Influence of hyaluronic acid transitions in tumor microenvironment on glioblastoma malignancy and invasive behavior

The extracellular matrix (ECM) is critical in tumor growth and invasive potential of cancer cells. In glioblastoma tumors, some components of the native brain ECM such as hyaluronic acid (HA) have been suggested as key regulators of processes associated with poor patient outlook such as invasion and therapeutic resistance. Given the importance of cell-mediated remodeling during invasion, it is likely that the molecular weight of available HA polymer may strongly influence GBM progression. Biomaterial platforms therefore provide a unique opportunity to systematically examine the influence of the molecular weight distribution of HA on GBM cell activity. Here we report the relationship between the molecular weight of matrix-bound HA within a methacrylamide-functionalized gelatin (GelMA) hydrogel, the invasive phenotype of a patient-derived xenograft GBM population that exhibits significant in vivo invasivity, and the local production of soluble HA during GBM cell invasion. Hyaluronic acid of different molecular weights spanning a range associated with cell-mediated remodeling (10, 60, and 500 kDa) was photopolymerized into GelMA hydrogels, with cell activity compared to GelMA only conditions (-HA). Polymerization conditions were tuned to create a homologous series of GelMA hydrogels with conserved poroelastic properties (i.e. shear modulus, Poisson’s ratio, and diffusivity). GBM migration was strongly influenced by HA molecular weight. While markers associated with active remodeling of the HA content, hyaluronan synthase and hyaluronidase, were found to be insensitive to matrix immobilized HA content. These results provide new information regarding the importance of local hyaluronic acid content on the invasive phenotype of GBM.


Introduction 54
Glioblastoma (GBM), a WHO grade IV astrocytoma, is the most common and deadly form of 55 brain cancer and accounts for more than 50% of primary brain tumors (Furnari et al.,56 2007; Nakada et al., 2007;Wen and Kesari, 2008). Unlike many other cancers that metastasize to 57 a secondary site, GBM instead is known to diffusely infiltrate throughout but rarely metastasize 58 beyond the brain, and this invasive phenotype contributes to poor patient prognosis (median 59 survival < 15 months and 5 year survival < 5%) ( The GBM TME is not homogeneous but a complicated heterogeneous environment, especially 67 on the tumor margins, where transitions between the tumor microenvironment and surrounding 68 brain parenchyma are characterized by transitions in structural, biomolecular, and cellular 69 composition. The matrix compositional transition from natural brain to tumor provides a 70 potential invasion path for GBM and, therefore, might contribute to poor patient prognosis 71 (Syková, 2002 . In this context, the amount and molecular weight distribution of HA, associated 77 with constant turnover from oligosaccharides to high MW HA, across the tumor 78 microenvironment is believed as an important regulator of GBM invasion (Itano and Kimata,79 2008). Hyaluronic acid, a negatively charged, nonsulfated GAG, is the main component of brain 80 ECM. HA is naturally produced by hyaluronan synthase (HAS) family and degraded by 81 hyaluronidase (HYAL) in mammalian animals (Misra et al., 2011). While the presence of HA 82 has been shown to be important to tumor progression (Toole, 2004;Stern, 2008; Kim and Kumar,83 2014), significant investigation is needed to explore the role of the molecular weight (MW) of 84 HA on processes associated with GBM invasion, progression, and therapeutic response. 85 Remodeling of hyaluronic acid in the context of GBM cell invasion requires the combined effort 86 of a range of degradative and biosynthetic proteins.

