Functional biomaterials for biomimetic 3D in vitro tumor microenvironment modeling

Abstract

The translational potential of promising anticancer medications and treatments may be enhanced by the creation of 3D in vitro models that can accurately reproduce native tumor microenvironments. Tumor microenvironments for cancer treatment and research can be built in vitro using biomaterials. Three-dimensional in vitro cancer models have provided new insights into the biology of cancer. Cancer researchers are creating artificial three-dimensional tumor models based on functional biomaterials that mimic the microenvironment of the real tumor. Our understanding of tumor stroma activity over the course of cancer has improved because of the use of scaffold and matrix-based three-dimensional systems intended for regenerative medicine. Scientists have created synthetic tumor models thanks to recent developments in materials engineering. These models enable researchers to investigate the biology of cancer and assess the therapeutic effectiveness of available medications. The emergence of biomaterial engineering technologies with the potential to hasten treatment outcomes is highlighted in this review, which also discusses the influence of creating in vitro biomimetic 3D tumor microenvironments utilizing functional biomaterials. Future cancer treatments will rely much more heavily on biomaterials engineering.

Introduction

Cancer continues to be a major global health concern despite decades of research and advances in cancer diagnosis and treatment methods [1, 2]. Using tumor cells and a matrix or scaffold to recreate the tumor microenvironment (TME) in vitro has helped scientists reduce the margin of error in their assessments of the efficacy of various drugs in recent years [3]. It is possible to conduct mechanistic studies into the biology of cancer as well as therapeutic development and tailored drug testing thanks to the recapitulation of patient tumors in vitro and in vivo as tumor models [4]. The implementation of tissue engineering technologies in cancer biology has been pushed by the significant roles played by the TME in regulating tumor genesis, progression, and metastasis. Recent research has demonstrated that the TME controls the behavior of cancer cells and the development, growth, and metastasis of tumors. It has also been demonstrated that the tumor environment has a significant role in encouraging tumor resistance and recurrence [5, 6]. Tumor cells communicate with their surroundings in a very dynamic and reciprocal manner, suggesting that researching cancer in specially constructed biomaterial scaffolds may more reliably result in the discovery of new therapeutic targets and treatment regimens [7]. Critical insights into the mechanisms of tumor resistance may be revealed when tissue engineering becomes increasingly successful in simulating the natural tumor environment, having an impact on clinical practice, medication development, and biological research [8]. These discoveries have motivated the use of tissue engineering techniques in cancer biology, with the hope that the ability to manipulate the tumor environment will allow for the discovery of therapeutic targets and the creation of novel treatment protocols [9]. Simple two-dimensional (2D) monolayer cultures, more lifelike three-dimensional (3D) spheroid as well as organoid cultures, intricate tumor xenografts, and genetically altered animal models are only a few examples of the models available [7]. Currently, no model systems exist that enable researchers to assess tumor genesis, development, and resistance to therapy in vitro under realistic settings [10].

The extraordinarily intricate microenvironment of the tumor, which includes biophysical signals (interstitial pressure and matrix mechanics) as well as chemical cues (growth factors and cytokines), is crucial to the tumor’s development and spread over time [11, 12]. This microenvironment exhibits a distinct set of significant traits at each stage of the disease (such as cellular and extracellular matrix (ECM) compositions, matrix stiffness, and degree of vascularization) [13, 14]. In order to understand how, why, and when these important factors contribute to the growth and migration of the tumor, it is crucial to break down all the important components that interact with the cancer cells. Additionally, there is a lot of variation not only across different cancer types but also within certain tumors (sometimes referred to as intertumor heterogeneity) (i.e., intratumor heterogeneity) [15–18]. Every patient needs individualized care because of the extreme complexity and diversity that this heterogeneity brings about [19]. In vitro designed human cancer models are greatly wanted in order to completely comprehend the complexity of cancer origins, progression, metastasis, and interactions between the numerous critical players in the tumor microenvironment, as well as the screening of potential anticancer medications. The creation of cancer medicines that work requires this knowledge. This is because the traditional animal-based cancer models, or xenografts, may not always accurately reflect human physiology or the effects that medications have on people [20–22]. A tool for the investigation of individualized anticancer treatments, three-dimensional (3D) cancer models are expected to accurately simulate the in vivo TME in human patients and, as a result, give accurate mechanistic studies. This is achieved by precisely mimicking the qualities and content of the tumor cell/matrix to match the illness type and stage.

Interactions among tumor cells and their surrounding environment influence cancer genesis at all phases of the process. Poor clinical outcomes are caused by significant variables that exacerbate tumor growth and metastasis, including the makeup of the TME and tumor–stromal interactions [6, 23–26]. The active stroma is a key role in cancer cell invasion/extravasation, migrations, angiogenesis, drug resistance, cancer stem cell maintenance, and immunosurveillance evasion, according to mounting evidence that it is a disease-defining factor [8, 27, 28]. Biological and biophysical signals, such as cell–cell and cell–ECM interactions, tissue architecture and dynamics, mass transfer, and tumor growth, among others, govern the connections between cells and the ECM [29]. When developed tumor models are employed in controlled and physiologically appropriate conditions, they provide a perfect platform for examining the effect of a variety of stimuli on tumor malignancy [30, 31]. Although anti-metastatic drugs and other treatments are required, a deeper understanding of tumor genesis, progression, and therapeutic resistance is vital for their successful development [32, 33]. When tumor cells multiply, they infiltrate the surrounding stroma, move via the circulation, and spread to other parts of the body [34]. Due to the focus of research on cancer cells, the genesis of tumors is not well understood. This is true even though the host tissues, the cancer, and the surrounding environment are constantly interacting with each other [35]. This is due to the fact that the molecular pathways behind tumor genesis are still poorly understood [36]. Stephen Paget realized in 1889 that cancer cells, which he termed “bad seeds,” require a favorable microenvironment, or “soil,” to proliferate and spread [8]. Thus, research has shown repeatedly that the TME influences not only metastasis but all stages of tumorigenesis [37, 38]. Research showing the crucial function of the microenvironment in influencing tumorigenesis suggests that cancerous cells can revert to normal when isolated from their environment [39, 40]. To fully understand how cancer develops, it is necessary to look into both the innate features of tumor cells as well as their microenvironment [41, 42].

