**1. Cellular Microenvironment**

The cellular microenvironment is characterized by a mixture of extracellular matrix proteins, soluble signaling factors, neighboring cells, and the physical properties of the niche that affect cell behavior through direct or indirect biomechanical and biochemical signals [1,2]. Properties of the microenvironment, such as stiffness and composition, have been shown to direct cell physiology and lineage [3,4]. Such findings have inspired research to define the cellular microenvironment and its links to cellular behaviors using in vitro tissue models that can mimic biomechanical conditions [5,6]. Here, we will discuss the cellular niche, namely, the biological and mechanical properties of the extracellular matrix (ECM), how cells sense these properties, and the dysregulation of cell-ECM interactions in various disease states. How these factors, which are involved in crosstalk with cells, contribute to cellular activities and overall health, will also be presented through a review of the various research publications on the topic. This review will provide an in-depth

**Citation:** Sahan, A.Z.; Baday, M.; Patel, C.B. Biomimetic Hydrogels in the Study of Cancer Mechanobiology: Overview, Biomedical Applications, and Future Perspectives. *Gels* **2022**, *8*, 496. https://doi.org/10.3390/ gels8080496

Academic Editors: Kiat Hwa Chan and Yang Liu

Received: 30 May 2022 Accepted: 2 July 2022 Published: 10 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

overview of what is known and what is unknown about the biomechanics involved in cellmicroenvironment interactions, how the use of biomimetic hydrogel models can fill these gaps in the knowledge, and the utility of biomimetic hydrogels in biomedical applications.

### *1.1. Extracellular Matrix*

Tissues may be described as having two components: cellular and non-cellular. The extracellular matrix is the non-cellular component and is composed of proteins, polysaccharides, growth factors, signaling molecules, proteases, and water [7] (Figure 1). These components are distributed heterogeneously rather than homogenously, resulting in unique niche microenvironments for each cell as well as tissue-specific mechanical, physical, and biochemical properties [7]. These play a large role in regulating and mediating cell behaviors. While the ECM is a mixture of many components, a large portion of it is composed of proteins. These include proteoglycans such as hyaluronan, and fibrous proteins such as collagens, elastins, fibronectins, and laminins [8]. These proteins function to anchor cells to the ECM via focal adhesions and aid propagating signals between cells [8]. ECM composition also affects physical properties such as elasticity, stiffness, porosity, static architecture, and dynamic deformations of the matrix [9]. For instance, based on their concentration, assembly, and crosslinking densities, the structural collagens and elastins of the ECM significantly alter its mechanical properties such as composite strength, elasticity, and mechanical resistance [10,11]. ECM properties are not static; they undergo dynamic changes as the ECM is continuously being remodeled through protein degradation, deposition, or modifications that can be self-contained or caused by cellular activity [10]. In cases such as tissue repair, the activity of growth factors and cytokines in the ECM cause matrix metalloproteinases to activate for ECM remodeling [12]. Wound healing and tissue remodeling processes activate growth factors through mechanical and biochemical stimuli to change ECM composition [13]. There are also specialized forms of ECMs that have proven to be important regulators of tissue and cell behavior. For instance, basement membrane is important structurally and functionally for blood vessels because of its involvement in angiogenesis [12]. The ECM has also been shown to mediate or regulate stem cell fate, cell proliferation, cell differentiation, cell migration, and tissue regeneration [11,14–16].

### 1.1.1. Cell-ECM Interactions

The cell and its ECM are involved in a dynamic reciprocity, through which cues from the ECM and cellular activities are in crosstalk to maintain a healthy state [17]. Signaling processes are one of the ways that the cell-ECM interactions are facilitated. Cell-matrix adhesion sites, or focal contacts, enable communication between cells and the ECM through physical connections of cellular integrins and cadherins to ligands in the ECM [15,16]. Focal contacts are important to cellular processes that require physical attachment to the ECM, such as migration and angiogenesis [18]. Engagement of integrins and cadherins to certain ligands can activate signaling pathways such as that of the Rho family of GTPases to stimulate structural changes in the cell or induce other processes, therefore serving as an important step in biochemical cell-ECM interactions [6,16].

Proteoglycans present in the ECM have functions such as inducing aggregation and participating in ECM structure by adhering to structural proteins [19]. Some proteoglycans reside on the surfaces of epithelial cells, where they act similar to cell-adhesion molecules and bind collagens and fibronectin to anchor cells to the ECM [20,21]. In addition to these vital functions, proteoglycans are active co-receptors that mediate cellular signaling by binding soluble ligands in the ECM and encouraging the formation of receptor complexes on cell surfaces [22,23]. Proteoglycan co-receptors are vital to various developmental processes, and the loss of co-receptor function or mutation has been implicated in diseases such as cancer and ischemic heart disease [23]. Therefore, the ECM also plays a crucial role in mediating cell–cell communication, which will be discussed more thoroughly in Section 1.2 of this review.

