Collagen

Despite collagen type I not being an abundant protein in the GBM and healthy brain ECM, it is often used in mechanics studies of these cells, due to its ability to easily form a gel. Even though collagen IV is upregulated and more abundant in the GBM microenvironment, there is a limited amount of studies in which collagen IV is used for hydrogel formation to investigate GBM mechanics [171]. There are commercially available pre-coated glass slides that make mechanics studies using collagen I simpler. In studies where glioma cells were seeded onto collagen I-coated substrates (glass or polystyrene), there was a trend of cells spreading more extensively in stiffer ECMs [76,273]. One group observed a nearly linear 80 µm extension called an "invadopodia" that stretched from one cell to another, which they hypothesized was a significant factor in cell signaling [273]. The drawback of studies using collagen-coated glass or polystyrene is that it forms a 2D culture, which does not accurately mimic the 3D environment that cells are exposed to in vivo. It was shown that GBM cells exhibited chemoresistance to sunitinib, a kinase inhibitor, in vitro in a 3D collagen-based environment, but not on plastic or collagen-coated surfaces [136]. Additionally, 2D cell migration does not require MMP activity, while 3D culture does, so MMP expression can be studied extensively in relation to mechanics only in 3D culture [274]. For greater insight into in vivo characteristics, it is more useful to employ 3D rather than 2D cultures.

Collagen fiber density or concentration is an important factor to consider in experimental design. A study confirmed that varying the collagen I concentration resulted in varying elastic modulus of hydrogels, but that the difference is not significant with collagen concentrations from 1.5 to 2.5 mg/mL [275]. They also found that a higher concentration of collagen inhibited growth of the GBM in vitro. Decreasing collagen gel stiffness was shown to increase migration distance and velocity of GBM cells [124]. Another study investigated the effects of gelation temperature on collagen I gel pore size and how this impacted glioma invasion. Gelation temperature caused variations in pore size for hydrogels composed of 1 mg/mL collagen and was a significant factor in determining speed of invasion of glioma cells with smaller pore sizes (5-12 µm) hindering cell motility [276]. A study by Hwang et al. showed that actin filamentation of migrating cells in collagen gels is dynamic and undergoes rapid changes, and produces of stress fibers and lamellipodia [277]. This study also drew attention to the migratory patterns of glioma cells, which was shown to mostly be composed of double-nucleated cells that migrated in clusters and had extensive interactions, through actin filament extensions, to collagen in the surrounding gel and nearby cells [277]. Distinct cell shapes, in terms of elongation and roundness, have been observed in different collagen gels [278]. Collagen gels were also used to study the effect of the microenvironment on gene expression and the effects of depletion or overexpression of certain genes on cell ability to retain mechano-responsiveness [278–280].

Three-dimensional culturing methods are being modified with novel technologies to model more than just a homogenous tumor microenvironment; they are being used to form models of interacting systems. In a study by Chonan et al., the tumor niche including ECM and blood vessels was mimicked in a microfluidic device using collagen I and human umbilical vein endothelial cells (HUVECs) [281]. Further studies with collagens are required for a more thorough understanding of the effect of these proteins on GBM cells.

### Hyaluronan

There is extensive research on the production of hyaluronan (HA)-based gels for studies of GBM mechanics. HA hydrogels have been used clinically as implants for neural regeneration and reduction of scar formation [282,283]. In in vitro studies, HA gels are generally functionalized with peptides or other ECM proteins, such as laminin, RGD (Arg-Gly-Asp peptides), poly-D-lysine, and poly-L-lysine, which allow for cell adhesion to the gel, since HA alone does not attach to the cells [283,284]. In a study with short Arg-Gly-Asp (RGD) peptides incorporated to HA gels, it was noted that glioma cells' actin stress fiber assembly and cell spreading was greater in stiffer gels [147]. Various HA crosslinkers can also be used to increase gel stiffness. Divinyl sulfone, for example, was used to crosslink HA carboxyl groups to varying degrees when different concentrations were used [285]. Chitosan-HA scaffolds, when compared to 2D surfaces, increased invasiveness

and chemotherapeutic resistance and were proposed as possible in vitro mimics of the tumor microenvironment for pre-clinical drug effectiveness studies [286].

### Other Proteins

Proteins such as fibronectin, laminin, and gelatin on their own are not commonly used for hydrogel formation. Rather, they are more frequently used to coat glass or polystyrene for two-dimensional cell-protein interaction studies. In the formation of hydrogels, they are generally used in combination with synthetic materials. These were also discussed previously in the synthetic hydrogels section of this review.

