Samenvatting
Introduction: The tumor promoting properties of a variety of cells in the tumor microenvironment pose a major challenge in the development of novel treatment strategies to fight cancer. Among the tumor promoting cells are myeloïd-derived suppressor cells (MDSCs). The accumulation of these cells is strongly stimulated during cancer development. MDSCs have various functions that promote tumor growth, including stimulation of neo-vascularization and suppression of antitumor immune responses. Consequently, there is increasing interest in manipulating these MDSCs.Aim: A major challenge for scientists that perform research on MDSCs, is the isolation of large numbers of MDSCs from cancer patients to study these MDSCs. Therefore, we will in this thesis project set up a flexible system to differentiate MDSCs in vitro that cloesely resemble the MDSCs found in cancer patients. Such in vitro MDSCs could significantly aid research on the function of MDSCs as well as strategies to manipulate them.
Methods: In this thesis, we sought for an efficient strategy to differentiate CD14+ monocytes, which are abundantly present in blood, into MDSCs. Monocyte to MDSC differentiation was mediated using “granulocyte macrophage – colony stimulating factor” (GM-CSF) rich conditioned medium that was harvested from lentivirally modified tumor cells. Furthermore, the effect of a variation in percentage conditioned medium versus standard medium (RPMI of X-vivo) has been investigated together with the supplementation of different cytokines (IL-6 and/or TGF- β) at different concentrations. Three days as well as seven days after the start of the culture, the cells were collected. Expansion, fenotypic and functional differentiation to MDSCs was evaluated using cell count, flow cytometry and T cell suppression assays, respectively. Via MTT-assays the effects of 5-FU, R848, MPLA, Ampligen, Decitabine and Quisinostat on MDSCs were investigated. Besides the generation of MDSCs from PBMCs, there has been a parallel study that generated MDSCs out of bone marrow of humanized mice (using the same techniques). ultimately, we search with a bioplex for an extra link between %MDSC in plasma of patients and 8 different cytokines.
Results: based on the different results, a protocol that resulted in the most optimal MDSC-generation could be set up. In this protocol RPMI was used with conditioned medium of the HCT-116-GM-CSF transduced cell-line in a 1:1 ratio, together with the supplementation of 20 ng/ml IL-6. Extra TGF-β did not made an important difference. Unfortunately the MDSC yield remained rather low. Starting from bone marrow of humanized mice, the results were better when looking at cell numbers and suppression.The MTT-assay confirmed th toxicity of 5-FU. Furthermore, the viability evolved in a downwards trend with increasing drug concentrations. The other drugs showed a rather stabile curve at high viabilities. We found no correlations with the Bioplex.
Conclusion: The PBMC-MDSCs seemed to be very sensitive and therefore more difficult to keep in culture. Frequently they even differentiated further to HLA-DR+-cells. Due to these variabilities, Further research on drugscreening will be held with the MDSCs obtained from bone marrow of humanized mice.
| Datum prijs | 2017 |
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| Originele taal | Dutch |
| Prijsuitreikende instantie |
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| Begeleider | Inès Dufait (Promotor) |
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