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Media type:
Doctoral Thesis
Title:
Glioblastoma Tissue Slice Tandem-Cultures for Quantitative Evaluation of Inhibitory Effects on Invasion and Growth
:
Dissertation zur Erlangung des akademischen Grades Dr. med. an der Medizinischen Fakultät der Universität Leipzig
Description:
A promising approach for the study of Glioblastoma are organotypic murine brain tissue slices as a substrate for the glioma cells to invade into. Current 3D assays based on this principle involved the use of tumor spheroids or cell suspensions in co-culture with the brain tissue slices. While this allowed for the study of glioma cell invasion, tumor spheroids lack the microarchitecture of patient-derived tumor tissue or glioma xenografts.This study has expanded this type of assay by investigating the viability of glioma xenograft slices in co-culture with organotypic murine brain tissue slices, proposing facile quantification methods of tumor growth and invasion and using this system for studying the effects of small molecule inhibitors.The organotypic murine brain tissue slices were prepared by slicing mouse brains in the coronal axis, using a vibratome, to a thickness of 300 µm. The slices were then transferred onto tissue culture membrane inserts for growth in air-liquid interface culture. A single mouse brain allowed for the production of multiple organotypic murine brain tissue slices, thus drastically reducing the number of animals needed for the study. GBM tumor xenografts from mice were sliced similarly on a vibratome, and circular portions with a diameter of 2 mm were placed on top of the murine brain tissue slices. After 7 days in culture, tissue slice co-cultures were analyzed by immunohistochemical staining of vertical sections, containing both the tumor and the murine brain tissue, for Type III intermediary filament proteins Vimentin and GFAP and the neural crest cell marker S-100. Independent of the cell line used for xenograft preparation, tumor tissues stained strongly positive for Vimentin, while the normal mouse brain tissue stained negative (in the studied region), so Vimentin was used as the primary tumor marker.For the quantification of the data acquired from micrographs, the tumor height, depth as well as the area of the invading cells and the tumor upper area (situated above the air margin of the brain tissue slice), the tumor lower area and the recipient tissue area were measured. From these parameters, a number of indices for each sample were derived, such as the tumor invasion index (TI-index), the tumor space occupying growth index (SOG-index), the tumor invasion depth index (TID-index), the space occupying growth depth index (SOGD-index) and the total tumor depth index (TTD-index). To validate the proposed quantification method, the results were compared with tumor spheroid tandem co-cultures.It was shown to be possible for GBM tumor xenografts to maintain their viability and invasive properties in co-culture with organotypic murine brain tissue slices. This was demonstrated immunohistochemically with xenografts from GBM cell lines G55T2, U-87 MG, LN-229 and T98G, displaying progressive tumor cell invasion from day 3 to 7 in co-culture. Tumor cell viability and proliferation in the ex vivo setting were also confirmed by Ki-67 staining.The usage of GBM tumor xenografts was also advantageous compared to spheroid-based assays. The direct comparison between the tumor spheroid and tumor xenograft co-cultures showed stark differences between assays even when using the same cell line. U-87 MG cells showed little or no invasiveness in the spheroid model but the glioma cells were diffusely spread into the murine brain tissue in the xenograft model. G55T2 xenograft co-cultures on the other hand displayed a significant increase of the SOG-index compared to their spheroid counterparts. Results were also compared to previous findings in an orthotopic tumor xenograft model, concluding that the proposed ex vivo model showed significant advantages compared to the orthotopic model by being more facile and cost-effective to implement and displaying comparatively more profound tumor invasiveness when studying the same cell lines. The strong increase of the invasive and space assuming properties of glioma cells in xenograft tissue slice tandem cultures also supports the hypothesis that they are superior to spheroid-based assays in studying tumor invasiveness.The developed GBM xenograft tissue slice tandem-cultures were also used for the ex vivo analysis of drug effects. The treatment with the Pim1 small molecule inhibitor SGI-1776 revealed after 7 days a decrease in the TI-index and SOG-index compared to the untreated group. A similar experiment was performed on spheroid co-cultures using a combination of SGI-1776 and the STAT3 inhibitor Stattic, also resulting in reduced TI- and SOG-indices.From this work, it can be concluded that the developed 3D ex vivo method is a facile and cost-effective platform to study the growth and invasiveness of GBM xenograft tumors in an in vivo-like environment. Owing to the large number of samples that can be generated from a single mouse, it has the potential to drastically reduce the number of animal experiments, addressing the 3R principle. It also showed more profound tumor cell invasiveness compared to spheroid-based ex vivo or orthotopic in vivo xenograft models and provides the quantification tools needed for preclinical drug testing. The model also has the potential to be expanded towards the usage of patient-derived tumor tissue as well as the preclinical testing of non-drug-based therapy options.