Matrix mechanophysical factor: pore size governs the cell behavior in cancer

ABSTRACT Cancer tissues are a heterogeneously multifaceted assembly. Understanding the relationship of tumors with their microenvironment is also required to understand the tumor progression and metastasis better. Like tumors, the tumor microenvironment (TME) is heterogeneous, offering numerous mechanobiological, mechanochemical, and mechanophysical cues. Biomaterials impersonating extracellular matrix (ECM) properties must provide the mechanical cues cells get from their 3D extracellular environment. Pore size is one imperative yet less studied ECM factor implicated in the invasion and migration of the tumor. Several techniques are used to control the pore size of biomaterials constructed for a distinct tissue. Electrospinning is one of the most steadfast techniques for producing scaffolds with the preferred pore size. A comprehensive interpretation of ECM pore size would contribute toward a better understanding of the reciprocal interaction between pore size and tumor progression and can be used as a promising target for cancer treatments. In this review, we abridged the knowledge pertaining to (1) ECM and pore size, (2) the importance of pore size and its interplay with cancer, and (3) current advancement in the field of biomaterials to study pore size. Overall, this review will cover the effect of pore size on tumor cell behavior concerning electrospinning. ABSTRACT


Introduction
Globally 19.3 million cases and 10 million cancer-related mortalities have been reported in 2020 [1].Cancer cell migration is a complex biological process influenced by the properties of migrating cells and their environment [2].Tumor prognostic methods are based on the clinicopathological features; tumor (T), node (N), metastasis (M) TNM grading.It is an effective prognostic strategy, yet the patients with the same prognosis showed varied overall survival [3].Despite significant breakthroughs in prognosis and treatment, higher tumor heterogeneity demands further exploration of tumor and its associated factors.There is still a need to understand better the role of major molecular players involved in tumors and their clinical relevance.
Extracellular Matrix (ECM) is one of the critical biological frameworks responsible for tissue homeostasis and plays a role in carcinogenesis.ECM is a non-cellular entity and maintains tissues' biochemical and physical permanence.ECM and cells exhibit bidirectional interactions and are responsible for cell adhesion and migrations [4].Given the importance of ECM, in the last decade, extensive studies have been carried out to understand the fundamental properties of ECM; dimensionality (2 dimensional 2D or 3 dimensional 3D), cell polarity, shape, cell adhesion surface, area, nano-topography, viscoelasticity, stiffness, porosity, static and deformation applied to matrix and pore size.All these constraints have influenced stem cell choices [5], and stem cell is an indispensable factor in tumor progression.
Tissue engineering intends to use 3D scaffolds to provide a suitable microenvironment for investigating cell behavior [6] and incorporate growth factors for tissue engineering.The scaffolds mimic the actual in vivo 3D microenvironment and provide suitable cell interaction and behavior cues.Hence material properties of the scaffolds are essential in determining the cellular behavior [7].The pore size and porosity of 3D scaffolds have direct implications for their biomedical applications.Pores are essential for cell nutrition, tissue vascularization, proliferation, and migration [8].A previous study demonstrated that permeability increases with increasing pore size due to a reduction in the specific surface area [9].If the pore size is small, it will restrict the cell migration, diffusion of nutrients, and removal of waste metabolites.If the pore size is too large, it will reduce the surface area for cell attachments.Despite the importance of pore size, the relationship between cell behavior and scaffold pore size is poorly understood, resulting in conflicting reports within the literature on the optimal pore size for studying cell activity.Herein, we critically assess the available literature for the optimal pore size and techniques being used or can be used to study the relationship between pore size and tumor cell behavior.

