*2.4. Clinical Applications of Hydrogels*

A clinical approach to hydrogel scaffolds is found tissue engineering applications, which have gained traction recently as potential solutions to donor shortage problems for tissue or organ transplantations [203,204]. These approaches generally combine cells from a donor with a hydrogel scaffold that is prepared to mimic the extracellular matrix of the tissue or organ being engineered [203]. Hydrogels are suitable tissue-engineering applications due to their ability to uptake water, to encapsulate cells, and to be bio-reactive [8]. Various hydrogels have been used as bio-ink for 3D organ or tissue printing applications in which tissues are built with direct deposition of cells with the bio-ink [205]. Studies have shown applicability of alginate, collagen, and various composite hydrogels for tissue printing methods [206–208]. For instance, natural protein and polysaccharide hydrogels have been used for articular cartilage-tissue engineering applications to promote cartilage regeneration [209]. Latifi and colleagues demonstrated the potential of an injectable hybrid hydrogel (collagen I and III) to be applied in soft-tissue engineering, specifically human vocal fold engineering [210]. A long-standing problem of the tissue engineering field is the need for in vitro tissue vascularization to be able to transplant or implant larger portions of tissue into patients. A couple of groups have recently made strides in promoting in vitro vascularization of hydrogel constructs of bone [211] and soft tissue [212].

Injectable hydrogels have been studied for drug delivery and wound healing or dressing applications [213]. Hydrogels have been applied in drug delivery applications due to their ability to give control over the time and/or site of drug delivery for enhanced treatment [214,215]. The biocompatibility, similarity to native tissues, and high-water content all contribute to great applicability of hydrogels for controlled drug release and delivery [216]. Naturally derived injectable hydrogels for the controlled delivery of small molecules to the central nervous system have also been extensively studied. Wang et al. have shown that an injectable hyaluronan-methylcellulose hydrogel enhanced delivery of growth factors [217]. Further, the great tunability of hydrogels has led to development of thermosensitive, pH-sensitive, and temperature-sensitive hydrogels that can be used for drug delivery in distinct biological environments [218–220]. Similarly, temperaturesensitive hydrogels have been developed for various wound-healing applications. A PEG-PLGA-PEG composite hydrogel has been developed for delivery of a growth factor linked to tissue repair for diabetic wound healing [221].

Tissue regeneration is another field in which hydrogels are being investigated and have shown to be promising for translation to the clinic due to their biocompatibility and ability to be fine-tuned or adapted for specific applications. For instance, Zheng et al. developed a polyacrylic, acid-alginate-demineralized, bone matrix hybrid double-network hydrogel, which was shown to promote vascular endothelial growth factor (VEGF) synthesis and basic fibroblast growth factor (bFGF) and alkaline phosphatase activity of MG63 osteosarcoma cells to enhance bone regeneration [211]. Stem-cell therapy is a promising solution for injuries that require tissue regeneration, but it is limiting since uncontrolled differentiation can lead to the presence of unnecessary cells at the site of injury and lead to stem cell metastasis and tumorigenesis [43,60,129]. Application of hydrogels can improve stem

cell therapy by introducing stem cells and differentiation factors to the injury in a sitespecific manner [5]. In a study of spinal cord injury, Mothe et al. investigated a hydrogelintegrated stem cell therapy by encapsulating neural stem cells and differentiation factors in a hyaluronan-based hybrid hydrogel and found that treatment enhanced graft survival, increased oligodendrocytic differentiation, and reduced cavitation in the injury site in rats [222].

Contact lenses are an example of the clinical application of hydrogels [223,224]. While hard contact lenses are composed of hydrophobic materials, soft contact lenses are hydrogelbased [225]. Owing to the wide variety in hydrogel-forming substances, attempts are continually being made to improve the physical and chemical properties of contact lenses [225]. Hydrogels have also been used clinically in dermatology applications such as wound healing and skin regeneration [226,227].

### **3. Cancer and the Tumor Microenvironment**

Biomechanical properties of the tumor microenvironment have been shown to be altered compared to the healthy state in many types of cancer to promote processes crucial to tumorigenesis, including cellular proliferation and migration. Since changes in extracellular mechanical properties can induce structural reorganization, morphological changes, and altered signaling, they can cause cancer cells to exhibit mechanical properties differently than healthy cells, e.g., stiffness [228]. This, in turn, can further promote invasive or metastatic phenotypes [229]. For instance, cancer cells also usually have a lower Young's modulus compared to healthy cells of the same type, which can influence deformability and influence migratory ability [230]. Cancer cells also have a more robust ability to respond to ECM conditions, and can alter cytoplasm viscoelasticity in response to increased ECM stiffness and collagen I deposition [231].

