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N. Barin
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Glioblastoma (GBM) is the most common and aggressive primary brain tumour with poor prognosis and no cure. To improve the efficacy of preclinical research, there is a need for reliable in vitro models that accurately recapitulate the tumour microenvironment. GBM cells and their protrusions use the brain tumour vasculature as scaffolds for migration, promoting tumour invasion. In addition, GBM cells form extensive cellular networks, which are associated to increased therapy resistance. In this study, we explored the use of two-photon polymerised scaffolds featuring elements that mimic brain tumour blood vessel geometries to study the intercellular networks of glioblastomas. We employed two distinct micro-scaffold designs to assess the effects of various geometrical design features on cell colonisation: the “Spider Web” scaffold and the “Grid-like structure”. The “Spider Web” design provides a 3D environment that consists of beams mimicking branching blood vessels. We first tested whether the presence of diagonal beams allowed for improved cell colonisation. For this, the vertical cell occupancy of cells on scaffolds with and without diagonal beams was quantified using confocal imaging. Results show that the presence of diagonal beams did not significantly improve cell occupancy of the top tier, although it led to a significant decrease in total cell count. The “Grid-like structure” was designed with pore sizes ranging from 4 μm to 75 μm. The expression of several immunofluorescent markers, as measure of cell occupancy across different pore size regions was compared. The area fraction of Hoechst, actin and tubulin expression was found to be significantly higher in the regions with larger pores compared to those with smaller pores. Despite a difference in cell density, the expression of gap junction protein CX43 was similar across pore sizes. On both scaffold designs, confocal and scanning electron microscopy revealed a variety of cellular protrusion morphologies, as well as punctate CX43 expression. This study shows that these biomimetic micro-scaffolds can be used to evaluate the effect of geometrical features on GBM cell colonisation, and hold potential for the investigation of the 3D GBM intercellular tumour network in vitro.
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Glioblastoma (GBM) is the most common and aggressive primary brain tumour with poor prognosis and no cure. To improve the efficacy of preclinical research, there is a need for reliable in vitro models that accurately recapitulate the tumour microenvironment. GBM cells and their protrusions use the brain tumour vasculature as scaffolds for migration, promoting tumour invasion. In addition, GBM cells form extensive cellular networks, which are associated to increased therapy resistance. In this study, we explored the use of two-photon polymerised scaffolds featuring elements that mimic brain tumour blood vessel geometries to study the intercellular networks of glioblastomas. We employed two distinct micro-scaffold designs to assess the effects of various geometrical design features on cell colonisation: the “Spider Web” scaffold and the “Grid-like structure”. The “Spider Web” design provides a 3D environment that consists of beams mimicking branching blood vessels. We first tested whether the presence of diagonal beams allowed for improved cell colonisation. For this, the vertical cell occupancy of cells on scaffolds with and without diagonal beams was quantified using confocal imaging. Results show that the presence of diagonal beams did not significantly improve cell occupancy of the top tier, although it led to a significant decrease in total cell count. The “Grid-like structure” was designed with pore sizes ranging from 4 μm to 75 μm. The expression of several immunofluorescent markers, as measure of cell occupancy across different pore size regions was compared. The area fraction of Hoechst, actin and tubulin expression was found to be significantly higher in the regions with larger pores compared to those with smaller pores. Despite a difference in cell density, the expression of gap junction protein CX43 was similar across pore sizes. On both scaffold designs, confocal and scanning electron microscopy revealed a variety of cellular protrusion morphologies, as well as punctate CX43 expression. This study shows that these biomimetic micro-scaffolds can be used to evaluate the effect of geometrical features on GBM cell colonisation, and hold potential for the investigation of the 3D GBM intercellular tumour network in vitro.
Glioblastoma is one of the deadliest types of brain cancer with patients having a poor prognosis. The development of effective treatments requires physiologically relevant disease model systems. Although there are a variety of 3D glioblastoma culture models available, they fail to capture a key characteristic, namely core and edge tumour regions. The tumour core refers to the central region of the tumour distinguished by densely packed quiescent cells. The edge is located at the periphery of the tumour, where infiltrating/proliferating tumour cells interact with normal tissue. In this study, we successfully designed and fabricated a two-photon polymerized 3D printed scaffold and developed a microinjection-based protocol for guiding the growth of biomechanically constrained glioblastoma spheroids. The formation of the spheroid was achieved by injecting nL-volumes of cells on the scaffolds either functionalized with an extracellular matrix (ECM) coating or embedded in Collagen hydrogel. An alignment protocol based on the use of fiducial cross-shaped markers was employed to align the microinjector needle to the desired position. The culture model was tested by examining cell colonization with a commercial glioblastoma cell line (U87) as well as with patient-derived glioblastoma cells. Immunofluorescence (IF) staining and scanning electron microscopy (SEM) were employed to characterize the model. Two core and two edge-specific immunofluorescence markers were tested to analyse the core and edge formation further. IF and SEM imaging revealed a dense core and protruding edge-like cells both in the ECM-coated scaffold and the collagen-embedded ones. Further, we observed a remarkable difference in terms of F-actin expression between the core and edge region. We compared these results with those obtained in a scaffold-free model, which showed that the presence of the scaffold influences the behaviour of glioblastoma cells, resulting in guided growth, along the microfabricated structures. The developed 3D culture model paves the way for further investigation of core and edge dynamics as well as for prospective in-vitro drug screening.
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Glioblastoma is one of the deadliest types of brain cancer with patients having a poor prognosis. The development of effective treatments requires physiologically relevant disease model systems. Although there are a variety of 3D glioblastoma culture models available, they fail to capture a key characteristic, namely core and edge tumour regions. The tumour core refers to the central region of the tumour distinguished by densely packed quiescent cells. The edge is located at the periphery of the tumour, where infiltrating/proliferating tumour cells interact with normal tissue. In this study, we successfully designed and fabricated a two-photon polymerized 3D printed scaffold and developed a microinjection-based protocol for guiding the growth of biomechanically constrained glioblastoma spheroids. The formation of the spheroid was achieved by injecting nL-volumes of cells on the scaffolds either functionalized with an extracellular matrix (ECM) coating or embedded in Collagen hydrogel. An alignment protocol based on the use of fiducial cross-shaped markers was employed to align the microinjector needle to the desired position. The culture model was tested by examining cell colonization with a commercial glioblastoma cell line (U87) as well as with patient-derived glioblastoma cells. Immunofluorescence (IF) staining and scanning electron microscopy (SEM) were employed to characterize the model. Two core and two edge-specific immunofluorescence markers were tested to analyse the core and edge formation further. IF and SEM imaging revealed a dense core and protruding edge-like cells both in the ECM-coated scaffold and the collagen-embedded ones. Further, we observed a remarkable difference in terms of F-actin expression between the core and edge region. We compared these results with those obtained in a scaffold-free model, which showed that the presence of the scaffold influences the behaviour of glioblastoma cells, resulting in guided growth, along the microfabricated structures. The developed 3D culture model paves the way for further investigation of core and edge dynamics as well as for prospective in-vitro drug screening.