In vitro models provide the capability to explore mechanistically the unique contributions of various microenvironmental components on tumor cell activity due to their ability to manipulate the biophysical and biomechanical properties. But while assessing them, care must be taken because in vitro models do not completely capture the tumor microenvironment. While it is possible to study the direct effects of interstitial flow on tumor cell invasion in a three-dimensional (3D) culture of tumor cells in a collagen gel with interstitial flow [43–45], this method ignores other factors that are prevalent at an invasive edge of the tumor cells, such as the architecture and composition of the ECM (that is highly heterogeneous), the variability of types of cells present in the tumor stroma and their physiological interactions, as well as gradients of oxygen, pO2, and One can only deduce from such research the direct impact of interstitial flow on equally dispersed tumor cells inside a homogenous collagen ECM because it is uncertain how significant interstitial flow is in contrast to other parameters that were not included in the in vitro model. One should not, however, presume that these results will automatically increase the tumor’s edge in invasiveness. Even as increasingly complicated models are developed, attention must be taken to carefully assess the questions they may be used to answer as well as the ways in which the data they generate can be analyzed. This has been true even for the initial two-dimensional (2D) monocultures [46]. It is certain that advances in TME modeling, an exciting and innovative area of cancer research, will enhance our capacity to discover and assess novel therapy choices [47, 48]. Academics are exerting enormous effort to design models that are applicable to as many situations as feasible, despite the fact that there is currently no ideal model [49, 50].

Tissue engineering continues to develop approaches to simulate various elements of cancer and has played essential roles in gaining a better knowledge of the disease. With the development of new biomaterials and three-dimensional in vitro models that can simulate some of the biological, chemical, and mechanical aspects of the tumor microenvironment.

This review focuses on the use of functional biomaterials to mimic the tumor microenvironment based on the recent advancements in the field of tissue engineering approaches towards modeling tumor microenvironment and tumor matrix engineering. The following section begins with a brief discussion about the 3D TME modeling then it focuses its shift on the engineering approaches of the tumor microenvironment. Next sections give an overview about the ECM models based on the engineered biomaterials and the lab-on-a-chip microfluidic platforms to model the tumor microenvironment as well as the 3D bioprinting techniques to mimic the tumor microenvironment. Finally, the paper addresses the limitations and the future prospects of using functional biomaterials to mimic the tumor microenvironment. Table 1 shows the comparison between the 2D and 3D models. The next section will discuss the three-dimensional modeling aspects of the tumor microenvironment.

Table 1.

A comparison of two-dimensional and three-dimensional models is shown [51]

SL no	Characteristics	Two dimensional	Three dimensional	References
1	Tumoral heterogeneity	The most fundamental; all cells receive the same quantity of nutrition. Misrepresentation of the TME during replication	Improved estimate and depiction of the tumor microenvironment; nutrients are not distributed evenly throughout the tumor	[52, 53]
2	Cell interactions	There are no cell niches formed when cells do not interact with one another or with the ECM	Cell junctions are widespread and enable for cell communication to take place between cells	[54–56]
3	Response to stimuli	Mechanical and biological inputs are inaccurately represented in the model	Cells develop in a three-dimensional environment and receive stimuli from all directions that are accurate representations of the stimuli they would experience in the environment	[57, 58]
4	Cost	Large-scale investigations are more affordable	Techniques that are more difficult and expensive	[59–61]
5	Cell differentiation and proliferation	Poor cell differentiation and unusually fast proliferation are observed	The proliferation of the cells is realistic and dependent on the interactions of the three-dimensional matrix	[54, 62–64]
6	Reproducibility	Highly repeatable	It is difficult to repeat several of the results	[6, 65]
7	Analysis and quantification	Results are simple to comprehend, and long-term cultures are preferable	Data analysis is difficult with various cell types or spheroid/organoid structures	[46, 51, 66]
8	Gene expression	Genes associated with cell adhesion; proliferation and survival are often altered	Representational accuracy of gene expression patterns	[67, 68]

3D tumor microenvironment modeling

Scientists may use three-dimensional models to conduct controlled research on the interactions of tumor cells, endothelial cells, immune cells, and fibroblasts in a controlled environment [69–71]. The ECM, growth factor proteins (GFP), and environmental variables (EVA) all combine to form a tumor, with each of these cell types altering or being altered on a regular basis [72]. By investigating the connections between various cell types and the TME, the relationship between fibrosis, poor vasculature, immunological control, and treatment complications associated with these disorders may be better understood [73, 74]. The easiest method to replicate the unpredictable behavior of the TME is to use a three-dimensional system. Because two-dimensional cell culture models have a limited surface area available for cell-to-cell contact and communication, reproducing interactions between cells and their microenvironments can be challenging [75]. Among the other indicators are alterations in growth, metabolism, and differentiation. For instance, the company is increasingly utilizing 3D culture technologies, which may be classified as aggregation cultures or encapsulation cultures. Aggregate cultures enable the growth of large amounts of tissue in a small region [76, 77]. Without connection or gravity in the culture environment, it is more difficult for cells in aggregate cultures to adhere to one another. Spheroids may be produced on custom plates, and spinning wall bioreactors can be employed in the laboratory to simulate microgravity [78, 79]. They are well-suited for high-throughput systems that require a high level of cellularity and can assist reduce the time required to configure the system before it can be used in most cases due to its basic structure [80]. When nutrition diffusion to cells inside the cultures is insufficient, the cultures’ very cellular character may also result in very limited control over noncellular mechanical components of the TME [81, 82]. Both outcomes are undesirable, depending on the culture size and cell count. When floating cells in a hydrogel are submerged in water for an extended period of time, they can develop into encapsulation cultures. Collagen, hyaluronic acid, and fibrin are all ECM components that may be employed in cell culture to promote cell proliferation and differentiation [83, 84]. This is a frequently used approach. Encapsulated techniques enable the production of multicellular models with advanced ECM structures that may be used for future study due to their ability to adjust all ECM properties, including self-organization potential [36, 85]. On the other hand, aggregate cultures have a lower throughput and take longer to form in their environment, resulting in a greater throughput. Lower initial cell density causes aggregate cultures to grow more slowly [86]. Traditional three-dimensional models lack the complexity of organoids, which are organotypic, tissue-equivalent models with a diverse array of cell types and the ability to aggregate or cluster cells in a manner comparable to that of an entire organ [65, 87]. To get the required outcomes, a variety of techniques must be applied, including aggregation and encapsulation, because organisms are frequently more complicated than three-dimensional models [88]. As previously demonstrated, progenitor cell fate-specification pathways may be used in a number of ways to govern the formation of organotypic structures [89].

Tissue culture plastic is used to cultivate tumor cell lines for the purpose of researching anticancer medicines. The primary goal is to inhibit tumor cell development or to cause cell death [90]. Dynamic microenvironments provide tissue-specific spatiotemporal modulation of communication between distinct cell types, which is required for tumor cells to form and spread in vivo [91, 92]. Paracrine loops between tumor cells and macrophages in a primary tumor are hypothesized to promote cancer spread by increasing both cells’ motility and preparing them for circulation. As a result of this paracrine loop, breast cancer metastasis is feasible. It has been demonstrated that a heterotypic cell contact pair may be created in vivo and in vitro using a range of 2D and 3D growth platforms employing intravital imaging and a variety of 2D and 3D growth platforms [93]. A significant progress has occurred in the realm of cancer therapy by deliberately targeting heterotypic interactions that occur prior to metastasis. When it comes to quantitative assessments of heterotypic signaling, there are relatively few in vitro systems capable of reproducible tissue morphology that can be used for a variety of other types of studies; this is in contrast to the relatively few in vitro systems capable of reproducible tissue morphology that can be used for drug development [94, 95]. Rather than the conventional 2D petri dish culture, 3D culture gives a more realistic portrayal of tissue and matrix structure [49]. When 3D systems were used instead of 2D systems, in vitro drug screening models were able to identify changes in cell proliferation, morphology, and responsiveness. This was a novel finding when it was made in the field [96, 97].

