# Elucidating the Role of Matrix Porosity and Rigidity in Glioblastoma Type IV Progression
## 5 Discussion  
In this work, the proliferation and migration propensity of human glioblastoma type IV cells (U87-MG) was studied within different tridimensional microenvironments: hyaluronic acid hydrogels (HA), whose density and rigidity resembled the gel-like properties of the brain parenchyma; collagen hydrogels (COL), whose spatial organization with fibers and pores resembled the ordered structures in the brain, such as the perivascular space and axonal fibers. Although none of these two simple hydrogels can precisely mimic the tridimensional complexity of a human brain tissue, relevant cell features, such as morphology, migration, clustering, and proliferation, were found to closely match in-vivo observations. In particular, glioma cells tend to spread more rapidly in matrices with organized fibers, such as COL, whereas cell clustering is typical of dense and poorly organized matrices, such as HA. This was documented in Figure 5. It was noticed that individual U87-MG cells rapidly assume an elongated shape, with cell membrane ruffling and long filopodial protrusions, on COL. This spindle shaped cell morphology is a clear sign of migratory phenotype and was observed as early as a few hours post-inclusion. Very differently, individual tumor cells on HA preserve their original rounded shape, which is more prone to form large and dense cell clusters. The different cell morphology could indeed be associated with ECM rigidity, as previously documented by [PERSON] and collaborators [47], which might induce a cell epithelial to mesenchymal transition and facilitate migration [48]. Furthermore, by tracking over time the motion of individual cell nuclei, U87-MG cells were confirmed to have higher motility and travel longer distances on COL as compared to HA. Overall this behavior agrees with in-vivo observations and the notion that glioma cells would more efficiently migrate along organized brain structures (blood vessel walls and axonal fibers) rather than across the dense extracellular space [3; 49; 50; 51]. This individual cell behavior is then reflected in the overall development of neoplastic masses, as documented in Figure 9. Tumor spheroids included into COL gels grow at faster rates and, more importantly, infiltrate more deeply the surrounding matrix, as compared to HA gels. At 8 days post spheroid inclusion, neoplastic masses in COL have a more developed infiltrating front as compared to HA. On the other hand, tumors in HA present a more dense and smaller infiltrating front again suggesting that cell clustering is preferred to individual cell migration within such matrices. This can be readily appreciated by comparing the individual pictures in Figure 9A,B: the infiltrating front is more diffuse and spread over the entire region of interest in the case of COL (Figure 9A), whereas it appears more compact and well defined in the case of HA (Figure 9B). At 8 days, the radius of the HA infiltrating front is still about two times smaller than for COL: the characteristic sizes are \(345.55\pm 139.09\)\(\upmu\)m for HA and \(661.65\pm 39.68\)\(\upmu\)m for COL. Supporting Movies S3 and S4 readily depict all these differences between COL and HA. It is worth noting the different evolution in time of the tumor core growing in collagen type 1 and hyaluronic acid: in the first case there appears an accelerated trend while in the second case some tendency to a stable state result. In the medium the growth pattern appears linear. This is more clearly shown in Figure 13, where a direct comparison with spheroids growing in cell culture media is presented too. It is evident that spheroids in suspensions do not develop as rapidly and aggressively as spheroids embedded into more realistic environments: at 8 days, tumor spheroids developing in media have roughly half the size of tumors growing in gels (\(551.22\pm 52.56\)\(\upmu\)m in COL, \(448.27\pm 69.53\)\(\upmu\)m in HA, and \(278.63\pm 17.08\)\(\upmu\)m in medium). Obviously, spheroids in suspension cannot develop a truly infiltrating front.