Breast cancer is a context in which mechanical properties of tissue and cells have been well described, and many biomechanical contributions to carcinogenesis and metastasis have been identified [96]. Many of the studies involving cell response to mechanical stimuli have been conducted with aggressive breast-cancer cells, especially the MDA-MB-231 cell line, due to their robust response to changes in extracellular mechanics. Clinically, tissue stiffness has served as an indicator of breast tumors and risk of breast cancer [232,233], and the biomolecular consequences of this phenotype has been studied rigorously in many types of 3D hydrogel models in the laboratory, especially in the last decade. Various mechanical stimuli can cause cellular stress and lead to carcinogenesis or increased invasiveness of cancer cells. For instance, several mechanotransduction pathways are linked to carcinogenesis and invasiveness and upregulated in cancer [95,100,234,235]. Application of mechanical load was shown to regulate breast-cancer cell proliferation independent of matrix deformations or stiffness [234]. Mechanical stretch, ECM stiffness, and fluid shear-stress all led to more invasive phenotypes of breast-cancer cells. At the level of response to treatment, ECM stiffness has been characterized to contribute to chemoresistance of breast-cancer cells to doxorubicin [235]. Therefore, a potential therapeutic approach may be to introduce proteinases or drugs that reduce ECM stiffness by degrading certain components to reduce the number of treatment-resistant cells. Lastly, certain microenvironmental properties have been correlated to improved prognosis in breast cancer and can be used to identify diagnostic and prognostic signatures. For instance, the tumor-associated collagen signature consisting of aligned collagen fibers in biopsied tissues from breast-cancer patients has been identified as a prognostic signature for survival [236].

The positive contributions that studying biomechanics has made to our overall understanding of breast cancer highlight the importance of incorporating a biomechanics-based approach to cancer biology in vitro studies. Inspired by the benefits of biomechanics studies on our understanding of breast cancer, we will present the current research in glioblastoma (GBM), which is a disease that has not been well-defined biomechanically. We believe that a better understanding of the biomechanical properties of GBM and its microenvironment

can produce translatable results that may contribute to the development of diagnostic and therapeutic approaches to improve the prognosis of this invasive cancer.

### *3.1. Glioblastoma and the Tumor Microenvironment*

Biomechanical and biophysical studies can help to glean valuable insight into a wide variety of diseases, since biomechanics is an integral part of cell proliferative, migration, and survival signaling, all of which are crucial to carcinogenesis and tumorigenesis. GBM is the most common and lethal form of primary brain cancer in adults [237], but there are only two clinically approved chemotherapies targeting it. It is an example of a disease that has not been traditionally studied in terms of biomechanics until approximately the last ten years but is one that may greatly benefit from such studies. Here, we will present an overview of the biomechanical properties known about GBM, the methods and contributions of available 3D culture and mechanotransduction studies of GBM, and the potential translational impact of those studies on the clinic and patient survival.

### 3.1.1. The Blood-Brain Barrier

The blood-brain barrier (BBB) is also an essential part of the brain microenvironment that is altered in the cancer state. The BBB is formed by vascular endothelial cells lining microvessels in the brain and is essential in regulating brain extracellular conditions to ensure neuronal signaling [238]. The endothelial cells of the BBB limit transcellular and paracellular transit into, and thereby protect, the brain by regulating permeability through tight junctions, adherens junctions, charged moieties, pericytes, etc. [239,240]. In tumor microvessels, however, loss of claudin-1 and claudin-3 and down-regulation of claudin-5 was observed, which correlated to increased permeability [241,242].

### 3.1.2. Extracellular Matrix of the Brain

The ECM of the brain (Figure 5) is altered when tumorigenesis occurs. Several ECM components, including HA, tenascin-C, and vitronectin, are upregulated in the tumor microenvironment [243]. Studies show that proteins of the tumor niche also tend to be different than healthy brain tissue and that tumor invasion alters ECM composition [244]. Basement membrane components such as laminin, fibronectin, and collagen type IV are more highly secreted by glioma cells and, in turn, alter composition of the local ECM [245,246]. Tumorassociated mesenchymal stem-like cells induce HA synthase 2 activity and lead to greater HA abundance in the tumor niche [247]. In addition to a distinct microenvironment, the GBM tumor has a hypoxic and necrotic core that aids in the cancer cell-induced blood vessel formation by increasing expression of pro-angiogenic VEGF, VEGFR2, and angiopoietin 2, which results in the disorganized network of blood vessels observed in GBM [243,248–250].