There is growing interest in the use of microfluidic devices such as cysts and tubules to create three-dimensional microenvironments analogous to the basic building blocks of epithelial tissue in order to enable the creation and maintenance of large-surface-area interfaces between chemically and physiologically distinct domains of tissue [31, 98]. Fibers’ chemical composition and topography may be adjusted throughout the extrusion process, allowing researchers to maintain control over the fibers’ production [99, 100]. This strategy was created lately by a group of academics. Onoe and colleagues have devised a hydrodynamically focused technique for industrial-scale manufacture of cell-encapsulated fibers for the first time [101]. However, because of the limited geometry of single-channel fibers, geometric flexibility and structural control can only be obtained by post-processing processes [102, 103]. Figure 1 illustrates the TME and the engineering aspects for the in vitro modeling of the TME. The following section will elaborate the engineering considerations for the tumor microenvironments.

Fig. 1.

Tumor microenvironment (TME) components (reproduced with permission from [28]). The TME is depicted schematically, showing essential mechanisms such as fibroblast recruitment, macrophage polarization, immunological suppression, and ECM breakdown that allow tumor cells to invade and spread

Tumor microenvironment engineering

Using biomimetic approaches to provide cells with attributes of their original microenvironment to recreate a unit of form and function for a given tissue is what has allowed for significant advancements in tissue engineering over the past several years. These advancements have been made possible by employing these strategies. As a result of the fact that researchers view the loss of integration of architecture and function to be the most important hallmark of cancer, it stands to reason that similar methodologies may be utilized in order to get an understanding of the biology of tumors [104].

To this day, the methods that have shown to be the most successful in the field of tissue engineering are those that attempt to replicate the composition, architecture, and chemical presentation of native tissue. For example, inducing the growth of new blood vessels for therapeutic purposes can be accomplished through the sequential release of vascular endothelial growth factor (VEGF) and platelet-derived growth factor, which works to both induce and stabilize the formation of new blood vessels [105]. This method mimics the process that takes place naturally during the physiological process of angiogenesis, which takes place as a result of heterotypic interactions between the stroma and endothelium [106]. Utilizing such biomimetic approaches has already resulted in significant progress made in the study of cancer. Table 2 shows the engineering and design consideration involved in the tumor initiation, progression, and metastasis. Figure 2 illustrates the in vitro tumor models engineered using hydrogels.

Table 2.

Various stages of tumor initiation, progression, and metastasis are represented by models and engineering design considerations [108]

SL no	Biological phenomenon	Engineering considerations	Models used	References
1	Tumor cell circulation	• Platelets, blood cells, clotting factors • Complex geometry (e.g., branching, narrowing capillaries) • Luminal shear stress	• Microfluidic flow chamber	[91, 97, 109]
2	Cell invasion and chemotaxis	• Chemokine gradients • Oxygen gradients/hypoxia • 3D matrix, stiffness, adhesion molecule density • Co-culture with stromal cells • Interstitial flow and ECM stress • Cell contraction and motility	• Vertical layered assay • Microfluidic devices • 3D cell tracking • Modified Boyden chamber	[1, 110]
3	Adhesion and extravasation	• Chemokines and adhesion molecules • Laminal flow • Co-culture with endothelial cells	• Parallel plate flow chamber	[111, 112]
4	Intravasation into blood or lymphatic vessels	• Luminal flow on endothelial cells • Trans endothelial cells (e.g., into lymphatic vessels) • ECM composition; presence of basement membrane • Interactions between tumor cells and endothelial cells	• Modified Boyden chamber • Microfluidic devices	[111, 113, 114]
5	Growth in ectopic site	• Features of the ectopic site that are tissue-specific, such as chemokines and ECM elements (e.g., lymph nodes, liver, brain, and bone)	• 3D cultures	[115, 116]
6	Tumor initiation and growth	• Adhesion molecule density • Compressive stress • ECM composition and stiffness	• Hanging drop assay • 3D spheroid assay	[40, 100, 117, 118]
7	Blood and lymph angiogenesis	• Role of macrophages, stromal fibroblasts, and other cells • Laminal and interstitial flow • Growth factor gradients • Endothelial sprouting into 3D ECM	• 3D flow chambers • Microfluidic devices • Tubulogenesis assays • Bead assay	[53, 61, 119]

Fig. 2.

In vitro tumor models engineered with natural and synthetic hydrogels (reproduced with permission from [107]). In physiological settings, hydrogel matrices are produced by a series of physical and chemical processes. These hydrophilic networks provide 3D microenvironments favorable to tumor development

Model systems that simulate the intricacy of tumor cells

Conventional methods for researching tumorigenesis in vitro

Preclinical models have been used to predict tumor growth for decades, and animal research continues to be the gold standard for identifying new treatments that are not yet available [120]. It has been challenging to gain a better knowledge of tumor development since animal models are prohibitively expensive and it is difficult to examine the spatiotemporal dynamics of tumor growth [121, 122]. A big advantage of using in vitro systems is that they may increase experimental variable control while maintaining physiologically relevant qualities, which is a substantial advantage. Most often utilized in vitro cancer models in the past were tumor-derived cell lines that had been immortalized histopathologically and grown as adherent 2D cultures [123, 124]. They can also be used to do fast drug testing owing of their broad availability and ease of upkeep. In clinical trials, several medication candidates have failed while displaying promising results in vitro and in vivo and in other research areas [125]. The following are a few examples: One or more of these issues may be to blame for translational research’s lack of predictive capabilities and other performance gaps [126, 127]. The absence of cellular heterogeneity in normal in vitro models contributes significantly to carcinogenesis in the same way that it does in tumors in vivo. Due to the lack of availability to cellular and acellular stimuli in two-dimensional culture, the inherent homogeneity of the cell lines is further exacerbated, which makes the problem even more difficult to deal with [128]. Some intratumor heterogeneity linked with clinical outcome may be reproduced in two-dimensional cell culture and in vivo models together, which may establish a beneficial bridge while avoiding some of the downsides associated with both techniques [129, 130]. The selection of biological components, the selection of appropriate ECM materials, the development of fluid flow, and the generation of biochemical gradients must all be considered when developing physiologically correct 3D models of the TME [131, 132]. Detailed explanations of these design concepts and examples of how they could be used in the future are provided in the following sections [133].

