# Elucidating the Role of Matrix Porosity and Rigidity in Glioblastoma Type IV Progression
## 6 Conclusions  
One of the key issues in glioblastoma progression is the highly infiltrating nature of the tumor mass, which is pathway dependent and marked by the ability of glioma cells to adopt different migratory strategies. In this work, for the first time, a set of quantitative studies was carried out, by means of in vitro and in silico tests, to elucidate the role of matrix porosity, rigidity, and morphology in the development of glioblastoma malignancy. Proliferation and motility of glioblastoma type IV cells were assessed in different extracellular matrices, namely made out of collagen type 1 and hyaluronic acid. Collagen matrices presented organized fibrous structures with a pore size ranging between 20 and 70 nm and a Young's modulus of 5000 Pa. Differently, hyaluronic acid matrices appeared as a compact and disorganized cluster of short fibers with no obvious porosity and a Young's modulus of 200 Pa. Glioblastoma type IV cells were observed to readily proliferate in both matrices, under similar culturing conditions. However, individual cells in collagen matrices exhibited an elongated morphology, similar to fibroblasts, documenting a propensity to migration whereas cells in hyaluronic acid matrices appeared globular. Culturing of tumor spheroids into the two different hydrogel matrices recapitulated the behavior manifested by individual cells. Glioblastoma type IV spheroids were documented to grow more rapidly in collagen matrices forming already after a few hours cell protrusions, possibly aligned with the collagen fibers. On the other hand, the same spheroids mostly developed as compact masses when cultured in hyaluronic acid matrices. Although infiltrating regions around the tumor core were observed for both matrices, this developed earlier and faster for spheroids included in collagen as compared to hyaluronic acid. A tumor growth multiphysics model, accurately predicting the different experimental behaviors in locally homogeneous environments, was able to match also the evolution in time of the tumor spheroids growing in the two different scaffolds by introducing some matrix heterogeneity: the plateau following the initial growth in hyaluronic acid and the faster trend in collagen. The computational results matching the realistic cases as well as those with different porosity morphology performed for investigative aim, confirmed that matrices with nonuniform porosities accelerate the development and tissue infiltration of neoplastic masses. All together, these data demonstrate that mechanical features of extracellular matrices, such as porosity and its spatial organization, are more relevant than matrix rigidity in supporting rapid tumor growth and infiltration. This experimental-computational framework can be used to quantitatively study the influence of porosity in tumor growth and to clarify the role of porosity morphology in the evolution of malignant masses. Furthermore, in silico tests allow to investigate many combinations of parameters and configurations, even in cases which would be impossible to reproduce in the laboratory. By exploiting the synergy of the tests performed in vitro and in silico, it is possible to predict the behavior of the neoplastic masses and their evolution over time, as well as to elucidate which are the most relevant biomechanical cues for combined therapies aiming simultaneously at killing cancer cells and modulating the tumor microenvironment. Finally, these first results about the importance of heterogeneity point to the need of investigating the impact of the stochastic heterogeneity of physical parameters on tumor growth because this heterogeneity is frequently attributed only to genetics [52; 53]. This paper has shown that this is not necessarily the case and paves the way to further investigation on this aspect. The following are available online at [[http://www.mdpi.com/2076-3417/10/24/9076/s1](http://www.mdpi.com/2076-3417/10/24/9076/s1)]([http://www.mdpi.com/2076-3417/10/24/9076/s1](http://www.mdpi.com/2076-3417/10/24/9076/s1)).