Combining organotypic tissue culture with multi-color fluorescence light-sheet microscopy (OTCxLSFM) – a novel tool to study glioma invasion/migration

Abstract

Glioblastoma is a very aggressive tumor and represents the most common primary brain malignancy. Key characteristics include its high resistance against conventional treatments, such as radio- and chemotherapy and its diffuse tissue infiltration, preventing complete surgical resection. The analysis of migration and invasion processes in a physiological microenvironment allows for enhanced understanding of these processes and can lead to improved therapeutic approaches. Here, we combine two state-of-the-art techniques, adult organotypic brain tissue slice culture (OTC) and light sheet fluorescence microscopy (LSFM) of cleared tissues in a combined method termed OTCxLSFM. Using this methodology, we can show that glioblastoma tissue infiltration can be effectively blocked through treatment with arsenic trioxide, as well as genetic depletion of the tetraspanin, transmembrane receptor CD9. With our analysis-pipeline we gain single-cell level, three-dimensional information, as well as insights into the morphological appearance of the tumor cells.

BRAT1 - a new therapeutic target for glioblastoma

“…Irradiated tumors appeared similarly invasive to solvent-treated tumors, but with decreased sizes. To confirm this observation we applied an imaging technique combining OTC culture with light sheet fluorescence microscopy (LSFM) called OTCxLSFM (Haydo et al, 2023) to measure the tumors and its particular single cell invasion in a 3D manner and display it in 90° angles (Figure 5D). This advanced microscopy approach confirmed that DMSO-treated tumors display diffuse 3D infiltration and spreading of cells into the surrounding brain tissue.…”

“…Figure 5A), similarly as genetic BRAT1-depletion. Likewise, migration of the GSC line GS-5 (Günther et al, 2008) on laminincoated plates was inhibited by CurD using a sphere migration assay (Haydo et al, 2023) (Suppl. Figure 5B).…”

“…Based on the above-presented data we inferred that 1) BRAT1-depletion might hinder tumor growth in more complex model systems and 2) BRAT1-depletion might also negatively impact tumor cell migration/invasion. In order to address both question we used an ex vivo approach consisting of organotypic tissue slice cultures (OTCs) of adult murine brains unto which GFPpositive tumor spheres are transplanted (Haydo et al, 2023;Gerstmeier et al, 2021;Linder et al, 2019;Remy et al, 2022). Fluorescently labeled tumor cells, in this case NCH644 GFP GSCs (Haydo et al, 2023) were placed onto murine brain slices and tumor development up to 17 d was monitored.…”

“…In order to address both question we used an ex vivo approach consisting of organotypic tissue slice cultures (OTCs) of adult murine brains unto which GFPpositive tumor spheres are transplanted (Haydo et al, 2023;Gerstmeier et al, 2021;Linder et al, 2019;Remy et al, 2022). Fluorescently labeled tumor cells, in this case NCH644 GFP GSCs (Haydo et al, 2023) were placed onto murine brain slices and tumor development up to 17 d was monitored. We applied 5 µM CurD to the established tumors on the OTCs at d0, which was followed by a fractionated radiation protocol consisting of 3 times of 2 Gy for two weeks.…”

“…Understanding the molecular mechanisms driving GBM invasion and migration is crucial for the development of novel therapeutic strategies. Moreover, advances in imaging techniques, like OTCxLSFM, provide valuable tools for studying GBM invasion dynamics and evaluating the efficacy of anti-invasive therapies (Haydo et al, 2023). By elucidating the complexities of GBM cell invasion and migration, may lead to the development of more effective therapeutic interventions for this devastating disease.…”