Creating multicellular spheroids

Multicellular aggregates called spheroids can be utilized to mimic the 3D cell–cell adhesions found in the human body. The adhesions between human cells can be mimicked in spheroid cultures. [134]. Inverted tissue culture plates can be used to promote cell–cell adhesion and spheroid growth. This is accomplished by placing a droplet of cell suspension on the plate’s lid and inverting the plate [135]. Rather than that, spherical aggregates form, which must be moved to a new culture tank in order for the organism to continue growing. Growing cells on non-adherent surfaces such as agar, agarose, or commercially supplied ultra-low attachment culture plates with preset well shapes can circumvent both of these constraints (spherical or tapered) [113, 136]. Utilize rotating culture and viscous media to expedite the process (cellulose). Large-scale drug screening requires precise spheroids, which can only be produced by contemporary micropatterning and microfluidic technology, as shown by the most recent investigations [137]. As a result, these systems are more expensive to operate and maintain than traditional ones. Researchers can use spheroid cultures to investigate three-dimensional cell–cell interaction and the creation of physiologically relevant structures such as tumor-like structures in mice and other animals [138]. A deficiency of nutrients and oxygen in the spheroid’s core is caused by the increased cell density and size of the spheroid, which restricts nutrient and oxygen diffusion. Apart from that, it has been demonstrated that the formation of distinct zones in spheres larger than 400–500 microns in diameter is congruent with the pathogenic gradients observed in real patients. In addition to monoculture spheroids, coculturing cancer and stromal cells in the same culture plate can form heterotypic cell–cell interactions [139, 140].

Organoid cultures

Cancer organoids produced from people are being used to further cancer research as a platform capable of imitating the cellular heterogeneity present in human malignancies [141]. A biomimetic matrix can be used to construct tumor organoids from fragmented tissue or circulating tumor cells, allowing the cells to self-assemble and develop into increasingly complex structures [142, 143]. The method most frequently employed to create organoids is matrigel (a bone marrow extract obtained from a mouse Engelbreth-Holm sarcoma that preserves the heterogeneity of cancer cell populations) [144, 145]. Matrigel preserves the heterogeneity of the tumor cell population when it is made from a mouse Engelbreth-Holm tumor, making tumor therapy more successful. The most often utilized method for synthesizing organoid derivatives is matrigel synthesis [146]. It is possible to increase physiological complexity and better comprehend cell–cell interactions in the TME by coculturing tumor organoids with stromal components such cancer-associated fibroblasts (CAFs), immune cells, and vascular networks, in addition to other tumor organoids [147, 148]. If you are seeking for an alternative method for developing organoids, you may use the transwell technique, which includes embedding tissues in collagen and exposing one side of each organoid to the medium while leaving the other open to the air [149, 150]. Thanks to biobanks, cancer organoids may now be employed in scientific study. These biobanks hold samples of malignancies and normal tissue from patients and animals [151]. With these biobanks, it will be easier to use patient samples in larger-scale research, which will benefit both researchers and patients. Due to the use of organoid cultures, ex vivo monitoring and assessment of biomarkers and other morphological changes in organoid cultures can give new insights into the dynamics of biological processes [152]. The capacity to generate matched organoids to assess cellular responses in tumor and normal tissue is crucial in cancer research and drug screening because it enables the construction of tumor and normal tissue matched organoids [35, 153]. This has a significant impact on cancer research and medication screening for individual patients. Additionally, normal tissue organoids produced from pluripotent stem cells may be transformed into tumor organoids, which can be utilized to track and understand the molecular pathways behind tissue morphogenesis and cancer progression, among other applications [33, 154]. Organoid cultures are more difficult to work with due to their great production efficiency and the considerable variety that may be gained by tissue harvesting. Alternatively, organoid proliferation may result in the selective loss or overgrowth of stromal populations, hence obstructing research into tumor-specific heterogeneity [57]. Another issue that may be addressed by the use of matched patient peripheral blood serum is the use of sophisticated media that often contain fetal bovine serum and require the addition of particular chemicals. This can be avoided by using patient-matched peripheral blood serum [155, 156]. For researchers, the addition of fetal bovine serum, a challenging medium, adds another hurdle (FBS). Apart from the fact that glioblastoma tumor organoids may be created from a variety of different types of tumors, the bulk of tumor organoids biobanks are focused on epithelial malignancies [157, 158]. Organoid cultures cannot all be sustained permanently, which is why this discrepancy exists [159]. According to some experts, entire organoid cultures may soon supplant cell line research as the primary method for cancer research [160, 161]. Figure 3 illustrates the development of tumor organoids via investigating the TME.

Fig. 3.

Investigating the TME and developing tumor organoids (reprinted from [162]). a Gastrointestinal tumor organoids and cancer-associated fibroblasts (CAFs) were co-cultivated to study the relationship between cancer cells as well as the tumor microenvironment. b Co-cultivating gastrointestinal tumor organoids with immune cells allowed researchers to investigate the interaction among immune cells and malignant cells, explore the interaction, and test immunotherapeutic techniques. c To research tissue-specific oncogenic pathway modifications, to monitor cancer start and progression, and to functionally examine (candidate) oncogenes, tumor organoids can be generated from wild-type organoids using CRISPR/Cas9 by creating tumorigenic mutations

Biomaterials-based ECM models of various types of cancers

Natural biomaterials

Researchers may investigate the functional significance of cancer development in a controlled environment by implanting cells into biomaterials that mimic the ECM found in tumors [163, 164]. Biomaterials, particularly those derived from mammalian tissues, have a broad appeal to a diverse spectrum of people. These materials, which include matrigel and cultrex, have been utilized in research to investigate organotypic tissue development and changed cell penetration into the bone marrow [165]. When oncoproteins such as Papillomavirus E7 and ErbB2 (erythroblastic oncogene B) are activated, cancer cell proliferation and morphogenesis can be accelerated. ErbB2 is an oncoprotein that has been discovered in a number of malignancies ErbB2 [166]. Following intravenous infusion, BM and Matrigel exhibit many of the same biological features, despite significant structural and mechanical differences [109, 167]. Due to the paucity of BM, large-scale examinations in its natural, complete condition are typically not possible (e.g., isolated from rat peritoneum) [168]. Examine the stiffness differential between normal and diseased tissues, as well as the organs most prone to metastasis (for example, breast cancer tissue is ten times stiffer than normal breast tissue: hundreds of pascals in the brain versus gigapascals in bones) [55, 169]. Additionally, commercial BM preparations contain growth factors and cytokines, which regulate cell activity throughout the body. When utilized in conjunction with one another, ECM components such as collagen, fibronectin, and HA can help ease these difficulties ECM. Collagen I, a kind of protein, is frequently employed in the ECM to form hydrogels [170]. Collagen type I hydrogels have aided in our understanding of how the ECM influences cell proliferation and migration, as well as the activity of stromal cells, tumor cells, and their combined activity [60, 171]. According to one study, when cells are transplanted into Matrigel, they develop in an ordered form; nevertheless, when cells are transplanted into collagen gels, they spread out [118, 172]. Additionally, the mechanical and structural properties of collagen hydrogels may be modified by adjusting the collagen content, the gelation conditions (e.g., temperature and pH), or the glycation of the gels [52]. The viscoelastic behavior of the gel varies according to the collagen fibrillar structure. Increased or reduced collagen fibrillar structure enables local strain-strengthening by cells in response to changing gelation circumstances. Numerous techniques exist for altering the collagen fibrillar structure [173, 174]. Electrospinning, magnetic fields, mechanical stretching, and shear flow are only a few examples. Using a strain device for linearized ECM (ECM) synthesis, fiber alignment boosted the directionality of breast cancer cells and increased directionality in a directed pattern [175–177].