**Figure 1.** Extracellular matrix (ECM) and cellular mechanotransduction. ECM components such as collagens can alter mechanical properties to induce changes in cellular signaling and gene or protein expression via mechanoreceptors and mechanotransduction proteins. Legend on the bottom left shows which ECM component is represented by each symbol. (Created with BioRender.com, ac-**Figure 1.** Extracellular matrix (ECM) and cellular mechanotransduction. ECM components such as collagens can alter mechanical properties to induce changes in cellular signaling and gene or protein expression via mechanoreceptors and mechanotransduction proteins. Legend on the bottom left shows which ECM component is represented by each symbol. (Created with BioRender.com, accessed on 29 May 2022).

### cessed on 29 May 2022)*.* Mechanobiology of the Cellular Microenvironment

1.1.1. Cell-ECM Interactions The cell and its ECM are involved in a dynamic reciprocity, through which cues from the ECM and cellular activities are in crosstalk to maintain a healthy state [17]. Signaling processes are one of the ways that the cell-ECM interactions are facilitated. Cell-matrix adhesion sites, or focal contacts, enable communication between cells and the ECM through physical connections of cellular integrins and cadherins to ligands in the ECM [15,16]. Focal contacts are important to cellular processes that require physical attachment to the ECM, such as migration and angiogenesis [18]. Engagement of integrins and cadherins to certain ligands can activate signaling pathways such as that of the Rho family of GTPases to stimulate structural changes in the cell or induce other processes, therefore serving as an important step in biochemical cell-ECM interactions [6,16]. Proteoglycans present in the ECM have functions such as inducing aggregation and participating in ECM structure by adhering to structural proteins [19]. Some proteoglycans reside on the surfaces of epithelial cells, where they act similar to cell-adhesion molecules and bind collagens and fibronectin to anchor cells to the ECM [20,21]. In addition The ECM is involved in mechanical crosstalk with cells [24], which relies on mechanotransduction proteins that help to regulate intracellular tensile response to mechanical forces from the ECM [25]. Mechanical stimuli that cells may receive include shear stress, membrane tension, force, strain, stiffness, and drag force [25]. These stimuli are listed in Table 1 with the mechanotransduction proteins identified to be involved in recognizing the stimuli and eliciting the response in cells. While the extracellular matrix of many cancers, including colon, breast, and prostate cancer, is stiffer than that of healthy tissues [26,27], cell deformability or reduced stiffness has been correlated to increased metastatic potential and invasiveness in cancer. In a study of ovarian cancer cells of varying invasiveness, Xu et al. found that the more-invasive ovarian cancer cell line was more deformable compared to the less-invasive cell line [28]. In another study, Hayashi and Iwata confirmed that cancer cells are softer (i.e., lower Young's modulus) than normal cells using atomic force microscopy [29]. Another study reported that cisplatin treatment caused decreased stiffness and invasiveness of prostate cancer cells [30], suggesting that various cell lines may have varied mechanical properties in the cancer state. Further controversy exists within the field of glioblastoma biomechanics. Gliomas are highly variable; therefore, measurements of tumor stiffness may vary depending on location of measurement [31]. Although some GBM tissues were stiffer than healthy reference tissues, GBM tissues, on average, were less

diseases such as cancer and ischemic heart disease [23]. Therefore, the ECM also plays a

to these vital functions, proteoglycans are active co-receptors that mediate cellular signaling by binding soluble ligands in the ECM and encouraging the formation of receptor stiff than healthy tissues [31–33]. The controversy regarding GBM tumor stiffness could by fueled by differences in methods of measurement, as there is currently no standard practice in the field [34–36].