Methacrylated gelatin (GelMA) has been used on its own as a scaffold for glioma cells in a study of gel biophysical properties and their effect on cell morphology, proliferation, motility, and gene expression. Pedron et al. observed that GelMA biophysical properties could be varied by the methacrylation degree and bulk density. Cell morphology, motility and expression of hypoxia markers (VEGF, MMP-2, MMP-9, and HIF-1) and the ECM protein fibronectin were affected by the environmental properties [287]. Ramamoorthi et al. used an alginate hydrogel with varying stiffness values and observed greater cell sensitivity to toxins in less stiff gels [133].

### Composite Biomaterial Hydrogels

There is a paucity of research involving composite hydrogels in the study of GBM mechanics. Researchers developed a composite matrix of HA and collagen oligomers with the addition of Matrigel-coated microfibers with tunable stiffness by varying component protein concentrations [124]. While low stiffness correlated to lower migration velocity and distance, collagen source and concentration was shown to affect these parameters variably. They also reported that in the HA hydrogel, cells exhibited collective migration while in collagen, they relied on single-cell migration [124]. These studies outline some of the different cellular responses to each of the ECM components and serve to emphasize the importance of studying specific cell-ECM interactions when attempting to describe GBM mechanics.

### Synthetic Hydrogels

There are several types of synthetic polymers that have been used in the study of GBM mechanics. In 2D culture, "poly-methylphenyl-siloxane film" with greater stiffness values resulted in increased cell spreading and migration compared to more compliant films [181]. Wan et al. developed nanotextured polydimethylsiloxane (PDMS) surfaces with aptamers overexpressing epidermal growth factor receptor (EGFR) to isolate human GBM cells from a mix with fibroblast cells [288]. PA hydrogels are commonly used in GBM mechanics studies. In a novel study of cell mechano-sensitivity to matrix confinement, cells that were seeded onto PA hydrogels in more narrow channels exhibited greater migration speeds when compared to cells seeded on PA hydrogels in wider channels or directly onto 2D surfaces. This effect was abrogated by the inhibition of non-muscle myosin II, implicating this protein as a mechanosensory [102]. In a study investigating patient-derived primary GBM cell sensitivity to gel stiffness or rigidity, cells were seeded onto PA gels with different rigidity measures and the migration rate was correlated to rigidity-sensitivity [173]. Human glioma cell lines U373 and U118 cultured on polyacrylamide gels of normal brain stiffness (1 kPa) and GBM tumor stiffness (12 kPa), had greater proliferation rates in the stiffer substrate [289]. Umesh et al. studied the effect of PA gel stiffness on expression of proteins related to the cell cycle and dependency on EGFR signaling in human GBM cells, and saw that increased stiffness caused increased expression and phosphorylation of EGFR and Akt. Conversely, loss of EGFR, Akt, or phosphoinositide 3-kinase (PI3K) function resulted in decreased stiffness-sensitivity of the cells [252].

### **4. Challenges and Future Directions** We have endeavored to provide a thorough explanation of the various hydrogelbased methods that are employed for biomechanical studies and an overview of how

**4. Challenges and Future Directions**

*Gels* **2022**, *8*, x FOR PEER REVIEW 23 of 36

We have endeavored to provide a thorough explanation of the various hydrogelbased methods that are employed for biomechanical studies and an overview of how studying cell-ECM interactions can lead to significant advancements in understanding the pathology of cancers, with a focus on GBM. It is abundantly clear from the state of the field that cellular response to biomechanical cues is a key player in maintaining health and homeostasis. To incorporate biomechanical effects into our current understanding of cell biology, hydrogel-based three-dimensional models of cell culture must become more widely used. The number of publications mentioning hydrogels has increased exponentially over the past 25 years (Figure 7). Furthermore, publications mentioning both hydrogels and cancer are making up a greater proportion of the hydrogel-related manuscripts published each year since 2012, indicating a growing interest in the development and use of hydrogels in the study of cancer. However, a major drawback of these approaches is that any one hydrogel model is not universally applicable due to the unique ECM composition and mechanical properties of the suite of cellular states that may be studied. Additionally, differences in hydrogel composition (and lot-to-lot variability of commercially available hydrogels), cell passage number, cell seeding density, hydrogel crosslinking density, time between cell seeding and microscopy, and other factors make it difficult to obtain consistent results [290,291]. It is worthy of note that ECM composition still has not been characterized based on the abundance of each component and elastic modulus for many tissues and conditions. Protocols unique to the disease model (cell type, microenvironment, perfusion, etc.) need to be established to enable wider adoption of hydrogel-based cell culture studies as an improved biomimetic replacement for conventional two-dimensional cell culture. studying cell-ECM interactions can lead to significant advancements in understanding the pathology of cancers, with a focus on GBM. It is abundantly clear from the state of the field that cellular response to biomechanical cues is a key player in maintaining health and homeostasis. To incorporate biomechanical effects into our current understanding of cell biology, hydrogel-based three-dimensional models of cell culture must become more widely used. The number of publications mentioning hydrogels has increased exponentially over the past 25 years (Figure 7). Furthermore, publications mentioning both hydrogels and cancer are making up a greater proportion of the hydrogel-related manuscripts published each year since 2012, indicating a growing interest in the development and use of hydrogels in the study of cancer. However, a major drawback of these approaches is that any one hydrogel model is not universally applicable due to the unique ECM composition and mechanical properties of the suite of cellular states that may be studied. Additionally, differences in hydrogel composition (and lot-to-lot variability of commercially available hydrogels), cell passage number, cell seeding density, hydrogel crosslinking density, time between cell seeding and microscopy, and other factors make it difficult to obtain consistent results [290,291]. It is worthy of note that ECM composition still has not been characterized based on the abundance of each component and elastic modulus for many tissues and conditions. Protocols unique to the disease model (cell type, microenvironment, perfusion, etc.) need to be established to enable wider adoption of hydrogelbased cell culture studies as an improved biomimetic replacement for conventional twodimensional cell culture.