On the other hand, the natural ECM is composed of a range of ECM proteins with a diversity of biochemical and physical properties that function in concert. Collagen is one of the ECM proteins [178]. Due to their inherent complexity in terms of composition, mechanical properties, and architectural design, it is challenging to recreate the multiple ECM components that affect cellular activity in more defined ECM cultures [59, 179, 180]. Researchers are examining the impact of diverse stromal cell types’ ECM on carcinogenesis and tumor formation using decellularized cells’ ECM. CAF-derived ECMs appear to promote cancer growth and treatment resistance in vitro [54]. Additionally, changes in the ECM have been demonstrated to contribute in the formation of tumors and the immune system in obese individuals, increasing their cancer risk and prognosis [181]. Regenerative medicine, proteomic mapping of the ECM composition, organoid culture encapsulation, and tumor engineering are only a few applications of this technique that have been successfully implemented [182].

Synthetic and composite biomaterials

Artificial materials are attractive as biomaterial substitutes due to their precision and repeatability, as well as the simplicity with which structural and mechanical modifications may be performed [183, 184]. Due to its potential to demonstrate a wide variety of stiffnesses while retaining a uniform surface chemistry, polyacrylamide (PA) hydrogels have been extensively investigated over the last several decades [185]. PA gels should not be employed in 3D culture due to their cytotoxic properties [186, 187]. To accurately model the interactions between the ECM and the cell, PEG gels must be covalently modified with adhesion ligands such as RGD in fibronectin or GFOGER in collagen, as well as MMP-degradable moieties [188]. It is possible to create adhesion and breakdown sites by cross-linking PEG hydrogels with endogenous proteins such as HA. It is now possible to investigate cell adhesion, motility, and ECM stiffness as a result of these alterations [189, 190]. Despite their adaptability, synthetic ECM models lack the stress-relaxation properties of genuine viscoelastic interstitial ECM. This is because synthetic ECM models lack the stress-relaxation properties of real viscoelastic interstitial ECM. On the other hand, viscoelastic PA gels may be formed by encasing a linear PA solution in an elastic network of PA hydrogels [191]. In other words, a dense network of elastic PA hydrogels isolates the linear PA solution from its surroundings. Reversible hydra zone connections may also be used to crosslink PEG hydrogels, as previously described [192, 193]. Dynamic stress-relaxing crosslinks in hydrogels preserve the benefits of covalently crosslinked hydrogels while allowing for complicated biological activities [194]. In other words, a composite system is one that incorporates both synthetic and natural components. The use of ionically crosslinked alginate hydrogels in conjunction with PEG spacers to develop viscoelastic qualities that were not dependent on the original elastic modulus or breakdown rate is an excellent illustration of this [195]. When heated, this 3D hydrogel displayed increased fibroblast spreading and proliferation, as well as better osteogenic differentiation of mesenchymal stem cells as a result of rapid stress relaxation [196]. Hydrogels may be modified to alter their viscoelastic characteristics, which aids in our understanding of how the ECM supports tumor growth and dissemination. Electrospun synthetic materials such as poly(e-caprolactone), poly(lactide-co-glycolide), polylactic acid, and polylactic acid can be used to build biocompatible and scalable porous scaffolds for a variety of applications (ester urethane) [197, 198]. Tumor-like tissues were generated in experiments using cancer cells placed on porous PLG scaffolds. Due to oxygen and feed supply constraints, tumors grown in this technique were more malignant than tumors produced in 2D culture or 3D Matrigel. According to the researchers, the ECM in bone metastases locations has an inorganic composition similar to that of plastionic acid (PLA), polycaprolactone (PCL), and polyurethane (PUR) scaffolds [62, 104, 199]. By implanting microporous PCL scaffolds into the lungs of mice, researchers can monitor the course of disease and the efficacy of various therapies [200]. While electrospun PCL fibrillar scaffolds resemble aligned ECM substrates, they have the additional benefit of inducing the epithelial-mesenchymal transition (EMT) in breast cancer cells, which is necessary for tumor invasion and proliferation [119, 201]. Another research examined the mechanical characteristics of single dextran methacrylate (DexMA) fibers and a bulk dextran methacrylate (DexMA) matrix. When present, softer fibers can help in the dispersion and multiplication of cells. This was done by recruiting fibers and forming localized adhesions [202]. Previous study with non-fibrous hydrogels suggested that the structure of the biomaterial plays a critical role in influencing cell activity. While synthetic scaffolds may have a number of advantages, they are not repairable by cells, and polymer breakdown can result in the formation of acidic byproducts that inhibit cell function [203, 204]. To get a better understanding of the composition, structure, and mechanical properties of tumor-relevant ECM, which will be utilized to further cancer research, biomaterial scaffolds must be manufactured utilizing carefully selected materials and manufacturing processes [205, 206]. The technique of replicating the cell-surrounding matrix in vitro is one of the most crucial steps in the creation of artificial tissue models. The qualities of the material utilized for the scaffold should correspond to those of the native ECM. ECM is sometimes replaced with hydrogels as discussed above, which are extensively used for 3D modeling. In the spaces between the crosslinked polymer chains, they create a network containing a sizable amount of water [207]. Soft tissues are routinely created in vitro using hydrogels made of polysaccharides [208]. This is mostly due to the resemblance between these hydrogels and natural glycosaminoglycans. The linear microbial exopolysaccharide gellan gum (GG) has many beneficial qualities, including biocompatibility, biodegradability, and a non-toxic composition. Guar gum is yet another name for it. The material’s great transparency, thermoresponsive capabilities, flexible mechanical properties, ease of production and cross-linking, stability under physiological conditions, and reasonable cost are further beneficial traits [208–210]. GG is viewed as a promising material in tissue engineering and regenerative medicine as a result of this. The extensive use of GG in the engineering of spinal cord injury regeneration and the regeneration of cartilage, bone, osteochondral, and other tissues is evidence of this [211–213]. Table 3 shows the use of biomaterials to generate 3D tumor models. The following section will briefly discuss the tumor microenvironment-on-a-chip and will explore the microfluidic design considerations and 3D bioprinting strategies involving TME. Figure 4 illustrates the engineering of ECM to model the TME.

Table 3.

