Paul-Flechsig-Institut für Hirnforschung
 Universitätsmedizin Leipzig

miRNA in Glioblastoma multiforme

The most frequent malignant braintumor “Glioblastoma multiforme” (GBM) is characterized by an extraordinary histomorphological and molecular diversity. While it is well known that tumorcells cause massive changes in their microenvironment, mainly in the extracellular matrix (ECM) and the local physiological cells, less is known about the reverse effect of the ECM on tumorcells and the malignant potential of bystander cells as part in creating the intratumoral heterogeneity.

Micro-RNAs (miRNAs) are short, non-coding RNA-sequences known to have significant impact on translation-levels via a post-transcriptional mechanism in physiological cells as well as in malignancies like GBM. Their indirect (via Exosomes) and direct (via Gap Junctions (GJ)) transferability make these molecules prime candidates for the regulation of the tumorcell-microenvironment-interplay.

The designation "Glioblastoma multiforme"is derived from histomorphological characteristics but also displayed on a molecular level: Verhaak et al. showed the existence of 4 molecular subtypes (pro-neural, neural, classic, mesenchymal) based on distinct genomic aberrations and mRNA-expressionprofiles. Both biopsies from different areas of single tumors as well as single cell experiments were able to show that cell populations with distinct alterations exist even within a single tumor It is however not yet known, which factors contribute to the occurrence of intratumoral heterogeneity and how local cells like reactive astroglia impact on the formation of different cell populations. MiRNAs are highly likely to be involved into this interplay due to their high transferability.

This projects aims at characterizing the direct transfer of miRNA via GJ in the tumorcell-tumorcell as well as in the tumorcell-local cell-setting. As initial steps the transfer of fluorescent miRNA-mimics from tumor cells to physiological cell types (glia, neurons) will be monitored on the basis of co-cultures and xenografts of tumor cells on mouse brain slice cultures (fluorescence microscopy, RT-PCR). Duration and extent of changes in the host-cells will be characterized by further cell culture experiments (resistance to apoptotic stimuli, proliferation, differentiation).

Furthermore we will evaluate the connectivity in slice cultures of freshly resected human GBM via calcium-imaging as well as by the application of GJ-permeable markers such as Neurobiotin. In a second step we will monitor the transfer of fluorescent miRNA-mimics within slice cultures.

A subproject will focus on intratumoral heterogeneity on the basis of miRNA-expression profiles. Histomorphologically characterized areas will be excised by laser-capture-microdissection (LCM) and will be submitted to massive parallel sequencing of miRNA-transcripts in order to compare their expression profiles. The goal is to provide a link between histological and molecular characteristics.

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Primary cells of GBM can be cultured in suspension as spheroids (Figure 1). These cells can be grafted onto organotypical brain slice cultures of immunocompetent mice by light-microscopical guidance in order to evaluate the interplay of tumor cells and their microenvironment. Figure 2 shows tumorcells (Vimentin, red/yellow) adjacent to local astrocytes (GFAP, green).

 
last update: 15.05.2020, 09:39 Uhr
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Paul-Flechsig-Institut für Hirnforschung