University of Twente Student Theses
Extracellular vesicle-associated DNA in neuroblastoma : hitching a ride for tumour progression?
Timmerije, Rick (2024) Extracellular vesicle-associated DNA in neuroblastoma : hitching a ride for tumour progression?
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| Abstract: | Introduction: Neuroblastoma, a prevalent and aggressive pediatric cancer originating from immature nerve cells in the sympathetic nervous system, poses significant challenges despite advancements in treatment. Recent studies highlight the role of extrachromosomal DNA (ecDNA) associated with extracellular vesicles (EVs) in neuroblastoma, particularly its association with oncogene amplification, tumour progression and drug resistance. While a further need for investigation is recognised, this study outlines three key research goals: (1) Investigate ecDNA morphology and quantity in the SHEP-2 neuroblastoma cell line using metaphase spreads. (2) Examine whether EVs released by SHEP-2 cells contain DNA and determine its location within or outside the vesicle. (3) Explore the transfer of EV-associated ecDNA between different cells. Results: Visualization of ecDNA in SHEP-2 cells, despite extensive optimisation of metaphase spreads of coverslip adherent cells, revealed challenges due to overlapping chromosomal fragments. Significant quantities of DNA were found in EV isolation fraction with and without enzymatic digestion of non-enclosed DNA. This indicates the presence of DNA both inside and outside of EVs. With bioorthogonal metabolic labelling, the nuclear DNA of SHEP-2 cells was successfully labelled by incorporating F-ara-EdU and later ’clicking’ a fluorophore to it using the CuAAC reaction. This could be used to follow the DNA from these cells when it is not known which genes are transferred, setting the stage for a subsequent detection and localization experiment for this DNA in EV fractions isolated using centrifugation and ultracentrifugation. However, the anticipated CuAAC click-labelling technique for detecting ecDNA from the isolated EV fraction in gel electrophoresis faced challenges. Next to this, the ecDNA from EV fractions isolated from bioorthogonal labelled SHEP-2 cell culture in different cell cultures could not be localized. Conclusion and discussion: Despite extensive optimization of the metaphase spreads for the observation of ecDNA, the overlapping chromosomal fragments posed a significant challenge, making it difficult to distinguish individual chromosomes and hindering a clear observation of ecDNA. The investigation into EVs secreted by SHEP-2 cells revealed the presence of DNA in the EV fraction and suggested the existence of both free or vesicle-associated DNA and membrane-enclosed DNA within the EV isolation fraction. However, the EV yield and exclusion of non-EV material remain unknown. Efforts to explore ecDNA transfer in cases where the genes are unidentified involved the effective use of a bioorthogonal metabolic labelling strategy within cells adhering to coverslips. However, specific detection of ecDNA in the EV fraction remained inconclusive due to challenges such as fluorophore binding issues or a potential absence of ecDNA in the sample. The experiment designed to explore the transfer of ecDNA through EVs did not yield usable results but the experimental set-up can contribute to future investigations. |
| Item Type: | Essay (Master) |
| Faculty: | TNW: Science and Technology |
| Programme: | Biomedical Engineering MSc (66226) |
| Link to this item: | https://purl.utwente.nl/essays/98008 |
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