ECM in tumor
ECM, a part of the tumor microenvironment (TME), is a highly intricate, complex dynamic structure constantly remodeled and differs from the normal organs.ECM controls the intratumoral signaling, metabolism, oxygenation, transport mechanisms, and immunogenicity.ECM regulations influence malignancy, tumor growth, and response to therapy.It generates the mechanical and biochemical properties of the organs, such as elasticity and tensile strength, extracellular homeostasis, water retention, binding of growth factors, signal transductions, and gene transcription regulations.Biomechanical, biochemical, and organizational properties of ECM differ between normal and cancerous tissues, as shown in Figure 1.Therefore, understanding the idiosyncrasies of ECM in tumors is essential to understanding the role of ECM in tumor progression and treatments [10,11].Components of ECM link to give stable composite structures for mechanical properties of tissues.ECM is vital in controlling the behavior and characteristic of cells, such as adhesion, migration, proliferation, differentiation, polarity, and apoptosis [12,13].
Since ECM is necessary, in the last decade, extensive studies have been carried out to understand the fundamental properties of ECM; dimensionality (2D or 3D), cell polarity, shape, cell adhesion surface, area, nanotopography, viscoelasticity, stiffness, porosity, static and deformation applied to matrix and pore size.All these parameters have been shown to influence stem cell choices [5], and stem cell is an essential factor in tumor progression.The mechanical properties of ECM have emerged as a critical cell fate regulator.These properties of ECM are significant in tumorigenesis, but the underlying mechanisms of these physical factors are still far from understood.
The development of material-based biomaterial mimic ECM conditions have been used to understand the role of physical properties of ECM factors in cell fate decision.Nevertheless, it is difficult to isolate each factor and study it independently.New biomaterials are being designed to study substrate mechanics in more complex ECM [15].Although all the physical factors of ECM have their role in tumor progression, pore size is crucial in tumor migration.Migration of tumor cells through the ECM depends more on pore size than on stiffness, with small and large pore sizes favoring protease-dependent and -independent migration, respectively [16].

Pore size; significant parameter in ECM and cell fate determination
ECM proteins are fibrous proteins (collagen, fibronectin, laminin, and fibronectin) and glycosaminoglycan (hyaluronic acid, keratan, chondroitin, and heparan sulfate).These molecules exhibit intricate mesh-like crosslinked distribution [17].Pore size is the gap between collagen fibrils or within the basement membrane [18].Pore size is an essential factor determining cell phenotype, cell behavior, and secretions on ECM on scaffold surfaces [19].Macropores with a pore size of >50 μm can determine cell proliferation and colonization, whereas micropores with a size <50 μm influence the adsorption of proteins on scaffolds [20].Rapid angiogenesis was observed in large pore sizes of 160-270 μm [21].The pore size of 0.849 μm with 30% porosity promotes metabolic activities and cell proliferation, whereas the pore size of 0.896 μm with 39% porosity showed higher cell aggregations and differentiation [22].Similarly, porosity is vital in neovascularization during tissue engineering survival after transplantation and is well documented [23].Suggesting pore size is an essential contributing factor in tumor development and progression.Despite recent studies, there are still conflicting reports about the optimum pore size and the relationship of pore size with cell behavior.
Since ECM is disorganized in a tumor, which presents physical barriers for the moving cells, this barrier can either oppose or facilitate the motility of tumor cells depending on the pore size.Cells navigating through these pores remodel their cytoskeleton or ECM.Stiff cytoplasmic organelles and relatively large nuclei limit the migrating cells' capacity to squeeze through ECM pores and physical barriers [24].The nucleus dictates the cellular movements [25] and negotiates how ECM will be remodeled.Nuclear structural proteins lamin-A and lamin-C are significant factors in sensing nucleoskeletal stiffness.The expression of these proteins correlates with the cell navigating ability through ECM pores [26].Overabounded collagen leads to smaller pore size [27,28].Invading cancer cells through these tiny pores squeezes these physical barriers and undergoes more physical damage [29].This squeezing would result in the isolation of nuclear proteins involved in DNA repair; hence this pore size contributes to the genomic instability of cells [30].Pore size is a decisive factor in cell trafficking.If the pore size is too smaller than nuclear dimensions, it will provide an impassable barrier to cells.
On the other hand, if the pore size is larger than nuclear dimensions, the physical barrier will no longer exist, and the cell will undergo invasion [31].
Tumor cells bypass these barriers either by; 1. Proteolytic remodeling of ECM or 2. Non-proteolytic remodeling or matrix plasticity [32] is shown in Figure 2. The above serves the Importance of ECM and the role of pores in tumorigenesis.Despite the growing focus on studying biomaterial synthesis to study the individual roles of pores in tumor development and progression, there are certain areas of neglect.Since ECM is a complex biological process, studying single factors is technically challenging.Similarly, technical limitations indicate that some scaffolds are insufficiently defined or not measured rigorously.The following section will discuss the porous materials and their physiochemical understandings in biomedicine.