Many cellular properties are altered in the cancer state. For instance, cell proliferation, migration, and deformability is increased in the GBM state when compared to healthy cells [67,138,251–254]. The impact of the extracellular niche on these cellular properties and the biology behind the changes have been elucidated by various in vitro and in vivo studies focusing on mechanical cell-ECM interactions [17,101,255–257].

### 3.1.3. Overview of Microenvironment and Biomechanics of Glioblastoma

A large body of research has been dedicated to studying the differences between the microenvironments, or niche, of tumor and healthy cells. These include studies on overall tissue stiffness, ECM composition, cellular signaling, and the presence/activation of mechanotransducers. There is a general consensus that while cancer cells are less stiff and more deformable than healthy cells [28,105,106,255,256], tumor tissue tends to be stiffer by variable magnitude compared to non-tumor tissues, a trend that has been shown in thyroid, breast, prostate, bladder, and kidney tissues [107–109]. The tumor niche is also characterized by altered ECM composition, which may lead to increased invasiveness and metastatic properties of cancer cells [257]. Characterization of the ECM, both biologically and mechanically, in GBM has not reached the depth of understanding

that has been achieved for several other types of cancer, such as prostate and breast cancer. However, there are certain differences that have been noted between healthy and cancerous tissue microenvironments, which can lay the groundwork for future studies of GBM microenvironment and mechanics. *Gels* **2022**, *8*, x FOR PEER REVIEW 18 of 36

**Figure 5.** Extracellular matrix (ECM) of the brain. The ECM of the brain has a unique composition, including hyaluronic acid, collagen IV, and other ECM components along with glial cells, neurons, and astrocytes. The blood-brain barrier (BBB) is a neurovascular unit composed of vascular endothelial cells with surface charge modifications, tight junction proteins, pericytes, astrocytes, and other components. The BBB is selectively permeable and can block solutes in the systemic blood from entering the environment of the central nervous system (Created with BioRender.com, accessed on 14 July 2022). **Figure 5.** Extracellular matrix (ECM) of the brain. The ECM of the brain has a unique composition, including hyaluronic acid, collagen IV, and other ECM components along with glial cells, neurons, and astrocytes. The blood-brain barrier (BBB) is a neurovascular unit composed of vascular endothelial cells with surface charge modifications, tight junction proteins, pericytes, astrocytes, and other components. The BBB is selectively permeable and can block solutes in the systemic blood from entering the environment of the central nervous system (Created with BioRender.com, accessed on 14 July 2022).

### 3.1.3. Overview of Microenvironment and Biomechanics of Glioblastoma Biomechanics of the Glioblastoma Extracellular Matrix

A large body of research has been dedicated to studying the differences between the microenvironments, or niche, of tumor and healthy cells. These include studies on overall tissue stiffness, ECM composition, cellular signaling, and the presence/activation of mechanotransducers. There is a general consensus that while cancer cells are less stiff and more deformable than healthy cells [28,105,106,255,256], tumor tissue tends to be stiffer by variable magnitude compared to non-tumor tissues, a trend that has been shown in thyroid, breast, prostate, bladder, and kidney tissues [107–109]. The tumor niche is also characterized by altered ECM composition, which may lead to increased invasiveness and metastatic properties of cancer cells [257]. Characterization of the ECM, both biologically and mechanically, in GBM has not reached the depth of understanding that has been achieved for several other types of cancer, such as prostate and breast cancer. However, there are certain differences that have been noted between healthy and cancerous tissue microenvironments, which can lay the groundwork for future studies of GBM microenvironment and mechanics. The mechanical properties of the tumor niche in GBM are different than those of the healthy brain ECM due to the altered composition, protein-protein, cell-protein, and cell-cell interactions. Even within the GBM tumor, there are distinct mechanical regions for necrotic and non-necrotic portions [250]. In general, GBM tissue has been found to be stiffer than healthy ECM, with increasing stiffness generally correlating to increased malignancy [109,147]. From a set of human brain biopsies, one study showed that increasing malignancy of tumors gave higher Young's modulus values, with primary GBMs exhibiting stiffness values varying from 70 to 13,500 Pa [258]. Stewart et al. showed that brain tumors had elastic moduli ranging from 170 to 16,060 Pa using a custom-built indenter [259]. Another study correlated increased ECM stiffness to decreased survival of human patients [260]. Altered stiffness results in changed mechanical cues that are relayed to the cell, which then impact cellular gene expression so significantly that the overall behavior of the cell can be drastically changed [9] (Table 1). Several studies have been published that note differences in cellular morphology, deformability, motility, proliferation, and signaling in response to changes in environmental stiffness [104,134].

