel and apoptotic cells in the upper panel. (c) A single-cell suspension was generated from slice cultures treated with PBS, AdEGFP, or AdsTRAIL and subjected to flow-cytometric analysis. The time course of appearance of the sub-G1 fraction indicating apoptotic cells was determined for the periods indicated
Mentions: Serially passaged cell lines are subject to genetic drift that often limits their utility in determining biological behavior and treatment response of human gliomas. Preclinical studies that rely solely on testing the effects of novel agents against such established cell lines or in vivo tumors derived from them retain the limitation of the cells. Although these limitations can be partly overcome by the direct use of human tumor tissue in preclinical studies, there are no models that currently allow such ex vivo studies in cancer. To address this, we developed a novel human glioma organotypic slice culture model based on similar cultures of normal rodent brain slices used in neurophysiological studies,17 which maintains genetic identity with the patient's tumor; this model was utilized to test the effect of AdsTRAIL in viable human glioma tissue. Freshly resected gliomas were processed to obtain organotypic slices, which were then treated with PBS, AdEGFP, or AdsTRAIL for up to 72 h. The slices were harvested, fixed in formalin, and embedded in paraffin. Slides were generated and used for immunohistochemical studies. Hematoxylin and eosin (H&E)-stained sections of the untreated control slices showed the characteristic features of malignant glioma. Slices treated with AdsTRAIL showed the presence of cells with condensed and pyknotic nuclei consistent with apoptotic bodies; these cells showed positivity for adenoviral hexon expression, indicating generation of viral protein in the transduced cells. The slices were also positive for cleaved caspase 3 (Figure 6a) and in situ TUNEL staining (Figure 6b), indicating activation of caspases and DNA fragmentation. In contrast, AdEGFP-treated cells, which also showed hexon expression, did not show positivity for either cleaved caspase 3 or TUNEL staining. To confirm these findings, the slices were disaggregated into single-cell suspension after treatment, fixed and stained with propidium iodide, and analyzed by flow cytometry for evidence of apoptosis. AdsTRAIL-treated cells showed a time-dependent increase in the sub-G1 fraction (Figure 6c). These results provide direct evidence of the ability of AdsTRAIL to induce apoptosis in human glioma tissue and strongly suggest that a similar effect could be expected in vivo if the agent is delivered to the tumor.

Bottom Line: It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays.Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity.Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro-oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77035, USA.

ABSTRACT
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

Show MeSH
Related in: MedlinePlus