# Elucidating the Role of Matrix Porosity and Rigidity in Glioblastoma Type IV Progression
## 1 Introduction  
Glioblastoma type IV (GBM) is the malignancy of the central nervous system with the poorest prognosis [1]. Standard treatments for glioblastoma type IV currently consist of surgical resection followed by radiotherapy and chemotherapy [2]. The median survival for GBM patients is nearly 14 months and the 5-year survival rate is only of 5%. A distinctive feature of GBM cells is their ability to infiltrate the host tissue forming multiple micro-metastases which cannot be surgically removed or efficiently killed by molecular anticancer therapies. Spreading of GBM cells is believed to occur through two major pathways: across the extracellular matrix (ECM), which is mainly composed of a densematrix of hyaluronic acid and proteoglycans; and along specific brain structures, called Scherer's structures, which include white matter tracts and blood vessels [3]. The migration mechanisms are pathway dependent and the ability of glioma cells to adopt different migratory strategies is key in priming the aggressiveness of this deadly disease. Tumor cell motility has been extensively studied using classical 2D assays, including the monolayer wound-healing assay, the radial migration assay, and the Boyden chamber assay [4]. Although these systems can be efficiently used to assess the role of a variety of biochemical cues (adhesion proteins, chemoattract, and so on) in modulating cell migration and proliferation, they cannot reproduce the complex set of biomechanical stimuli typically found in three-dimensional microenvironments [5; 6; 7]. The picture is further complicated by the fact that gel-like parenchyma coexist with ordered and more rigid structures within the brain tissue [8; 9]. On the other hand, animal experiments and ex-vivo assays based on tissue slices, which would more closely recapitulate the biophysical features of a brain tumor microenvironment, are technically cumbersome, costly, and introduce additional variability which discourage any systematic analyses [10; 11; 12]. In this context, scaffolds made out of natural and artificial polymers offer a simple and reproducible solution for studying tumor cells in realistic microenvironments. Collagen (COL), poly(ethylene glycol) (PEG), hyaluronic acid (HA), chitosan, and combinations thereof, are among the most extensively used polymers for these applications [13; 14; 15; 16; 17; 18; 19]. For instance, COL and PEG-based hydrogels were used to demonstrate that HA inclusions tend to inhibit migration and favor aggregation of brain tumor cells [14; 15; 16]. Similar hydrogels were also employed to demonstrate that matrix porosity supports the rapid migration of GBM cells and that ECM remodeling favors cell tumor spreading more than proliferation [17; 18; 19]. In particular [PERSON] et al. [17] suggested within the framework of biochemistry of glioma invasion in different collagen gels that pore size is a key parameter determinant of glioma invasive speed in these gels, independent of global collagen concentration and both local and bulk stiffness. On the other hand, [PERSON] et al. [20] have shown that Hyaluronan can support formation of cancer cell aggregates. [PERSON] et al. [20] deal with Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. However, it is found that solid stress facilitates the formation of spheroids and that relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. [PERSON] et al. [20] suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. In the case of the glioblastoma cells however, [PERSON] et al. [21] have shown that these cells grow as spheroids in free suspension.