Mechanical stimuli coming from the extracellular environment can be processed by cells through mechanotransduction pathways. The proteins of these pathways translate mechanical cues to induce biochemical and genetic responses. For instance, integrins and focal adhesion proteins are mechanotransduction proteins that communicate mechanical forces to the cell cytoskeleton, and there have been some studies that show force-dependent integrin activation [37–39]. Yes-associated protein (YAP) and other transcription factors have been shown to translocate to the nucleus in stiffer substrates [40]. Similarly, E-cadherin is a mechanotranducer of shear stress [41–43]. Many mechanotransduction proteins have been studied in the context of specific cell types, including platelet endothelial cell adhesion molecule-1 (PECAM-1) in skeletal muscle cells, G protein-coupled receptors in endothelial cells, and vascular endothelial growth factor receptor 2 (VEGFR2) in chondrocytes [44–47]. Cell surface receptors also transduce mechanical signals to cells upon recognition of a ligand that sustained force from the ECM, which can then cause conformational changes in the mechanoreceptor to activate a protein signaling pathway to alter cellular processes [48]. Such membrane proteins are not the sole propagators of mechanical force; studies have shown that there are mechanotransduction systems in cells that enable the progression of force through a long distance [49]. Src and Rac1 have been shown to be activated at distances from 30-60 µm from the original area the force was applied to, via cytoskeletal mediation of the force [49,50]. Mechanical signals can also be transmitted throughout the cytoskeleton to the nucleus via proteins such as linker of nucleo- and cyto-skeleton (LINC) complex 9 to change chromatin structure and cause nuclear stiffening [49,51]. Mechanotransduction systems translate the numerous mechanical cues from the ECM into biochemical signals interpreted by the cell that lead to signaling cascades that control transcription, proliferation, migration, and many other cellular processes [52–55] (Figure 2). For instance, smooth muscle cells can migrate along gradients of substrate stiffness through durotaxis [56]. ECM biomechanics is not only vital for cellular processes, but also regulates tissue and organlevel processes such as tissue differentiation, morphogenesis, and development [57–60]. Therefore, elucidating the interplay of ECM biomechanical and biochemical signals with mechanotransduction proteins and pathways is critical to understanding diverse aspects of cellular and tissue health. *Gels* **2022**, *8*, x FOR PEER REVIEW 5 of 36 ECM biomechanical and biochemical signals with mechanotransduction proteins and pathways is critical to understanding diverse aspects of cellular and tissue health.

**Figure 2.** Application of extracellular stress leads to increased intracellular calcium concentrations. Matthews et al. applied high levels of stress to cells (**A**) and found that when imaged via Fura-2AM ratio imaging, it led to a transient increase in calcium concentrations, as shown in pseudocolor images ranging from blue to yellow (**B**) that is quantified (**C**) as a function of time for control and gadolinium chloride-treated cells. (Figure reprinted/adapted with permission from Ref. [61]. Copyright 2006, National Academy of Sciences*.* **Figure 2.** Application of extracellular stress leads to increased intracellular calcium concentrations. Matthews et al. applied high levels of stress to cells (**A**) and found that when imaged via Fura-2AM ratio imaging, it led to a transient increase in calcium concentrations, as shown in pseudocolor images ranging from blue to yellow (**B**) that is quantified (**C**) as a function of time for control and gadolinium chloride-treated cells. (Figure reprinted/adapted with permission from Ref. [61]. Copyright 2006, National Academy of Sciences.

**Table 1.** Mechanotransducers of various mechanical properties and human cellular responses.

**ducers Mechanical Signal Examples of Cellular Responses**

creased cell stiffness [62,63]

maturation [40]

Implicated in development of mechanical unloading-induced diaphragm weakness [64]

regulate assembly of filaments [65]

membrane fusion promotion [67]

signaling [15]

Force Transmit tension to other proteins,

Force Regulate junction dynamics [68]

Shear Stress Altered cell morphology [69]

Integrins Force RhoA activation leading to in-

Vinculin Force Transmit tensile force [66]

Piezo1 Force Vascular structure [70] Lamin A Rigidity Nuclear mechanics [71,72]

Focal Adhesions Force Integrin convergence [73] Fibronectin Force Altered integrin binding [74]

Integrins Force Tyrosine Phosphorylation, MAPK

Myosin II Force Increased cortical tension and cell

Focal Adhesions Force Actin polymerization [55]

Stress Fibers (actin filaments, myosin II, etc.)

Vasodilator stimulated phosphoprotein (VASP), zyxin, and Testin LIM domain protein (TES)

Neurogenic locus notch homolog protein 1 (NOTCH1)

Titin Force

**Mechano-Trans-**

**Functional Category**

Cell Mechanical and Physical Properties

Alters Signaling Pathways



**Table 1.** Mechanotransducers of various mechanical properties and human cellular responses.




**Table 1.** *Cont.*


### 1.1.2. Cell-ECM Interactions in Cancer

Problems in mechanotransduction can result from changes in ECM mechanical properties and defects in proteins involved in mechano-sensitivity [101]. Since mechanotransduction is essential for modulating cellular homeostasis, its failure is linked to metastasis and cancer progression [96]. For instance, there are many proteins implicated in mechanotransduction in glioblastoma (GBM). Talin1 inhibition has been observed to decrease cell spreading and limit cell stiffness changes of glioma cells in response to ECM stiffness, proving its role as a mechanosensory [76]. Non-muscle myosin II depletion reduced the effect of matrix confinement on GBM cell motility [102]. Constitutive activation of RhoA GTPase caused lower sensitivity to matrix stiffness of GBM cells in toxicity assays [55]. Increased matrix stiffness was correlated to Hras, RhoA, and rho-associated, coiled-coilcontaining *protein* kinase 1 (ROCK1) upregulation, which are mechanosensor proteins that are implicated in migration and proliferation in cancers in general [61,87]. Integrins are also particularly important in mechanotransduction by relaying signals from the ECM to the cell actin cytoskeleton and are essential to cell migration and cell-matrix adhesion in cancer [103].

