# Elucidating the Role of Matrix Porosity and Rigidity in Glioblastoma Type IV Progression
## 4 Results  
### Morphological and Mechanical Characterization of Hydrogels for the Inclusion of U87-MG Tumor Cells  
Collagen is the most ubiquitous protein in the ECM and forms random networks of fibers depending on the assembling conditions [38,39]. In the brain, collagen is mostly found along the vascular network together with other proteins, such as laminins and fibronectin, and provides mechanical support as well as important cues for tumor cell dynamics [3,14,17] Another major component of the brain ECM is hyaluronic acid. This is a naturally occurring glycosaminoglycan that has the capability to bind to cell specific surfaces receptors (CD44), which are necessary for cell anchorage, homeostasis, and proliferation [40, 41, 15, 42]. For this, both COL and HA have been used in different forms to reproduce different portions of the brain tissue. Cells of glioblastoma type IV transfected with green florescence protein--U87-MG GFP\({}^{+}\)--were included into hydrogels of COL and HA either individually or as preformed spheroids (Figure 3). For individual cells, these were first expanded on a dish, then resuspended and added to the natural polymeric hydrogel. For spheroids, cell clusters of about 200 \(\upmu\)m were preformed and eventually included in the polymeric hydrogel. Finally, the COL and HA mixtures were deposited at the bottom of polystyrene multiwell plates, precoated with agarose, polymerized, and left in cell culture media for up to 8 days. Knowing that the microstructural and biomechanical features of the extracellular matrix may severely affect the growth and spreading of malignant tissues, the morphology and deformability of the hydrogel scaffolds were characterized. First, scanning electron microscopy (SEM) was used to assess the microstructure of COL and HA. Figure 4A and its lateral insets show the organization of collagen fibers within the hydrogel. COL fibers appear entangled, with a diameter of approximately  
Figure 3: Schematic representation of the experimental plan. Glioblastoma type IV cells transfected with green florescence protein (U87-MG GFP\({}^{+}\)) are encapsulated into 3D hydrogels, made out of collagen type I (COL) or hyaluronic acid (HA), either individually or as preformed spheroids. Representative images of the two different hydrogels and cell culturing conditions (suspension and spheroids, at day 4 of culture) are displayed on top and bellow, respectively. Some distinctive features of U87-MG spatial organization and morphology in the two different hydrogels are already visible in the four central insets: cell elongation and roundness; compact tumor core and infiltrating region. 10 nm, and randomly arranged to form a matrix with pores varying in size between about 20 and 70 nm, as from SEM images. Differently, HA presents a more compact organization of thinner (about 5 nm) and curly fibers (Figure 4B). Importantly, the magnified SEM image for HA does not reveal any obvious, regular porosity of the scaffold. This supports the notion by which HA hydrogels can be used for mimicking the soft and dense brain parenchyma, whereas fibers within COL tend to resemble morphological features of ordered structures in the brain [3]. Subsequently, the mechanical deformability of the hydrogels was assessed under wet conditions by standard compression testing. As expected, COL presented a higher Young's modulus \(E\) as compared to HA. Specifically, as documented in the chart of Figure 4C, \(E\) was \(5.16\pm 1.40\) kPa for COL and \(0.20\pm 0.03\) kPa for HA, assuming a Poisson coefficient \(\
u=0.45\). These values are in good agreement with previously reported characterizations for human brain tissue [43; 44; 45]. Figure 4: Mechanical characterization of natural hydrogels. (**A**,**B**) SEM micrographs of COL and HA, respectively, showing very distinct morphologies. Magnified images are presented on the right. COL presents a fibrous structure with a variable porosity. HA presents a complex dense structure. (**C**) Young’s modulus of COL and HA hydrogels.