Young's modulus 148
The compressive modulus of each hydrogel variant was measured using an Instron 5943 149 mechanical tester. Hydrogels were tested under unconfined compression with a pre-load 0.005N 150 at the rate of 0.1 mm/min, with their Young's modulus obtained from the linear region of the 151 stress-strain curve (0-10 % strain). 152

Diffusivity 153
The water diffusivity of each hydrogel was measured through indentation tests using atomic 154 force microscopy (AFM, MFP-3D AFM, Asylum Research) (Figure 1). The stiffness of the 155 cantilever used in the measurements is 0.6 N/m. A spherical polystyrene probe of 25 μm 156 diameter was attached to the tip (Novascan). Three separate measurements of different 157 indentation depths were taken. After surface detection, the spherical indenter was pressed into 158 the sample to a certain depth in the rate of 50 μm/s and was held for a period of time until the 159 force on the indenter reaches a constant value. The force on the indenter was measured as a 160 function of time F(t). The time-dependent response of hydrogels is due to solvent migration. The 161 poroelastic relaxation indentation problem has been solved theoretically by Hu  properties from the relaxation indentation measurement. According to this method, the 164 normalized force relaxation function is a function of a single variable: the normalized time 165 = / 2 , with D being the diffusivity, t being time, and a being the contact radius that is related 166 to the radius of the spherical probe R and indentation depth h by a=√Rh: 167 (1) 168 This master curve has been derived numerically as 169 Normalizing the experimental data and fitting it with the theoretical curve (Eq.2), we can extract 171 the single fitting parameter diffusivity D. More details can be seen in references ( HAMA solution, photopolymerized and cultured following the same method previously 195 described. Cell invasion into the hydrogel was traced throughout seven-day culture by taking 196 images on days 0 (immediately after embedding), 1, 2, 3, 5 and 7 using a Leica DMI 400B 197 florescence microscope under bright field. Analysis of cell invasion distance (d i = r i -r 0 ) was 198 quantified via ImageJ using the relative radius (cell spreading shape ~ πr i 2 ) compared to day 0 (r 0 ) 199 using a method previously described by our group (Chen et al., 2017a). 200

Analysis of cell metabolic activity 201
The total metabolic activity of cell-containing hydrogels was measured immediately after 202 hydrogel encapsulation (day 0) and then subsequently at days 3 and 7 of hydrogel culture. ImageJ and normalized to β-actin expression. Buffers and antibodies used in each condition are 234 listed (Table S1). 235

Statistics 236
All statistical analysis was performed using one-way analysis of variance (ANOVA) followed by 237 Tukey's test. A minimum sample number of n = 3 (MTT, ELISA, Western), n = 6 (Young's 238 modulus, diffusivity, invasion) samples were used for all assays. Statistical significance was set 239 at p < 0.05. Error is reported as the standard error of the mean. 240

Metabolic activity of GBM39 PDX cells cultured in GelMA hydrogels is sensitive to the 255 molecular weight of matrix bound HA 256
The metabolic activity of GBM39 PDX cells encapsulated within the homologous series of 257 GelMA hydrogels (-HA, 10K, 60K, 500K) was traced through 7 days in culture, with results 258 normalized to day 0 values for each group. The groups with matrix-bound HA (10K, 60K and 259 500K) showed a significantly higher metabolic activity compare to -HA group (p < 0.05), with 260 the 60K HA group showing the highest metabolic activity amongst all groups (Figure 2). 261

The molecular weight of matrix-bound HA significantly affects invasion 262
The invasion of GBM39 PDX cells into the surrounding hydrogel matrix was measured via a 263 previously reported spheroid assay through 7-days in culture. GBM39 invasion was strongly 264 influenced by hydrogel HA content. The highest level of invasion was observed for GelMA 265 hydrogels either lacking matrix bound HA (-HA), or those containing mid-range (60K) 266 molecular weight matrix-immobilized HA (Figure 3). At early-to-mid time points (up to day 5), 267 GBM cell invasion was significantly depressed in the low molecular weight 10K group, but 268 GBM invasion increased steeply at later time points (day 7), matching the highest invasion 269 groups. GelMA hydrogels containing the largest molecular weight HA (500K) showed 270 significantly reduced invasion compared to all other hydrogel groups (-HA, 10K, 60K) 271 throughout the entire period studied. Interestingly, the presence of soluble HA for hydrogel groups containing matrix-immobilized 277 HA was found to be strongly associated with the molecular weight of immobilized HA, with 278 500K group showing significantly upregulated secretion compared to GBM cells in 10K and 279 60K HA hydrogels as early as day 3. Significant increases were observed in soluble HA 280 production in 60K vs. 10K hydrogels appeared by day 7 of culture ( Figure 4). compared to all other hydrogel conditions. 290