The various categories of biomaterials that can be utilized to produce 3D tumor models, along with the positives and negatives associated with each category [46]

SL no	Biomaterial	Examples	Advantages	Disadvantages	References
1	Synthetic	• PLG • PLA • PEG • PLGA	• Wide range tunable material properties • Can be structurally complex or isolate individual microenvironment cues	• Fabrication methods may be cytotoxic • Limited bioactivity	[71, 215]
2	Naturally derived	• Alginate • Silk • Fibrin • Collagen • Matrigel • Decellularized ECM	• Recapitulate cell-material interactions present in native tissue • Biomimetic	• Limited cell adhesion site number • Can only fabricate in limited range of material properties • Batch to batch variability	[1, 216, 217]
3	Hybrid and semi synthetic	• Peptide hydrogels • Chitosan-PCL • Hyaluronic acid	Combine bioactivity of natural materials with structural complexity and tunable properties of synthetic materials	• Bioactivity of natural components may be affected • Fabrication methods may be cytotoxic	[1, 71]

Fig. 4.

Engineering the ECM to model the TME (reproduced with permission from [214]). The cancer cell ECM could be engineered following approaches such as micropatterning, modification of the ECM, synthetic biomaterials, etc.

Modeling tumor-associated ECM changes using biomaterials

Understanding how changes to the ECM affect tumor progression is crucial. To learn more about the role that changes in the composition of the ECM play in the development of cancer, biomaterials-based techniques have been used to create in vitro models of the tumor ECM milieu. The process of creating and exploiting 3D scaffolds made of intact ECM molecules, decellularized ECM molecules, and functional ECM molecules with minimum adhesion sequences is covered in this section. To create a scaffold, these elements are either added to materials that have been changed to allow cross-linking or directly gelled. Gelatin, crosslinker-modified poly(ethylene glycol), and hydroxyapatite are a few examples of such substances (HA).

Matrix in decellularization

It is possible to decellularize animal models, patient samples, and cell cultures to investigate natural matrix elements [218]. Early research relied on decellularized matrix isolated in separate labs, but now commercial vendors supply reconstituted decellularized matrix for use as a surface coating or to make a 3D hydrogel (e.g., Xylyx Bio). These matrices, which can be used to study early tumor formation or metastasis, are produced from healthy tissues. Researchers often need to create their own decellularized matrix to solve biological challenges. Cancer-related disorders may result from ECM changes. Breast cancer cell proliferation was increased by decellularized adipose stromal cells isolated from obese and lean mice [219]. This finding is in line with obese people having an elevated risk of or propensity for breast cancer [220]. Hepatocellular carcinoma cells were driven to undergo EMT by decellularized ECM from healthy or cirrhotic livers in a Smad-dependent manner [221]. These studies highlight the value of applying ECM that is specific to the area of study and show how ECM connects pre-existing issues to cancer risk. The impact of decellularized tumor matrix on the activity of tumor cells has been investigated. ECM that is produced from tumors is typically thought to promote tumor growth. In contrast to collagen gels, glioblastoma cells moved more freely in decellularized tumor matrix [222]. Spheroids of colon cancer that were cultured on ECM from liver metastases expanded more quickly than spheroids of healthy colon [223]. The question of how decellularized matrix from the same tissue distinguishes between tumors from different patients or between normal and malignant states remains unanswered.

Isolated elements of the ECM

Decellularized ECM maintains the complexity of the native ECM environment, although it is challenging due to the scarcity of available tissues and the difficulty in identifying the contributing factors. Scaffolds can be constructed out of ECM components. To investigate the metastasis of breast cancer, collagen I gels were used [224]. Structural scaffolds were created using collagen I and pure ECM. Adding fibronectin to collagen I gels accelerated the migration of breast cancer cells [225]. Increased tumor cell mobility was seen in multicellular models of breast cancer cells and fibroblasts in collagen I or collagen I and fibronectin. The production of extra matrix metalloproteinases (MMPs) by fibroblasts in collagen I plus fibronectin is possible [226]. In an in vitro model of cortical inclusion cysts, the addition of collagen III to collagen I hydrogels boosted fallopian tube epithelial cell invasion [227]. Since full-length ECM proteins play numerous roles, controlling cancer cells is challenging. They are expensive and challenging to employ in scaffolding due to solubility difficulties. Biomaterials can be created from RGD ECM fragments. Glioma cells become chemoresistant due to RGD in HA [228]. To investigate their effect on the size of breast cancer cell clusters, the adhesion peptides RGDS (fibronectin/vitronectin), GFOGER (collagen I), and IKVAV (laminin) were used to make photopolymerizable PEG-based hydrogel systems [229]. (MDA-MB-231) triple-negative and luminal in response to peptide identity, A cells responded differently (T47D). MMP-responsive scaffolds may be required for monitoring cancer cell invasion. PEG hydrogel linkers can be broken down by MMP. Glioblastoma cells expanded and protruded more when cultured in hydrogels that are MMP-degradable [230]. ECM concentrations, not ECM components, frequently fluctuate in malignancies. Many systems function. 3D collagen I gradients show that menaINV enhances breast cancer cell migration to high-fibronectin regions [225]. Breast cancer cells lost their ductal appearance when the density of the collagen I gel was raised and glutamine entry into the TCA cycle increased. This suggests that the ECM plays a role in energy management [231]. Ovarian cancer cells were insensitive to HB-EGF when collagen I density matched omentum values [225, 232]. Ovarian cancer cells multiplied on gels that simulated omentum metastases as a result of HB-EGF [225]. By adjusting the concentration of peptides produced from the ECM, synthetic scaffolds can mimic changes in the ECM’s density. Reducing RGDS or IKVAV density induced an increase in breast cancer cell clustering in the PEG-peptide experiment, indicating a quicker rate of proliferation. GFOGER density is dependent on cell line [229]. Tumors run into new ECM-containing tissues as they spread. To study tumor cell tropism, in vivo replicas of environmental factors were created [233].

