Article
Human organotypic glioblastoma tissue slices for therapy improvement can be analyzed by transcriptome-wide next-generation sequencing, whole slice immunochemical analysis and immunoblotting
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Authors
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Susann Haehnel
- Universität Leipzig, Medizinische Fakultät Leipzig, Institut für Anatomie, Leipzig, Deutschland
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Kristin Reiche
- Fraunhofer Institute for Cell Therapy and Immunology, Department of Diagnostics, Leipzig, Deutschland
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Henry Oppermann
- Klinik und Poliklinik für Neurochirurgie, Universitätsklinikum Leipzig, Universitätklinikum Leipzig, Leipzig, Deutschland
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Karsten Winter
- Universität Leipzig, Medizinische Fakultät Leipzig, Institut für Anatomie, Leipzig, Deutschland
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Jürgen Meixensberger
- Universität Leipzig AöR, Universitätsklinikum Leipzig, Klinik und Poliklinik für Neurochirurgie, Leipzig, Deutschland
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Ingo Bechmann
- Universität Leipzig, Medizinische Fakultät Leipzig, Institut für Anatomie, Leipzig, Deutschland
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Frank Gaunitz
- Medizinische Fakultät, Klinik und Poliklinik für Neurochirurgie, Forschungslabore, Leipzig, Deutschland
| Published: | June 9, 2017 |
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Outline
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Objective: The individual treatment of brain tumor patients requires experimental models that can be used to propose the outcome of a certain treatment strategy. Today, cell culture or mouse models are frequently used for the development of test systems that poorly reflect the tumor’s biology. Hence, we asked whether organotypic slice cultures can be used for an in depth analysis of tumor response to treatment. Here, we describe an automated analysis of whole slices subjected to immunochemistry and the isolation of proteins for immunoblotting. Most importantly, we demonstrate that transcriptome-wide next-generation sequencing of RNA isolated from slices can successfully be applied.
Methods: Tissue slices (~350 µm) were obtained from surgically removed glioblastoma and normal brain tissue using a tissue chopper. Slices were treated by irradiation (4 Gy) and Temozolomide (200 µM). Caspase-3 and Ki-67 expression within the tissue were determined from whole slices using an automated scanning technology. In addition, protein was isolated for immunoblot analysis and RNA was processed for strand-specific transcriptome-wide NGS sequencing.
Results: Normal brain tissue that had to be removed to get access to the tumor and corresponding glioblastoma tissue from the same patient were used for organotypic slice cultures. Agilent BioAnalyzer quality control revealed high quality RNA and libraries could be established and subjected to transcriptome analysis. A total of ~10.000 genes were detected with at least 1 transcript per million in all samples at a moderate sequencing depth (5 to 8x106 counts per sample). Principal component analysis of annotated genes revealed expression variance between normal and tumor tissue as well as between treated and untreated samples. Automated quantification of whole slices revealed a high correlation with data obtained by manually counting cells. Moreover, we could identify components of signal transduction pathways and their phosphorylation by immunoblotting.
Conclusion: Here we demonstrate that tissue slices obtained from surgically removed glioblastoma tissue can be analyzed by transcriptome-wide RNA sequencing after treatment in culture. In addition, automated quantification of immunohistochemistry is possible and proteins isolated from slices can be used for immunoblotting. This offers the opportunity for an in depth analysis of the response of individual patient-derived glioblastoma tissue to treatment, which is an important step towards personalized treatment of patients.