### The mechanical properties of the tumor niche in GBM are different than those of the Modification of the Extracellular Matrix in the Brain by Glioblastoma Cells

healthy brain ECM due to the altered composition, protein-protein, cell-protein, and cellcell interactions. Even within the GBM tumor, there are distinct mechanical regions for The tumor also modifies the local ECM through protein degradation. Some of the most studied cell-secreted proteins to be up-regulated in the cancer state are matrix metal-

necrotic and non-necrotic portions [250]. In general, GBM tissue has been found to be stiffer than healthy ECM, with increasing stiffness generally correlating to increased ma-

ing stiffness values varying from 70 to 13,500 Pa [258]. Stewart et al. showed that brain

Biomechanics of the Glioblastoma Extracellular Matrix

loproteinases (MMPs), which are proteases that remodel the ECM by degrading certain component proteins [261]. The hypoxic core of GBMs has been shown to be a significant contributor to the increased MMP activity [262]. Various MMPs that are up-regulated in GBMs have also been shown to aid in glioma cell invasion [263]. Plasminogen activation by the urokinase pathway, which includes urokinase (uPA), urokinase receptor (uPAR), and plasminogen, is also prominent in GBMs. The urokinase pathway aids in ECM degradation and remodeling by converting plasminogen into active plasmin, which is a serine kinase that degrades certain ECM proteins, and activates MMP-2 and MMP-9 [264]. MMP-2 is a type IV collagenase that has been implicated in invasion and metastasis of GBMs [263]. Lastly, various cathepsins, which are lysosomal cysteine proteases that can be secreted into the ECM, are also up-regulated in GBMs and have been linked to tumorigenesis and invasion [265–267].

### 3.1.4. Glioblastoma Migration, Invasion, and Mechanotransduction

Some hallmarks of GBM are enhanced cellular migration and aggressive invasiveness [268]. These properties are achieved through complex mechano-chemical signaling mechanisms that enable crosstalk between the tumor cells and the tumor microenvironment. The proteins that sense and translate mechanical cues from the ECM or microenvironment and relay them to the cell are called mechanotransducers [54]. Interestingly, one study found that keeping the mechanotransducer RhoA GTPase constitutively active in vitro in U87 cells caused similar toxic responses in 3D environments with varying stiffness, indicating the importance of mechanotransduction in cell response to environmental conditions [133]. Another study found that by altering mechanotransducers in GBM tumor-initiating cells, they were able to alter cell motility and invasion in 3D cultures [67]. Knock-down of CD44, a transmembrane glycoprotein receptor for HA and other ECM components, resulted in decreased microtubule and vimentin expression, hampered migration, and decreased nuclear stiffness compared to control cells [104]. Integrins are well-studied mechanotransducers that are linked to malignancy and are primary communicators in cell-ECM adhesion and signal transduction [134,245,263]. Clinical nuclear medicine studies have evaluated the role of various positron emission tomography (PET) radiotracers (e.g., [18F]Galacto-RGD [269], [ <sup>68</sup>Ga]PRGD2 [270], and [18F]FPPRGD2 [271]) to enable molecular imaging of integrin avb3, a member a class of adhesion molecules that mediate cell–cell and cell-ECM interactions, and which plays an important role in cancer metastasis and angiogenesis [272]. These studies reveal the importance of understanding how mechanotransducers process mechanical cues from the ECM to influence cancer-cell properties [93,94].

Cellular structure and cytoskeletal alterations are under the direct influence of ECM mechanical cues and cellular mechanotransducers [93]. Pathak and Kumar found that culturing cells in narrow versus wide channels of various extracellular stiffnesses led to altered cell morphology, migration, and myosin alignment (Figure 6), underlining the importance of extracellular culture conditions in determining cell behavior. Various integrins serve to form attachments to proteins of the ECM. Once an integrin is bound to an extracellular ligand, focal adhesion clusters form at the surface of the integrin receptor within the cell, which link to the cytoskeleton and function in cell motility by recruiting proteases for ECM degradation and activating signaling pathways that induce cytoskeletal rearrangement [55]. Cytoskeletal rearrangement as orchestrated by cell-ECM interactions are vital to increasing cell deformability and, in turn, enhancing migration.

