Development of a 3D Cell Culture Model for Breast Tumor Cells Disseminating to the Brain

Over the last decade, advanced biofabrication technologies became one of the most powerful tools to replicate complex human tissues. Unlike traditional two-dimensional (2D) cell cultures, three-dimensional (3D) technologies offer significant advantages for cancer research. Integrating advanced 3D bioprinting and biomaterials enables recapitulation of the tumor microenvironments by embedding cells within a custom-tailored bioink. Our aim was to develop a 3D hydrogel-based cell culture disease model that reflects characteristics of native brain extracellular matrix (ECM) in order to investigate the progression of breast-to-brain metastatic tumor cells. Since the brain is one of the softest tissues in our body, developing ultra soft matrices causes physical and biological challenges. To address this, melt-electrowriting (MEW) technology was used to fabricate microfiber scaffolds, enabling a reinforcement of the matrices to allow an easy handling of the 3D composites. The developed 3D hydrogel model consists of hyaluronic acid (HA), a major glycosaminoglycan (GAG) highly expressed in the brain. The first part of this thesis investigates how matrix stiffness and ECM components affect the survival and morphology of both primary and secondary brain tumors in our developed 3D cell culture model. In the developed model, thiolated hyaluronic acid (HA-SH) was used, enabling crosslinking through its thiol (-SH) groups. Both breast tumor and glioblastoma cells formed spheroid-like aggregates on soft and stiff hydrogels, regardless of the scaffold geometry. However, the addition of ECM components to HA-SH, as well as co-culturing breast tumor cells with primary neurons and astrocytes in HA-SH without ECM components, effectively reduced spheroid-like formation. This reduction is likely attributed to enhanced cell-matrix and cell-cell interaction, suggesting a more physiologically relevant microenvironment. In the second part of this thesis, cell–cell and cell–matrix interactions were examined in detail by co-culturing breast tumor cells with primary neurons and astrocytes in ultra-soft 3D HA-SH, both with and without ECM components. The incorporation of ECM components into HA-SH enhanced functional interactions such as GluA2, vGlut1, Cx43 expression, and calcium (Ca²⁺) transients activity between metastatic triple-negative breast cancer (TNBC) cells and neuronal cells. In contrast, brain-derived Luminal B/HER2-positive subtypes exhibited more pronounced physical contact with neuronal cells in the absence of ECM components. This model effectively demonstrates how distinct breast cancer subtypes adapt to and interact differently within a brain-like microenvironment. In the final part of the thesis, a 3D cell culture model was utilized to investigate the pathophysiology of murine glioblastoma, a primary brain tumor. The tumor microenvironment was effectively mimicked using 3D HA-SH/laminin composite hydrogels, reinforced with 3D printed MEW scaffolds. Glioblastoma cells exhibited functional interactions with primary neurons and astrocytes through GluA2 and Cx43 expression. Furthermore, calcium Ca2+ imaging revealed that the presence of tumor cells induced a hyperexcitable neuronal phenotype, which correlates with neurological symptoms observed in glioblastoma patients. Overall, this 3D hydrogel-based cell culture platform enables us to investigate both primary and secondary brain tumor progression in physiological and biologically relevant surroundings.