Tumor-microenvironment-on-a-chip

Over the past few decades, the cost of medication development has increased significantly due to the inefficiency of preclinical drug screening techniques [215]. Consequently, there is a growing demand for new therapies for people with long-term health conditions [234]. Traditional techniques to drug screening have a number of flaws, the most notable of which are the differences between two-dimensional (2D) in vitro cell culture systems and in vivo models, as well as the evolutionary distance between humans and other animals [235]. Reproducibility has been improved in new 3D cell culture models in addition to delivering more physiologically relevant surroundings and improved prediction capabilities [236]. The fields of microfluidics and cell biology have reached a tipping point with the emergence of “organs on a chip” technologies, which may simulate organ-level in vivo properties in a laboratory environment using a microfluidic substrate that is becoming increasingly popular [237, 238]. These cutting-edge technologies may be used to build better in vitro models for cancer research, expediting the discovery of novel treatments and the transition from laboratory to clinical testing, according to our predictions [239]. It is during the growth of the tumor that the TME is created, which includes both cellular and noncellular components such as blood vessels that encircle the tumor and immune cell populations, cancer stem cells, and ECM [240]. Biological interactions inside the TME are still a significant challenge in cancer treatment research and development because of their complexity [241]. Cancer cannot be examined as a collection of homogenous neoplastic cells, but as a complex multicellular system in order to fully depict the interactions between malignant and nonmalignant cells, according to the most recent study. In particular, this is true as our knowledge of cancer continues to grow [242, 243]. According to cancer biology’s tumor–stroma interaction, this contact between the tumor and the surrounding stroma is crucial to distinguishing the tumor–stroma interaction from other interactions in cancer biology (TMI). Due to the complexity of the TME cancer patients are exposed to, animal models have long been regarded as the gold standard for cancer therapy screening [244, 245]. Even in a laboratory setting, it is nearly impossible to exactly replicate human carcinogenesis. The validity of currently existing in vivo models for the transfer of treatment success data into clinical practice is being questioned as a result of this [38, 246, 247]. Organ-on-a-chip technology has made considerable advances in TME microengineering in recent years. Consequently, it is now possible to create pathophysiologically realistic human cancer models [204, 238].

In the context of tumors, it is vital to keep in mind that they are constantly interacting with their surroundings [195, 197]. Cancer cells and stromal cells (fibroblasts and immune cells) are often found together in a tumor, as are vascular networks in the surrounding tissue that contribute in the tumor’s growth [61, 248]. When developing cancer medicines, it is essential to understand the interplay between the tumor, its stroma, and its circulatory system (blood vessels) [241, 249, 250]. This section focuses on microfluidic devices that are designed to mimic the TME. These systems may be used to investigate the interactions between tumor cells and the stroma, endothelium, and extracellular components that surround them [251]. Table 4 shows the ECM in some common forms of cancer.

Table 4.

ECM deposition in tumors for prevalent kinds of cancer [9]

SL no	Types of cancer	Tumor-specific ECM deposition	Reference
1	Glioblastoma	• Collagen IV • Procollagen III • Laminin • Fibronectin • Hyaluronic acid • Fibrillar collagens (collagens I, III)	[240, 252]
2	Lung cancer	• Collagen types I and III • Laminin • Fibronectin	[166, 192]
3	Pancreatic cancer	• Collagen I • Hyaluronan collagens I, III, and IV • Laminin • Tenascin • Vitronectin	[54, 56, 181]
4	Colon cancer	• Chondroitin sulfate proteoglycan • Hyaluronic acid • Laminin • Collagen IV Heparan sulfate proteoglycan	[191, 253, 254]
5	Hepatocellular carcinoma	• Collagen IV and laminin	[48, 255]
6	Breast cancer	• Collagen I • Collagen IV • Collagen V • Fibronectin • Laminins Entacin	[52, 256–258]

Microfluidic design considerations

Several key criteria must exist for a chip-based TME to correctly imitate a medically relevant TME [40, 259]. The following conditions apply: TME microfluidic models will be constructed in vitro for drug delivery experiments utilizing data from the study’s in vivo characteristics [249].

Malignancies are hypothesized to stimulate the development of nanoparticles due to their abnormal tumor vasculature and ineffective lymphatic arteries inside the tumor tissue [107, 260]. Their contributions to the EPR phenomenon, which involves the utilization of magnetic fields to transport nanoparticles to solid tumors, are inextricably intertwined [261, 262]. In the laboratory, EPR events have been demonstrated to be hard to adequately replicate using conventional in vitro 2D models. When a solid tumor becomes fibrotic, it develops an increased stiffness, which the illness exacerbates [95, 263].

Collagen and other scaffolding proteins have been connected to the illness’s invasiveness and metastatic potential [103]. Hypoxia and necrosis are more likely to occur in tumors with reduced blood vessel constriction, which decreases oxygen transmission [264, 265]. Because restriction decreases blood flow, it also decreases the effectiveness of therapeutic therapies. As previously stated, it also directly affects cancer cells, triggering death and lowering growth [93, 258, 266]. Long before biomechanical models of tumor formation were developed, mechanical stress was recognized as a factor in tumor growth. To build physiologically appropriate in vitro cancer models, solid tumor stress must be added [267].

Fluid pressure within the tumor cavity increases as a result of the tumor’s highly variable blood flow, both in time and location [268]. Apart from complicating the treatment of malignant tumors, these abnormalities also promote tumor formation. Researchers can employ microfluidic devices to investigate how an aberrant vasculature can be restored to functional normality by increasing pericyte and basement membrane coverage and decreasing vascular permeability [247, 269]. By normalizing the tumor’s vascular system, the tumor’s angiogenic signaling pathways, as well as its abnormal form and function, are restored [270, 271]. When the nanoparticles’ size is raised to compensate for the EPR effect, they become more capable of reaching the tumor site. To provide the most effective and balanced distribution of nanomedicine to a cancer site, it is envisaged that tumor vascular function would need to be normalized [272]. Historically, mutations in tumor vasculature have been explored mostly through computer simulations and mouse models [273, 274]. If researchers employ microfluidic devices, they may get a better understanding of the molecular, cellular, and functional ramifications of dynamic tumor vascular normalization [269, 275].

TME modeling using 3D bioprinting

Extrusion, inkjet printing, stereolithography, and laser-assisted bioprinting are the four primary categories of the currently accessible 3D printing methods that can be employed for 3D in vitro TME modeling. The traditional 3D printing method relies on extrusion, where the material is fed through a nozzle in a continuous stream that can be precisely controlled. A reservoir holds the substance in a specified volume. The extrusion method is a straightforward and reasonably priced procedure. This method’s simplicity allows for any necessary modifications to the printing system, including cross-material printing as well as co-extrusion printing. It is likely that the extrusion process will cause harm and injury to the cells due to the amount of force used. Therefore, the stability and survival of the cells could be jeopardized if the bioprinting parameters, such as the quantity of biomaterial, pressure, and nozzle diameter, are not precisely tuned [276]. In stereolithography bioprinting, bioink is frequently made of light-sensitive polymers, and the fundamental method of this technique is photopolymerization of the bioink. The 3D biomaterial is carefully constructed layer by layer to create the desired pattern. The biomaterial is subjected to light to produce the desired pattern, typically by raster scanning a laser spot or by 2D light projection. Relative to line-by-line raster scanning, 2D light projection allows for speedier printing. It is clear that stereolithography offers superior resolution as compared to bioprinting when extrusion is used. A UV light source is frequently used because the high-energy light required to quickly cure the photopolymer requires it. Stereolithography bioprinters offer good cell viability (> 85%). In contrast to the extrusion procedure, stereolithography bioprinting’s light-induced bioink cross-linking does not subject the cells to shear forces. The third technique, known as inkjet bioprinting, is based on how paper is typically printed in offices. It operates by dropping bioink drops on demand from controlled jets onto a surface. In this trajectory, either heat or piezoelectric actuation controls the bioink’s deposition. The temperature of the bioink increases after the nozzle has been heated, which causes a bubble to form in the thermal actuation. Finally, the force generated by the bubble expansion caused the bioink to be ejected from the nozzle. In piezoelectric actuation, the inkjet printer is under pressure created by a piezoelectric device, which forces the ink out of the nozzle opening. The volume of bioink discharged can be significantly adjusted using the piezoelectric dispensing technique. This technology can provide a greater pattern resolution while employing the least number of cells and biologics since the control can vary from nanoliters to picoliters of bioink. Inkjet bioprinters are thought to be affordable and simple to produce, similar to extrusion bioprinters. As opposed to extrusion bioprinters, which use print heads in sequence, inkjet printers may operate many print heads simultaneously, making this approach speedier and able to deposit multiple cell types quickly. The precise control over the drop-by-drop deposition of bioink is what gives inkjet printers their high resolution (30 mm) [277]. A recent development in tissue engineering is laser-assisted bioprinting. This method’s physical process makes it possible to print liquid materials and cells at the cell level. Laser-assisted bioprinting offers great promise for the production of living tissues with physiologic functionality by offering tissue engineers command over cell density and organization of 3D tissue constructions [278].

