Nanostructured Biomaterials for In Vitro Models of Bone Metastasis Cancer

Abstract

In recent years, tissue engineering approaches have attracted substantial attention owing to their ability to create physiologically relevant in vitro disease models that closely mimic in vivo conditions. Here, we review nanocomposite materials and scaffolds used for the design of in vitro models of cancer, including metastatic sites. We discuss the role of material properties in modulating cellular phenotype in 3D disease models. Also, we highlight the application of tissue-engineered bone as a tool for faithful recapitulation of the microenvironment of metastatic prostate and breast cancer, since these two types of cancer have the propensity to metastasize to bone. Overall, we summarize recent efforts on developing 3D in vitro models of bone metastatic cancers that provide a platform to study tumor progression and facilitate high-throughput drug screening.

Introduction

According to the World Health Organization, cancer is the second leading cause of death globally, resulting in an estimated 9.6 million deaths in 2018. Often, no effective treatment is available when cancer spreads to distant organs through a process called metastasis [1–3]. Preclinical studies on cancer drug developments often rely on conventional two-dimensional (2D) monolayer cultures, which do not faithfully recapitulate the three-dimensional (3D) tumor microenvironment, thus failing to capture realistic drug response, leading to ineffective translation of preclinical studies to clinical trials [4]. In 2D monolayer cultures, cell-cell, cell-matrix interactions are limited compared to in vivo, leading to poor phenotypic retention [5]. 3D models can mimic the inherent native milieu, which is comprised of the extracellular matrix, cell-cell interactions, and cell-matrix interactions. Recently, 3D in vitro model systems have been at the forefront of cancer research due to attempts to mimic in vivo tumor growth, invasion, and resistance to drug therapy while showing a good correlation with clinical outcomes [6] and also replicate many characteristics of bone metastasis [7].

The tissue engineering approach is commonly used to isolate and culture the specific cell types onto 3D scaffolds for generating tissues for implantation into the patient to restore or augment tissue function [8]. The scaffolds can provide biophysical and biochemical cues to the cells to facilitate tissue formation, while the porous microstructure helps enable nutrient supply and waste removal throughout during tissue growth [9]. Overall, tissue engineering approaches improve cell attachment, proliferation, differentiation, and extracellular matrix (ECM) formation in vitro [10–12]. Natural materials such as chitosan, alginate, silk have been successfully used as biomaterials for tissue engineering applications. Synthetic materials such as polycaprolactone, polyurethane, polyethylene glycol, poly-L-lactide have also been used to develop scaffolds with improved mechanical properties compared to natural materials. Some studies have incorporated nanoparticles within the polymer matrix to mimic the nanocomposite structure of biological tissue while improving the mechanical properties of scaffolds. Nanostructured biomaterials have been shown to replicate tumorigenic phenomena by simulating the signaling cascade of cancer progression [13–15], which can be used for the discovery and development of anti-cancer therapeutics [16]. A recent review summarizes applications of nanostructures and nanoobjects for diagnosis, treatments, and managing different aspects of bone malignancies [17].

Increasing evidence supports that the epithelial monolayer significantly impacts the progression of many cancers, including prostate and breast [18,19]. At the primary site, cancer cells undergo epithelial to mesenchymal transition (EMT), which drives cells to take up the mesenchymal phenotype and invade the basement membrane, leading to the initiation of the metastatic cascade. These metastatic cancer cells get chemoattracted by a gamut of cytokines or growth factors secreted by the organs that can provide a suitable milieu for cancer survival, growth, and secondary metastasis. Secondary metastasis is also known as mesenchymal to epithelial transition (MET). MET amplifies the intensity of cell-cell adhesion, leading to the generation of large tumors that interfere with the normal physiological function of secondary site tissue, and eventually cause death of the patients. Studies on cancer have predominantly been focused on EMT even though MET has more severe consequences. A recent review summarizes the use of various 3D tissue engineering scaffolds for the development of bone-like structures to evaluate the ‘homing’ of several cancer types to bone [12]. In the current review, recent examples of how nanostructured composites and biomaterials are utilized to create metastatic microenvironment to recapitulate tumorigenic phenomena, including EMT, MET, and chemoresistance, are discussed. Also, we shed light on the usage of tissue-engineered bone as a tool for recapitulating metastatic phenomena of breast and prostate cancer owing to the inherent tendency of these two cancers to metastasize to bone. We also discuss how material properties such as stiffness or composition influence cell behavior, highlighting the role of scaffold properties in designing a suitable 3D in vitro model for bone metastatic cancers.

Calcium Phosphate based in vitro models of bone metastasis

The calcium phosphates based biomaterials can be classified into several classes based on their chemical composition, morphology, and crystal structure [20]. Among them, hydroxyapatite (HAP) and tri-calcium phosphate (TCP) has gathered more interest over time due to their tissue engineering applications.