Techniques for controlled porosity
Tissue engineering applications encompass 3D scaffolds to provide a suitable microenvironment for incorporating cells or growth factors to regenerate damaged tissues or organs.These scaffolds mimic the actual in vivo microenvironment where cells interact and behave according to the mechanical cues obtained from the surrounding 3D environment.Hence, the material properties of the scaffolds are vital in determining cellular response and fate [7].There have been rapid advances in bone tissue engineering with the development of porous, biocompatible, 3D scaffolds.Regardless of the application, the scaffold should be biocompatible and imitate both the physical and biological functions of the native ECM.
The ECM provides a substrate with specific ligands for cell adhesion and physical support [33].
Current tissue engineering techniques involve using controlled porous 3D templates to study the main biological functions.The development of new techniques enabled us to design customized biomaterials with controlled porosity.Currently, used techniques are listed in Table 1.

Factors affecting pore size
Under stiffer conditions, the pore size of the ECM is stiffer [27]; there are several factors influencing pore size.The primary factor is the overabundance deposition of ECM protein [28].Collagen fiber diameter, concentration, gelation temperature, and pH are the main factors manipulated to tune the pore size of biomedical materials to study the biological implications of these matrix pores in tumorigenesis [51] [52,53].

Pore size with biological response
Since pore size is an essential factor in ECM and governing biological processes, the design of 3D scaffolds with controllable multiscale pore size architecture currently represents a significant challenge.In recent years biomaterials have been manipulated to achieve optimal templates of scaffolds to study their impact on cell activities.Accurate control of the pore size is hugely demanded as the property of a material not to compromise the mechanical stabilities and properties of the scaffold [54].Figure 3 depicts the pore size of various scaffolds required for various cellular activities for their biomedical use.

Electrospun 3D scaffolds to mimic ECM
Various techniques have been used for the fabrication of 3D scaffolds.Approaches that mimic how tissues are formed in the body and their
nanofibrous structure can be found in modular assembly and electrospinning methods.William Gilbert was the first to observe that liquid droplets will deform when electrically charged amber is brought closer to the droplet [70].This later became 'Taylor Cone' and was the first recorded electrospraying observation.Rayleigh, in 1897 was the first to observe the amount of charge required for droplet deformation, and the first fabricated artificial was patented in 1934 by Fromhals [71].
Electrospinning is a versatile fiber fabrication technique, as demonstrated [72].It has become the most reliable way to fabricate continuous fibers scaled from nanometer to micrometer, much smaller than the fibers obtained by the conventional techniques, such as phase separation and selfassembly.The electrospun fibers have high porosities and a high surface-tovolume ratio [73], and they can perfectly mimic the native ECM.Electrospun fibrous scaffolds have been investigated as potential scaffolds for biomedical applications [74,75].
When designing scaffolds for any tissue engineering application, the mean pore size is a significant consideration.Scaffolds must be permeable with interconnecting pores to facilitate cell growth, migration, and nutrient flow.A previous study demonstrated that permeability increases with increasing pore size due to a reduction in a specific surface area [9].If pores are too small, cell migration is limited, resulting in the formation of a cellular capsule around the edges of the scaffold.This, in turn, can limit the distribution of nutrients and removal of waste products resulting in necrotic regions within the construct.
Conversely, if pores are too large, there is a decrease in a specific surface area [76].Initial cell adhesion mediates all subsequent events such as proliferation, migration, and differentiation within the scaffold.As a result, the mean pore size within a scaffold affects cell adhesion and ensuing proliferation, migration, and infiltration.Therefore, maintaining a balance between the optimal pore size for cell migration and the specific surface area for cell attachment is essential [33].
As the ECM matrix is a complicated structure with multivariate mechanophysical factors influencing the response of cells to ECM, the relationship between scaffold pore size and cell activity is not extensively studied or fully comprehended.As a result, there have been conflicting reports on the optimal pore size over the years.Figure 4 highlights the studies investigating the behavior of cells in response to matrix pore size, and Table 2 summarizes the behavior of cells cultured on electrospun with varying pore sizes.