Identifying proteins involved in mechanotransduction and their roles in cancer progression can be an essential part of developing therapeutic strategies to hinder cancer progression and malignancy. Certain studies have shown the potential effects of targeting mechanotransduction proteins on cancer cells. Knock-down of CD44 led to decreased structural microtubule, vimentin, and glial fibrillary acidic protein expression and decreased migration and cell stiffness [104]. Targeted inhibition of integrins in the tumor microenvironment has been shown to reduce angiogenesis and inhibit tumor growth [103]. While these studies show partial inhibition of mechanical sensitivity as decreasing invasive properties of tumor cells, other studies have shown that tumor initiating cells are mostly insensitive to mechanical cues from the ECM and that mechanically-insensitive cells have increased motility and invasiveness in vitro [28,105–109]. A complex approach is needed to target mechanical sensitivity in cells through mechanotransduction-based therapeutics for it to become a promising mode of cancer treatment. Therefore, extensive research in mechanotransduction and cell-ECM crosstalk is essential.

### *1.2. Neighboring Cells and Secreted Factors*

In addition to the ECM components, cells are surrounded by heterogeneous populations of neighboring cells that are unique to the tissue and sub-location within the tissue [110]. How cells communicate and influence one another is crucial to maintaining homeostasis and coordinating processes that require several cells, such as tissue formation and regeneration.

Cells can interact through secreted signals that are recognized by membrane-bound receptors through either paracrine signaling, between cells, or autocrine signaling, which is from one cell to itself. In fact, cancer cells are often able to "override" signals from neighboring cells through autocrine pro-survival and proliferation signals. It is crucial to better understand the specific interactions between cells that promote healthy conditions or lead to disease states. For instance, Zervantonakis et al. found that fibroblasts in the tumor microenvironment of HER2 positive breast-cancer cells reduced drug sensitivity through paracrine signaling that activates mechanistic target of rapamycin (mTOR, anti-autophagic) and anti-apoptotic signals [111]. In addition to elucidating mechanisms of tumor resistance, cell–cell interaction dynamics can provide insights to developing self-assembled multicellular structures in vitro. Mueller et al. demonstrated that by utilizing pulsed light activation to control engineered photo-switchable cell-cell interactions, they were able to control the spatial organization of multicellular structures without a scaffold [112]. This highlights the importance of maintaining cell interactions in efforts to mimic physiological conditions in vitro.

Cells are also involved in communications through physical contacts with one another. This is another way that mechanical stimuli may play a role in influencing cellular processes. Physical contacts are also involved in collective migration, which has been exhibited by metastatic and invasive cancer cells [113]. The cytoskeletal tension at cell-cell contacts can serve as a significant regulator of mechanotransduction pathways, and there is a wide field of study on the mechano-sensing implications of cell-cell contacts such as focal adhesions and adherens [114]. One method in particular, physically interacting cell sequencing (PICseq), is a novel sequencing approach that combines cell sorting, RNA-sequencing, and computational modeling to describe complex cellular interactions in different contexts [115]. However, the heterogeneity and complexity of the interactions between cells and with the ECM need to be better recapitulated in vitro to provide a more accurate understanding of cellular communication networks.

### *1.3. Hydrogels as In Vitro Models of the Cellular Microenvironment*

Three-dimensional hydrogel models of the cellular microenvironment have gained interest in recent years for their improved mimicry of in vivo conditions as opposed to two-dimensional cell cultures (Table 2). These hydrogel models have been extensively studied and compared to 2D culture and in vivo conditions. Cells have been shown to have differing spatio-physical properties in 3D and conventional 2D culture conditions [116–119]. Hsieh and colleagues showed that cells cultured in 2D exhibit greater drug sensitivity than in vivo, and 3D cultures exhibit chemosensitivities comparable to solid tumors provided that they have similar cell density [120]. Expanding the cellular environment from 2D to 3D has also been shown to affect proliferation and metabolism [120–122]. Using 3D cultures has led to significant advancements in the understanding of cell migration strategies as well, since some of these could not be observed in 2D cultures [123–125]. For instance, fibroblasts have several different migration strategies that are utilized in different microenvironmental conditions, demonstrating cellular plasticity [125]. Such findings prompt questions about the mechano- and bio-sensing abilities of cells in relation to their environments. In this section, we will elaborate on the different methods utilized thus far to improve cell-microenvironment mimicry in the form of hydrogel models, the cellular response to such models in contrast with conventional cell-culture methods, and factors to be considered in crafting a hydrogel model.


**Table 2.** 2D versus 3D cultures [117–119,123,126–128].
