AbstractsBiology & Animal Science

Influence of the extracellular matrix on cellular behaviour – Development and application of a bioengineered 3D cell culture system for cancer research and high resolution microscopy

by Elke Kämmerer

Institution: Technische Universität Darmstadt
Department: Membrane Dynamics
Degree: PhD
Year: 2014
Record ID: 1105720
Full text PDF: http://tuprints.ulb.tu-darmstadt.de/4181/


The aim of this thesis was the application and establishment of a semi-synthetic, bioengineered model system for ovarian cancer and a naturally based, optimised hydrogel system for non-invasive high resolution microscopy analysis of plasma membrane dynamics in a physiological relevant microenvironment. Providing ex vivo models for cancer research with physiologically relevant 3D conditions combined with controllable physical and chemical composition is an ongoing challenge in the field of tissue engineering and cell biology. In this study a semi-synthetic GelMA-based hydrogel system was successfully applied to the study of ovarian cancer cells. Thereby GelMA provided a biomaterial approach combining natural binding and cleavage sites with tuneable properties in an easy to handle and cost-effective hydrogel system. Using this model system, the unique metastatic pattern of ovarian cancer, found in patients with late stage disease, was replicated in vitro and in vivo. The polymer concentration (2.5-7% w/v) did directly impact the hydrogel stiffness (0.5± 0.2 kPa – 8.9 kPa ± 1.8 kPa) but had only minor impact on solute diffusion. Diffusion of FITC labelled 70 kDa dextran was in all tested hydrogels (2.5-7% w/v; 29.9±3.3 till 16.9 μm2/s) close to the diffusion coefficient measured in water (39.2 ± 2.8 μm2/s). In the stiffest tested hydrogel the diffusion coefficient was decreased by only 2.3 times. Spheroid formation, occurring in the tumour fluid (ascites) of ovarian cancer patients with late stage disease, combined with the highest metabolic and proliferation rates was reflected in medium stiff hydrogels (5%, 3.4 kPa). Inhibition of hydrogel degradation, with a MMP inhibitor, reduced spheroid growth and metabolic activity. Additional ECM components, Laminin-411 and hyaluronic acid increased spheroid growth, metabolic and proliferation activity significantly. Furthermore this hydrogel system allowed bio-molecular analysis of mRNA and protein levels. Ovarian cancer cells showed an increased mRNA level of integrin β1 compared to monolayer or other 3D cultures. Next to the ex vivo experiments it was shown that GelMA-based hydrogels can be successfully used as spheroid-based cancer cell carrier system for in vivo studies. Spheroid-seeded hydrogels were intraperioteneally implanted into female NOD/SCID mice, resulting in tumour development and metastasis, known from the clinical sequence of the disease. The developed tumours showed a response rate of 33% to the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. Combined treatment using both therapeutics resulted in 37.8% treatment response, while treatment with ATN-161 alone had no effect.Collagen-based hydrogel systems are widely used for investigations of cell-ECM crosstalk; however, they often lack reproducible, well characterised properties and are mostly not suitable for high resolution microscopy. A small volume collagen-based hydrogel system provided a 3D cell culture model utilised for high resolution microscopy…