Biologically engineered ECM a way forward? Future perspectives
Since cancer is a combination of more than 100 diseases, ECM combines complex mechanophysical characteristics that provide tumor development and progression cues.ECM is an active 3D structure continuously undergoing remodeling to facilitate tissue homeostasis.It is a remarkably active 3D structure continuously undergoing remodeling to regulate tissue homeostasis [96].Our understandings are far from comprehension regarding the role of ECM in tumor progression.A better comprehension of ECM remodeling's effect on cancer progression and development may improve understanding of tumor and therapeutic strategies.
Over the past several decades' 2D culture models have been widely used for 3D tumor microenvironment in research.2D culture models provide limited success [97], failing most therapeutic drugs in clinical.One primary reason for the failure was to use the inappropriate culture model, which could not recapitulate the accurate tumor microenvironment [96].
Compelling evidence suggests the use of 3D systems is the better biological and clinical model for understanding cancer.3D culture systems provide more diverse phenotypes and heterogeneous growth and mimic a better microenvironment for gene and protein expression and metastatic and drug-resistant models.Recent years have seen more focus on understanding how the 3D microenvironment affects cell behavior and how it is different from the cell behavior studies in 2D culture models [2,[98][99][100].
ECM is a 3D culture system comprised of various physical and biological cues that constitute its overall properties and effect on tissue homeostasis.Emerging technologies can better design and mimic the native tumor microenvironment for cancer studies [101].Such technologies/platforms can structure cancer-specific architecture with relevant stromalcancer cell interaction, physiological cues, molecular signaling, vascular and immune components, and cell-Cancer interactions [97] mimicking patient tumors.Significant studies proposed that ECM stiffness promotes invasion; the limitation lies in the pore size is the current hypothesis [102].Pore size smaller than 5 μm (smaller than the epithelial cell nucleus) is a significant barrier and a limiting factor in cell migration/invasion.Tumor progression and ECM porosity is another area of research that may provide better insight into tumor progression and metastasis.It is reasoned that increased ECM deposition decreases the porosity of tumors [103].Porosity is the ability of the matrix to provide homeostasis [104].The effect of tumor pore size has yet to be evaluated alone.
Currently, tuneable engineered natural biomaterials are being used to investigate the role of ECM physical properties, especially pore size.However, the current 3D cultural systems are defective and do not mimic the optimal biophysical and biochemical signals for cell survival and growth.A greater comprehension of ECM pore size regulation and its function in  Techniques/Systems Uses in a study of Cell Behavior Transwell [105] Commercially available transwell with customized pore size.Use to study migration and invasion of tumor cells.Matrigel [106,107] Solubilized gelatinous protein.
Isolated from Engelbreth-Holm-Swarm mouse sarcoma cells.Used to study the formation of epithelial structures, 3D cell culture characterization, and in vitro recreation of in vivo architecture of tissue complexity.Artificial ceiling (PDMS) [108,109] An artificial ceiling provides a confined environment by introducing spacing pillars with the desired height and ceiling to make the customized pore size.Environmental confinements are introduced by externally applied pressure.Used to investigate the height threshold for cell migration and nuclear rupture.Used for artificial ceiling showed the transition of cell movement from mesenchymal migration to amoeboid movement.Microfluidic channel [110] [111]

PDMS soft lithography can help design channels and pillars with different tunable geometric dimensions. To study how cells squeeze through smaller pores or how cancer cells migrate on linear tracks. To study how cancer cells squeeze through small constrictions. MDA-MB-231 breast cancer cells showed mesenchymal to ameboid
transition to facilitate their invasion in the confining matrix.It can also be used to evaluate the tumorigenic potential of surviving cancer cells after transmigration [112].Hydrogel 3D [113] Collagen hydrogel self-assembling can make artificial ECM with tunable pore size, stiffness, and fibrillar structures.Perfusion systems [114] To investigate circulating tumor cancers and their properties necessary for transmigration to blood vessels, this in vitro system can mimic blood circulation.Perfusion-based systems recapitulating the mechanical stress experienced by cancer cells in these situations can play an essential role in the research conducted in this field.Blood vessel-on-a-chip [115] Microfluidic devices mimicking human micro-circulation can provide a platform to investigate the migration and formation of metastasis lesions in vitro.Extensive efforts are being made to develop in vivo microcirculation on-chip model for cancer studies [116].3D Scaffolds [117]harvested from human gingiva Humanized 3D scaffold designed to understand the cell behavior.
Matrix was suitable to study blastema cell migration.This scaffold has the potential to be used in tissue engineering.Breast Cancer Patient-Derived Scaffold [118] Matric cellularized with cancer cells.
It may potentially be an ECM testing model for drug resistance.Patient-derived Scaffold PDS can also be used to study the ECM and chemotherapeutic response in vivo.Tissue matrix scaffold (TMS) system fabricated using native tissue ECM [119] The structural composition of TMS favors robust cell survival, proliferation, migration, and invasion in culture and vascularized tumor formation in animals.