on expression of proteins related to the cell cycle and dependency on EGFR signaling in human GBM cells, and saw that increased stiffness caused increased expression and phosphorylation of EGFR and Akt. Conversely, loss of EGFR, Akt, or phosphoinositide 3-ki-

nase (PI3K) function resulted in decreased stiffness-sensitivity of the cells [252].

**Figure 7.** A bar chart showing the number of publications containing the key word "hydrogel" or ("hydrogel" AND "cancer") from the years 1998 to 2021 found in the PubMed database. **Figure 7.** A bar chart showing the number of publications containing the key word "hydrogel" or ("hydrogel" AND "cancer") from the years 1998 to 2021 found in the PubMed database.

The use of hydrogels as model systems for studying cell response to biomechanical stimuli has been discussed in this review, with a focus on GBM as a case study for what has been achieved versus what has yet to be understood in the field. Breast cancer is an exemplar of the impact that biomechanical studies can have on diagnostic, prognostic, and therapeutic approaches to a disease. Therefore, we believe that future studies to understand how cells respond and adjust to mechanical stimuli, and how these responses may be dysregulated in various pathologies, are of utmost importance to craft a more The use of hydrogels as model systems for studying cell response to biomechanical stimuli has been discussed in this review, with a focus on GBM as a case study for what has been achieved versus what has yet to be understood in the field. Breast cancer is an exemplar of the impact that biomechanical studies can have on diagnostic, prognostic, and therapeutic approaches to a disease. Therefore, we believe that future studies to understand how cells respond and adjust to mechanical stimuli, and how these responses may be dysregulated in various pathologies, are of utmost importance to craft a more systematic understanding of diseases that have been difficult to treat and cure, such as GBM. An improved understanding of tissue and cellular mechanics would facilitate the development of mechanotherapies for regenerative rehabilitation [292–295]. Not only will such studies further our understanding of the disease, but they may also provide clues for how to take advantage of mechanical stimuli to treat them. Hydrogels are used in contact lenses and as vehicles for drug delivery; they also have the potential to be used as therapeutic agents in cases where mechanical cues such as stiffness or stress can influence therapeutic resistance

in certain cancers. For example, hydrogels with low stiffness may be implanted after tumor removal in patients with invasive cancers to reduce the ability of cancer cells to migrate. Such innovative uses for hydrogels and applications of knowledge that can be gained by biomechanical studies would improve our understanding and treatment of cancer.

**Author Contributions:** Conceptualization, A.Z.S., M.B. and C.B.P.; Methodology, A.Z.S. and C.B.P.; Validation, A.Z.S.; Investigation, A.Z.S.; Resources, A.Z.S. and C.B.P.; Data Curation, A.Z.S., M.B. and C.B.P.; Writing—Original Draft Preparation, A.Z.S. and C.B.P.; Writing—Review and Editing, A.Z.S., M.B. and C.B.P.; Visualization, A.Z.S.; Supervision, M.B. and C.B.P.; Project Administration, C.B.P.; Funding Acquisition, C.B.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. The APC was funded by the McNair Medical Institute at The Robert and Janice McNair Foundation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** CBP is a McNair Scholar supported by the McNair Medical Institute at The Robert and Janice McNair Foundation.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Abbreviations**



### **References**

