Glioblastoma multiformae (GBM) is one of the most lethal cancers, the median survival being about 12 months. Novel concepts of tumour origin propose that a tumour initiating cells with stem cell-like characteristics – GBM stem cells (GSC) are giving rise to malignant tissue. Selective targeting of these cells, including infiltrating mesenchymal stem cells (MSC), emerged as a new approach in GBM treatment. Part A:GSC proteome – PM associated protein markers For research and clinical application we need novel GSC protein markers, which would distinguish the GSCs from normal neural stem cells (NSC). These markers will also be useful for diagnosis and prognosis of patients’ survival and serve as potential targets for more efficient therapy. As no reliable marker(s) exist up-to-date, we propose to use the proteomic approach on plasma membrane (PM)–associated proteins to better define the GSC fingerprint. Specifically: 1. We aim to reveal the PM proteome by label-free geLC-MS-based proteomics in GBM spheroids (tumorospheres) derived from human GBM biopsy, which are highly enriched in GSCs and compare it with the NSC spheroids (neurospheres). Spheroids grown in serum-based and serum-free media will be tested. 2. The most significant differentially expressed proteins in tumorospheres vs neurospheres will be validated on GBM tissue microarrays (TMA), constructed from glioma patients, to reveal their diagnostic and predictive value. Most relevant markers will be subjected to functional assays in vitro and in vivo. Part B:Phenotypic characterisation of GSC Infiltrative invasion of GBM cells is the major hallmark of this tumour. It has been postulated that GSC also develop a migratory phenotype associated with epithelial-to-mesenchymal-transition (EMT), similar to epithelial cancers. We hypothesize on a reversible switch from stationary to migratory (invasive) phenotype of GSC and propose to define transcriptome and degradome alterations of EMT, associated with PM-proteome in GSCs. Specifically: 1. We aim to compare the transcriptome of GSC tumorospheres with the transcriptome of highly migratory NSC neurospheres. The “stationary” transcriptome of the cells will be compared to their “activated migratory” transcriptome (migratome) upon the environmental stimulation of EMT with (a) the addition of serum and (b) exposure to hypoxic conditions. 2. Particular emphasis will be given to the part of GSC migratome, associated with the degradome (proteases) to reveal signalling pathways linking protease transcriptome to the process of EMT. 3. Novel GSC markers (GSC-specific & EMT-relevant) will be validated and functionally assessed by silencing/knockdown and biological/chemical inactivation in the invasion studies. Particular emphasis will be given to comparison of PM-associated protease profile based on PM-associated proteome of GSC (Part A). Part C:GSC microenvironment – infiltrating MSC Tumour microenvironment has an impact on EMT/MET. As part of it, endogenous GBM associated MSC (gbMSC) may have an impact on GSC. The interactions of bone marrow derived (BM)MSC, gbMSC and GSC will be studied in vitro and in vivo in animal model. We aim to resolve the transcriptomic, metabolic and paracrine response of co-cultured cells in vitro.
Significance for science
This project combines tumour biology and stem cell research with systems biology what is an important novel scientific approach. Following changes of a great number of expressed genes, proteins and regulatory molecules at different in vitro conditions, which mimic those in the tumour in vivo , we aim to better understand the characteristics of glioblastoma (GBM) in comparison to normal neural stem cells. The appropriate selection of methods and partners will ensure the link between experimental research on clinical samples (providing the data of so called “wet laboratory research”) bioinformatics, using tools (“dry laboratory”) that would select relevant results from a large number of data and this contributing to understanding of the cell processes under investigation. Proteomic approach will be used to define differentially expressed proteins on the surface (plasma membranes) of GBM vs neural stem cells, that will reveal new proteins, which would more selectively as presently known, characterise tumour stem cells. New biomarkers may impact prognosis and predict the response to therapy and by the same token represent new targets for drug design. Following the proteins and their regulatory molecules (cytokines, microRNA),as well as metabolic state of GBM stem cells at various environmental conditions (for example hypoxia), investigations will be focused on their variability in invasive properties which are crucial for tumour spread. Identification of genes and proteins/proteases, activated at the reversible transition from stationary to migratory phenotypes (EMT transition) is important for understanding, and for nhibition of invasion. It is even less known, how mesenchymal stem cells (MSC), infiltrate into GBM from bone marrow and thereby become a part of tumour microenvironment and effect behaviour of tumour cells, in particular the GBM stem cells. Using the same systems approach, as indicated above, we will follow their mutual interactions by identifying release of regulatory molecules (cytokine, microRNA) that mediate the cross-talk between GBM stem cell and MSC. Several even contradictory models on cancer initiation by GBM stem cells exist. With the proposed experimental approach we may even find the new hypothesis on the initiation and invasive progression of GBM as the result of stem cells. By the proposed is original approach we aim to reveal the group of molecules and point to important signal pathways in the initiation and progression of GBM, as originating from tumour stem cells and their interactions with changing environment (such as hypoxia) and other micro environmental factors, and on the other hand infiltrating mesenchymal stem cells (MSC). Taken together, the proposed project predicts to reveal novel biological markers of tumour stem cells, important to application in clinical oncology as targets for treating GBM