Projects / Programmes
Dual nature of stem cells in cancer and their application in therapy
| Code |
Science |
Field |
Subfield |
| 1.03.00 |
Natural sciences and mathematics |
Biology |
|
| Code |
Science |
Field |
| B001 |
Biomedical sciences |
General biomedical sciences |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
Bioinformatics (Degradome, Migratome, Metobolomics, Proteomics, Transcriptomics); Glioblastoma;Proteases; Stem cells (cancer, neural, mesenchymal, neural)
Researchers (19)
Organisations (5)
Abstract
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. Specifically:
1. The impact of co-culture conditions on metabolic, drug resistance fingerprint of (BM)MSC, gbMSC and GSC will be revealed. We hypothesize that their differential response will reveal conditions in which MSC display most detrimental effect on GBM cell invasion.
2. Transcriptomic, miRNA, cytokine and proteomic analysis (studied in Parts A, B, C) will focus on migratome and degradome changes in GSC after EMT/MET induction by MSC.
3. To elucidate the efficiency of MSC/gbMSC/GSC interactions observed in vitro the experiments with iv injection of labelled MSC to experimental animals with GBM xenograft will be performed, where the expected GBM regression will be mo
Significance for science
This project had combined tumour biology and stem cell research with systems biology to approach the identification of novel cell specific biomarkers. 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, it is namely possible to get insight into the characteristics of glioblastoma (GBM) cells in comparison to normal neural cells. With the appropriate selection of methods and the linkage of experimental research on clinical samples and cell culture (providing the data for so called “wet laboratory research”) with bioinformatics tools (“dry laboratory research”) we managed to identify novel biomarkers of GSC and GBM cells at transcriptome, proteome and cytokine profiling level. The revealed biomarkers CD9, TRIM28, Steffin A and Cathepsin K were also characterized and in vivo validated in small patients’ cohorts. Those are awaiting large cohort screening prior clinical translation. We also identified new biomarker GNAO1, for which we confirmed the diagnostic and prognostic value in the plasma of GBM patients. This type of diagnostic marker and its detection in the patient’s blood are designated as less invasive methods compared to GBM biopsy retrieval. The above mentioned new biomarkers with the impact on prognosis may even predict the response to therapy and by the same token represent new targets for future anti GBM drug design. Following the proteins and their regulatory molecules (cytokines, microRNA),as well as metabolic state of GBM and GSC in various environmental conditions (for example hypoxia), it is possible to develop regulators to control their invasive properties are crucial for the GBM spread. Identification of genes and proteins/proteases, activated at the reversible transition from stationary to migratory transition in GSC is important for understanding and inhibition of their invasion. We have presented a proof of principle that targeted lowering CatL activity could, because of their synergistic activity with known cytotoxic drugs i.e. Arsenite be a useful strategy when combined with the chemotherapy. As we succeeded in proving the enhanced apoptotic activity in the CatL silenced U87 and astrocytic pilocytoma spheroids cells arising from the arsenite treatment, we feel confident to ambitiously predict that targeting CatL expression in tumours during chemotherapy would indeed enhance the selectivity of tumour cells’ response to therapy. This would allow for a decrease of the applicable therapeutic doses of toxic drugs, resulting in elimination of the toxic side effects and thereby in the increase of the quality of the GBM patient’s life. It was also suggested that mesenchymal stem cells (MSC) after GBM infiltration may become a part of the GBM microenvironment and effect behaviour of GBM (stem) cells. Using the same systems approach, as indicated above, we follow the GBM/GSC and MSC interactions in direct and in direct cocultures and revealed MCP1/CCL2 and BDKRB1 molecular mechanisms that mediate their cross-talk and invasion, which may serve in clinical oncology as targets for treating GBM
Significance for the country
The completed project involved the so called “bench to bed approach” of medical research, which focused on translating laboratory result into clinical application for diagnosis, prognosis and prediction of response to treatment, as well as towards the targeted drugs development for GBM patients. Our results are therefore relevant from three aspects: - for the GBM stem cells biomarkers’ use in clinical oncology as pattented diagnostic kits which may even evolve into indicating therapy response kits. An attractive though distant clinical application of this research shows as possible use of normal neural and mesenchymal stem cells (MSC) for GBM therapy. The behaviour of normal stem cells in the tumour microenvironment has been investigated by many, confirming their suitability for drug delivery to cancer tissue. The number of biobanks, which store the tissue, enriched in MSC for their subsequent transplantation, is increasing as well as the number of donors and patients, who all expect and demand the development of this knowledge, as it raises the hope for treatment of hard to treat, incurable diseases, such as GBM. - the new applications in the field of stem cells are most attractive for small biotech companies, developing equipment, tools, reagents and technologies, useful for stem cells research. This is a particular good model for Slovenia. The results of this project shall contribute to better international business recognition of our institute (NIB) and enhance the possibilities of Slovenia to take part in the regional European biotech market. The performed research was also supported by the Centre for Stem Cells (established by our initiative, see: http://www.nib.si ), that shal also in future act as well recognised forum of opportunities for new business connection, as well as the partnering network of the international Interreg GLIOMA project (International Centre for Genetic Engineering and Biotechnology (ICGEB – Area di Recerca, Padriacano), University of Udine, University of Trieste and Oncology Centre in Aviano). - the third aspect of this project addressed expansion of knowledge on normal stem cells as therapeutic tools for »advanced cell therapy«. The term was coined in the Regulation (EC) No 1394/2007 of the European Parliament and of the Council on Advanced Medicinal Products and also includes cell therapy and tissue engineering. Advanced cell therapies are actually a continuation of treatment by bone marrow transplantation. An upgrade of such approach is the application of MSC derived from bone marrow /or other organs/ into tumour, combined with gene therapy approach (for example genetically modified MSC). In the majority of the developed countries, advanced cellular therapies became recently an integral part of transplantation services, due to their common technical and logistical demand. In Europe, clinical trials for stem cell therapies are still in early stages (clinical trials I, II, III) which is mostly due to the insufficient quality and stability of therapeutic stem cells available. In USA, the non- pharmacological status of stem cell therapies means that the Food and Drug Administration clearance process takes less time for commercialization than drug therapy. As the number and range of clinical studies is increasing worldwide, we can expect that at least a few cancer therapies will succeed in near future and will be marketed within the next 5-7 years also in Slovenia. Due to the demanding technology of above mentioned application, we therefore urgently need to develop the relevant knowledge, resulting from our internationally integrated investigation in this field that will contribute to application and biosafety of stem cell based therapy in future.
Most important scientific results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