The porous and hydrogel TMS combination allows the compartmental culture of cancerous and stromal cells, which are distinguishable by biomarkers. The response of the cancer cells grown on TMS to drugs well reflects animal and clinical observations. 3D in vitro collagen models
Provide a Sterric barrier for cell movement as well as an adhesion substrate.Rattail collagen [120] and their pore size are adjusted by controlling pH [121], temperature [122], fiber density, and ionic strength during polymerization [123]. (Continued) the development of cancer progression may lead to improved understanding, drug discovery, and treatments [96].A summary highlighting the engineered system with pore properties, pore size and potential cellular behavior observed by various approaches, is presented in Table 3.However, until now, there is no single biomaterial accepted for all the applications, and there is no single material fabrication approach that can potentially fill all the aspects of tissue bioengineering scaffolds.

Conclusions
Highly remodeled ECM provides a variety of physical characteristics that can either oppose or facilitate the moving cells.Tumor cells have enormous potential to adapt to that mechanobiological plasticity of ECM and migrate by well-defined locomotive strategies.ECM properties are essential, and they should be studied in 3D cultural models rather than 2D cultural models.ECM pore size governs the escape of human breast cancer (MDA-MB231 cells).More studies are needed to significantly comprehend the importance of pore size tumor metastasis and invasion.Tunable engineered cultural models can provide more accurate and fateful models to identify the role of ECM pore size in tumorigenesis.

Article highlights
• Tumors can remodel ECM components.
• ECM component collagen density defines matrix pore size.
• Pore size dictates a variety of cell behavior.
• ECM and pore size interaction can be important in understanding cancer and therapeutics.

Table 3. (Continued).
Techniques/Systems Uses in a study of Cell Behavior Interstitial in vivo animal models [124] To investigate how the structure of in vitro lattices corresponds to interstitial tissue in vivo.3D in vitro reconstitution of collagen can be used to compare connective tissues from mouse or chicken embryos.Animal experimental models microvascular structures, cell dynamics, or tumor metastasis.Combinations of fluorescence and SHG microscopy can help to evaluate cell morphology and movement concerning connective tissue spacing in the living animal 3D Bio-Printer [125] [126].
3D bioprinter offers the potential to design scaffolds with tunable properties for in vitro modeling of BC ECM.Pore size, fiber density, and orientation of the layer can be adjusted to understand the role of each factor.

Figure 1 .
Figure 1.Schematic of healthy and diseased tissues.The BM serves as a structural barrier to cancer cell invasion, intravasation, and extravasation derived from[14].

Figure 2 .
Figure 2. A. Schematic of the effect of pore size on the tumor.ECM pore size is one of the key factors in inducing ECM stiffness and ultimately leads to the downstream cascade in tumor progression.B. Migration of cells in the confined environment.Left panel: Shows the established and reported models of cell migration in the confined environment, including squeezing through micropores or protease degradation dependent on pore size confinement.The middle panel shows how cells modify the ECM by proteolytic degradation and migration.Right panel: it is a new model floated by wisdom et al., If the pore size is larger than 3 μm, it can still help migration through squeezing without protease degradation of ECM.This model of cell migration is a plasticity-mediated model independent of protease activity.If the membrane is smaller than 3 μm and has plasticity, cells progressively make their way by widening pores in the tissue matrix [32].(Image taken from the open-source paper with proper citation).

Figure 4 .
Figure 4. Timeline for the effect of varying matrix pore size of electrospun on the tumor cellular behavior.BC, Breast Cancer; LC, Lung Cancer; CRC, Colorectal cancer; FB, Fibroblast Cells.

Table 1 .
Techniques used to design Biomaterials.

Table 2 .
The behavior of cells cultured on electrospun with varying pore sizes.

Table 3 .
Engineered system to manipulate ECM components for studying the tumor cell behavior.