Details

Autor(en) / Beteiligte

• Wieland, Annalena

Titel

The Influence of Ultra-Soft 3D Hydrogels and Fiber Scaffolds on the Migration Behavior and Gene Expression of Glioblastoma and Breast Cancer Cells In Vitro

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2023

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Glioblastoma Multiforme (GBM) is the most malignant human brain tumor characterized by its high proliferation rate, aggressive invasion of surrounding tissues and resistance to conventional therapies leading to poor patient survival. Triple negative breast cancer (TNBC) is also associated with a bad prognosis as this subtype is defined by an aggressive phenotype with high rates of recurrence and metastases. Therefore, regarding both tumor types it is crucial to gain a better understanding of tumorigenesis, progression as well as migration mechanisms to develop new therapeutic strategies. In-vivo cells grow within a complex tissue-specific 3D microenvironment, the extracellular matrix (ECM), which has a major influence on cell behavior. Thus, one could predict that the ECM also plays an essential role in tumor cell migration and invasion. It is known that conventional 2D cell culture models cannot mimic a 3D microenvironment. That is why the development of physiologically relevant 3D models play a fundamental role in biomedical research. The aim of this project was to mimic the ECM of the brain. However, the brain is one of the softest organs of the human body, which features low mechanical properties with a low Elastic Modulus. Therefore, the establishment of 3D models with comparable biological as well as mechanical properties can be challenging due to the instability of soft hydrogels. To mimic the mechanical properties of the brain and overcome these challenges, ultra-soft hydrogels were reinforced with meltelectrowritten (MEW) Poly-ε-Caprolacton (PCL) - scaffolds. The GBM-cell lines U87 and LN18 as well as the TNBC cell line HTB26 were grown in these 3D composites and assessed for 3D-induced changes using qPCR, fluorescence staining and live cell imaging. Especially U87 showed a high affinity towards the PCL-scaffold as well as an interaction with the PCL-fibers, which also had a prominent influence on gene expression. In comparison to the PTEN wildtype GBM cell line LN18, which did not show an active interaction with the PCL-scaffold, U87 harbored a loss of function mutation in the PTEN gene, an aspect, which gained great importance in the later part of this project. It was previously shown that GBM cells and possibly TNBC-derived brain metastases use existing brain structures, such as white matter tracts and blood vessels as migratory cues. Here we developed a MEW-PCL based 3D migration model, so-called PCL-tracks, to mimic the migratory cues of the brain. These 3D PCL-tracks were used to analyze the migratory behavior of GBM- as well as TNBC-cells by live-cell imaging. Moreover, by using different inhibitors, we identified a new PTEN-dependent RHOB-ROCK migration signaling pathway, which was further confirmed by RNA-sequencing. We could show that the two cell lines U87 and HTB132, with PTEN loss of function and high RHOB expression, showed an active migration towards and along the PCL-tracks with an amoeboid morphology, whereas LN18 and HTB26, carrying the PTEN wildtype gene, did not show a directed migration and displayed a stretched mesenchymal-like morphology. Lastly, we unraveled a PTEN-RHOB-dependent caterpillar-like cell movement composed of a dynamic sequence of different plasticity modes by high-resolution live-cell imaging.In a further part of this thesis, we implemented different hydrogels as 3D matrices and analyzed their influence on GBM cell morphology and gene expression. In addition to Matrigel (MG), ALG, ADA-Gel, HA-SH and Collagen were used to establish ultra-soft 3D GBM-models, with a stiffness between 50-200 Pa (Elastic Modulus), which were analyzed by immunofluorescent staining and RNA-sequencing (RNA-Seq). We showed that U87 formed complex astrocytic 3D networks in MG, whereas in ALG, ADA-Gel and HA-SH a round cluster- or spheroid formation was observed. Additionally, RNA-Seq data showed significant differences between 2D and 3D cell cultures. Finally, it was shown that some hydrogels, particularly ALG, induced a (pro-) inflammatory phenotype, indicated by increased expression levels of IL-6, IL-8 and TNFa. The ALG-induced cell behavior and morphology as well as expression of (pro-) inflammatory genes, strongly suggest an induction of cellular senescence. This hypothesis was supported by the detection of senescence biomarkers, such as ß-Galactosidase (ß-Gal) and DNA-damage. Overall, this study showed that not only the cellular genotype of tumor cells drastically impacts the cell behavior in 3D, but also individual hydrogels actively change the overall cell functionality. Therefore, it is crucial to consider these interactions for future 3D cell culture models.