Discussion

Although tumor engineering has improved our knowledge of cancer initiation, progression, and therapeutic response, there are still numerous opportunities to advance research and improve treatments. 3-D tumor models must be mechanized and standardized for general use. Hundreds of thousands of spheroids can be produced by bioprinting biomaterials; however, efficiency and size consistency are problems. The size of the spheroid impacts how nutrients diffuse inside the core, which leads to uneven cell growth. In 3-D scaffold-based models, the scaffold material affects medicine absorption and adherence. The choice of the scaffold and the materials should be based on the screening of drugs and their interactions. Even though co-culture of many cell types can simulate the tumor microenvironment in 3-D tumor models, a thorough series of studies must be designed to look at how one cell population affects other cell populations. Different culture media are required for various cell types. In a 3-D in vitro TME model, maintaining distinct growth environments for various cell types may necessitate challenging peripheral component design. In-depth analysis should be done of the ratio of one cell type to other cells in a co-culture setting, and tests should be run to determine not just cell proliferation and invasiveness but also genetic and epigenetic alterations throughout time. Pharma companies may use 3-D TME models that exhibit strong performance in in-depth cross-pharma validations. Imaging and automated readout are required for 3-D tumor models. Limitations of collagenous scaffold autofluorescence 3-D examination of cancer cell images. Cancer cells from 3-D TME models are required for biochemical and cell analyses. It is impossible to gently remove synthetic scaffolding made of polystyrene from growing cancer cells. Enzymatic destruction of PLG-based 3-D scaffolding can liberate cultured cancer cells without hurting the cells. Similar techniques are required for other 3-D TME scaffold models. Despite the fact that they are more automated, scaffold-free 3-D TME models have variable composition and batch-to-batch volatility (basement membrane extract). The composition of the 3-D model can be balanced by allowing stromal cells to deposit extract from the basement membrane before cancer cells are expanded. The development of 3-D tumor models with functional biomaterials using co-cultures of the patient’s cancer, immune, and stromal cells will personalize cancer treatment and establish a new branch of precision medicine. This approach might result in the development of a new class of efficient, specialized drugs that target the cancer cells in the patient. This tactic might also lessen the harmful impacts of drug testing. Finally, by extrapolating observed processes to timelines pertinent to human tumor evolution, computational techniques can support in vitro tumor models. The development, invasion, and treatment outcomes of tumors caused by metabolic heterogeneity can be predicted using computational approaches over timescales of days to years. In silico models can be enhanced by microenvironmental data such as cell deformation, spatial organization, and chemical gradient alterations. Experimentation and computer modeling need to be iteratively integrated to increase relevance. Cancer is a complex disease in which interactions between tumor cells’ microenvironment and their genetic makeup are equally important. Science continues to evolve thanks to in vitro tumor models that focus on the impact of cellular and acellular microenvironmental factors on the development of cancer. Engineered tumor models can offer unique cancer treatment and precision medicine insights that enhance patient outcomes when paired with contemporary imaging, multi-omics, and computational techniques.

Conclusion

Simulating the tumor environment enables the researchers to closely study the tumor progression in relation to its microenvironment. It allows the preclinical testing of new drug candidates for the toxicological and efficacy studies. Using engineered tumor microenvironments based on functional biomaterials will enable researchers to mimic the real TME as closely as possible. Accurate modeling of TME will significantly accelerate the cancer drug development process and will establish a close relation to the results of preclinical studies and the clinical trials since engineered TME contained patients own cells. Therefore, a more personalized form of cancer therapy could be developed and tailored for specific patients. Today’s technological advances have made many aspects of 3D modeling, such as spheroid formation, available to a wider range of researchers and have allowed the creation of multicellular models with increasing complexity. The variety of model systems, cellular sources, and methods for analyzing data may seem daunting, but each has benefits. As a result, the choice of model should be carefully studied in order to meet the goals of the research. Explants give scientists functional units to study TME biology at the sacrifice of tractability, whereas hydrogels give researchers control over the gel’s composition but might produce biomimetic structures that do not exactly replicate the physiology of the host. In the end, 3D modeling is a delicate balance. A balance between usability and model system compatibility must be struck by the ideal cell. Additionally, the 3D system must effectively facilitate the appropriate level of comprehension while accurately displaying the studied biology. With greater access to clinical tissue and improved tools for observing cells in situ and interpreting heterocellular communication, the use of 3D models to understand the TME holds enormous promise for resolving translational research problems across biomedicine.

Abbreviations

ECM

Extracellular matrix

TME

Tumor microenvironment

CAF

Cancer-associated fibroblast

HOXD10

Homeobox D10

p53

Tumor suppressor protein

EMT

Epithelial-mesenchymal transition

AKT

Protein kinase B

mTOR

Mammalian/mechanistic target of rapamycin

MMPs

Matrix metalloproteinases

AMPK

AMP-activated protein kinase

LKB1

Liver kinase B1

ROCK

Rho-associated protein kinase

HIF-1

Hypoxia-inducible factor-1

PI3Ks

Phosphoinositide 3-kinases

MAPKs

Mitogen-activated protein kinases

TGF

Transforming growth factor

PGDF

Platelet-derived growth factor

IGF

Insulin-like growth factor

IL

Interleukin

CXCL-8

C-X-C motif chemokine 8

VEGF

Vascular endothelial growth factor

IGFBP-3

Insulin-like growth factor-binding protein 3

ZEB1

Zinc finger E-box binding homeobox 1

Author contribution

Tanvir Ahmed: conceptualization, writing—original draft, and writing—review and editing.

Data availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Declarations

Ethical approval

Not applicable.

Conflict of interest

The author declares no competing interests.