**Figure 6.** Cell migration, cell area, actin alignment, and myosin alignment when exposed to different channel widths and hydrogel stiffnesses. (**A**–**C**, **left panel**) Migration speed was quantified for cells cultured in narrow or wide channels with varying stiffness. With increased stiffness but decreased channel width, cell migration speed was higher. \* *p* < 0.05. (**A**–**D**, **right panel**) Cell area, actin alignment, and myosin alignment were quantified from cells imaged after culture in wide or narrow channels with varying stiffness. Cells cultured in stiffer conditions with narrow channels exhibited increased actin and myosin alignment but lower cell spreading than those in narrow channels. Figure reprinted/adapted with permission from Ref. [102]. Copyright 2012, National Academy of Sciences. **Figure 6.** Cell migration, cell area, actin alignment, and myosin alignment when exposed to different channel widths and hydrogel stiffnesses. (**A**–**C**, **left panel**) Migration speed was quantified for cells cultured in narrow or wide channels with varying stiffness. With increased stiffness but decreased channel width, cell migration speed was higher. \* *p* < 0.05. (**A**–**D**, **right panel**) Cell area, actin alignment, and myosin alignment were quantified from cells imaged after culture in wide or narrow channels with varying stiffness. Cells cultured in stiffer conditions with narrow channels exhibited increased actin and myosin alignment but lower cell spreading than those in narrow channels. Figure reprinted/adapted with permission from Ref. [102]. Copyright 2012, National Academy of Sciences.

extracellular ligand, focal adhesion clusters form at the surface of the integrin receptor within the cell, which link to the cytoskeleton and function in cell motility by recruiting proteases for ECM degradation and activating signaling pathways that induce cytoskeletal rearrangement [55]. Cytoskeletal rearrangement as orchestrated by cell-ECM interac-

tions are vital to increasing cell deformability and, in turn, enhancing migration.

### 3.1.5. In Vitro Studies of Mechanotransduction in Glioblastoma 3.1.5. In Vitro Studies of Mechanotransduction in Glioblastoma

Studies of GBM cell-ECM interactions and mechanics rely on in vitro matrix-mimetics that are composed of either biologically occurring proteins or synthetic materials. In 2D cultures, this is accomplished by coating glass or polystyrene with bio- or synthetic materials and culturing cells on top of the coating. In 3D cultures, hydrogels, defined as crosslinked polymer networks that can retain water, are composed of one or several synthetic or biomaterials to mimic the cell microenvironment. Changes in composition, component concentration, and crosslinking density lead to changes in rheological properties such as elasticity and stiffness[177]. Cell migration, morphology, cytoskeletal structure, Studies of GBM cell-ECM interactions and mechanics rely on in vitro matrix-mimetics that are composed of either biologically occurring proteins or synthetic materials. In 2D cultures, this is accomplished by coating glass or polystyrene with bio- or synthetic materials and culturing cells on top of the coating. In 3D cultures, hydrogels, defined as crosslinked polymer networks that can retain water, are composed of one or several synthetic or biomaterials to mimic the cell microenvironment. Changes in composition, component concentration, and crosslinking density lead to changes in rheological properties such as elasticity and stiffness [177]. Cell migration, morphology, cytoskeletal structure, invasiveness, and signaling are some of the properties characterized in gels of varying stiffness.

invasiveness, and signaling are some of the properties characterized in gels of varying stiffness. It is common practice in the study of GBM mechanics to use various naturally occurring proteins of the brain ECM to construct hydrogels in which cells are seeded. While most studies cite hydrogel formation from one ECM protein, there are several that utilize hydrogels mixed with several proteins, which are discussed in the next sections. Either protein concentration or crosslinking density is altered in the hydrogels to change Young's modulus of the gels and study the effect of elasticity or stiffness in the microenvironment on the cells. The use of biomaterials in hydrogels offers the additional variable of gel It is common practice in the study of GBM mechanics to use various naturally occurring proteins of the brain ECM to construct hydrogels in which cells are seeded. While most studies cite hydrogel formation from one ECM protein, there are several that utilize hydrogels mixed with several proteins, which are discussed in the next sections. Either protein concentration or crosslinking density is altered in the hydrogels to change Young's modulus of the gels and study the effect of elasticity or stiffness in the microenvironment on the cells. The use of biomaterials in hydrogels offers the additional variable of gel degradation by cells or over time in incubation, which adds an important dimension to understanding tumor-cell function in remodeling the ECM.