Discussion 291
The heterogeneity of GBM tumor microenvironment complicates its study both in vivo and in 292 vitro. Within that high diversity, the extracellular HA has been widely associated with cancer 293 invasion and response to treatment (Park et  framework to adjust the relative ratio of GelMA to HA content as well as manipulating the 312 crosslinking conditions to generate a series of GelMA hydrogels containing increasing wt% of a 313 single MW HA (Pedron et al., 2013a). We therefore adapted this approach to create the 314 homologous series of hydrogels described in this study, that contained a conserved wt% of HA 315 but that varied the MW of matrix-immobilized HA. We then employed a series of biophysical 316 and biochemical characterization protocols to describe poroelastic features of these hydrogels. 317 Crosslinking density can be preserved by adjusting the photoinitiator concentration in the pre-318 polymer solution (Table 1), and therefore maintaining the Young's modulus between different 319 hydrogels. Moreover, the deformation of the gel in contact with the AFM tip results from two 320 simultaneous molecular processes: the conformational change of the network, and the migration 321 of the solvent molecules (Hu et al., 2010). In this case, the poroelasticity of the hydrogels, 322 characterized by the diffusivity (Figure 1D), stays unchanged for all samples used. Both 323 Young's modulus and diffusivity showed no significant difference among all groups suggesting 324 these hydrogels were able to provide similar culture conditions for cells while providing the 325 opportunity to adjust the molecular weight of bound HA. we found GBM invasion in GelMA hydrogels lacking matrix bound HA was greatest. However, 334 invasion was strongly influence by the MW of immobilized HA with GBM cell invasion in 335 hydrogels containing 60kDa being equivalent to hydrogels lacking matrix bound HA. Further, 336 this invasive potential of GBM39 cells within -HA and +HA hydrogels is not associated to their 337 metabolic activity profiles (Figure 2). Although migration and proliferation are considered to be 338 circumscribed phenotypes that do not co-occur with each other in GBM, the complex 339 microenvironment of PDX suggests that both can coexist. Moreover, GBM cells adapt to the 340 different phenotypes by using regulatory signaling from the local microenvironment (Xie et al.,  341 2014). Interestingly, while invasion was initially significantly reduced in low MW HA hydrogels 342 (10K), GBM invasion increased significantly at later time points. However, GBM invasions was 343 strongly reduced in GelMA hydrogels containing high molecular weight HA (500K) throughout 344 the entirety of the study, suggesting more mature HA matrices will inhibit GBM invasion. While 345 recent studies have begun to examine the design of implants to reduce GBM invasion (Jain et al.,346 2014), these findings suggest an interesting line of future studies that wound center on the 347 incorporation of hydrogels into the resection margins containing attractive biomechanical and 348 biomolecular properties to potentially recruit nearby GBM cells as a means to reduce invasive 349 spreading. Regardless, the presence of both fibrillar and HA associated features of the TME in 350 these HA decorated GelMA hydrogels may be particularly useful in the context of GBM 351 invasion in perivascular niches that contain such matrix diversity (Ngo and Harley, 2018). 352 Studies have shown that HMW HA could inhibit tumor invasion by inhibiting MMPs production 353 and down-regulating invasion related pathways such as MAPK and Akt (Chang et al., 2012), 354 while LMW HA may promote these invasion related pathways (West et al., 1985;Lam et al., 355 2014). We hypothesized that the significant decrease of motility in PDX cells in 500kDa 356 hydrogels is due to the down-regulation of invasion related pathways, induced by the local 357 extracellular microenvironment. We observed endogenous HA production was significantly 358 elevated without the presence of matrix-bound HA (-HA) (Figure 4)

Conflict of Interest 414
The authors declare that the research was conducted in the absence of any commercial or 415 financial relationships that could be construed as a potential conflict of interest. via Western Blot at day 7 (n=3). β-actin is used as loading control. ^ p < 0.05 significant increase 665 between different groups. 666 667