Hydroxyapatite:

Synthetically created hydroxyapatite (HAP) is primarily known for its role in bone tissue engineering due to its similarity with the mineral in human bone. The current literature also suggests some application of HAP for 3D in vitro cancer models, particularly for bone metastatic cancers such as breast cancer [21] and prostate cancer [22]. It is challenging to fabricate in vitro models from HAP directly since HAP is a ceramic material. Thus, HAP is frequently combined with other polymeric materials to construct composite scaffolds. HAP shows excellent biocompatibility at the nanoscale since the nanosized HAP possesses high surface area as compared to micro-sized HAP. HAP nanoparticles with a diameters ranging from 20 nm to 80 nm have shown to promote adhesion, proliferation, and mineralization of osteoblasts [23]. Several current studies have made efforts to evaluate the effect of nanosized HAP bone minerals on the behavior of breast cancer cells. A recent study indicated that altering the nanoscale properties of HAP could help create a bone niche suitable for breast cancer metastasis and growth. Studies with poly(lactide-co-glycolide) (PLG) scaffolds coated with HAP nanoparticles of varying crystallinity showed that smaller, poorly crystalline nano HAP particles promot adsorption of serum proteins onto the scaffold surface, thus leading to increased breast cancer cell adhesion and growth for MDA-MB-231 cells. In contrast, larger crystalline HAP nanoparticles stimulate pro-tumorigenic cytokine interleukin-8 (IL-8) expression of breast cancer cells [24]. A similar study showed that nanocrystalline HAP stimulated the malignancy potential of ductal breast carcinoma cells (MCF10DCIS.com) by enhancing their IL-8 expression (Figure 1). MCF10DCIS.com is a clonal breast cancer cell line derived from a xenograft originating from premalignant MCF10AT cells injected into immunologically deficient mice. The enhanced malignancy of breast cancer cells was attributed to non-stoichiometric hydroxyapatite associated microcalcification that triggers their malignant phenotype [21].

Figure 1.

Hydroxyapatite (HA) loaded poly(lactide-co-glycolide) (PLG) scaffolds stimulates the malignancy of ductal breast carcinoma cells (A) Surface mineralization shown by Alizarin Red S staining in PLG scaffold (left) and HA containing PLG scaffold (right). (B) MicroCT cross-sections display mineral distribution as a function of the attenuation coefficient, which increases with the atomic number. (C) Schematic showing breast cancer cells seeding on the scaffold and then dynamically cultured. (D) Enhanced calcium phosphate mineralization (black) measured by Von Kossa-staining in breast cancer seeded (cell nuclei stained with pink) HA scaffolds (right) compared to PLG control scaffolds (left). Reprinted from [21] with permission from Elsevier.

Tricalcium phosphate:

Tricalcium phosphate (TCP) has received significant attention, owing to its unique characteristics such as excellent biocompatibility, high osteogenic potential, and high bioactivity, making TCP a superior candidate for bone tissue-engineered constructs. These properties are mainly attributed to its Ca/P stoichiometry ratio of 1.5 that closely resembles the ratio for bone minerals. Generally, TCP is comprised of α-phase and β-phase. Although the β-TCP possesses higher solubility and degradability than HAP, it exhibits lower bioresorbability than α-TCP [20]. Among the two phases, β-TCP is commonly used as a biomaterial for tissue engineering applications. In comparison to β-TCP microparticles, and nanoparticles exhibit excellent mechanical properties with tunable degradability [25]. Recently TCP has been investigated extensively for creating 3D in vitro and in vivo bone metastatic cancer models [26,27]. It is well known that TCP holds an ability to form a robust interlocking network with living bone tissue. A recent study showed excellent integration of polycaprolactone (PCL)-TCP electrospun nanofibrous scaffold into host bone tissue of NOD/SCID mice, leading to the formation of new bone. Further, these bone tissue-engineered constructs were utilized to study invasive and osteoclastic behavior of breast cancer cells in mice [26]. Recently, the antitumor effect of TCP nanoparticles has also been investigated on MCF-7 breast cancer cells by treating them with different dosages. The results showed that nano HAP particles of around 70 nm inhibited MCF-7 cell growth by inducing oxidative stress [28].

In order to improve pore size and pore interconnectivity of TCP scaffolds, various fabrication techniques have been introduced that allow improved cell growth and differentiation. A recent study showed the influence of β-TCP scaffold pore geometry on the behavior of bone marrow metastasized neuroblastoma cells. The β-TCP scaffolds with tailored pore geometry trigger the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and neuroblastoma cancer cell growth. Figure 2 represents the 3D wax printing and a slip casting fabrication technique used during the study [27].

Figure 2.

3D wax printing and a slip casting fabrication technique utilized to tailor the pore geometry of β-TCP scaffolds (A) Fabrication of different β-TCP scaffold geometries using a wax casting mold. (B) Representation of the β-TCP scaffolds. (C) SEM representation of the β-TCP scaffolds. (D) Schematic showing mesenchymal stromal cells (MSCs) culturing on β-TCP scaffolds; MSCs (pink), β-TCP (blue), and collagen I/III (green). (E) Table showing porosities of scaffolds. (F) Immunostaining of neuroblastoma cells (GFP tagged -green) and stromal cells (F actin-red) showing their interactions. Adapted from [71] Copyright (2016) Springer Nature. Reprinted from [27] with permission from Elsevier.

Biobased in vitro models of bone metastasis

Alginate:

Alginates are biobased materials obtained from extracts of brown algae that are block copolymers of β-D-mannuronate (M) and α-L-guluronate (G) linked via (1,4) linkage. The ratio of M/G plays a critical role in tuning the mechanical properties of alginate-based hydrogels. Alginate gels with high G-block concentration are usually stiffer and exhibit little or no immune response in vivo than high M-block alginates [29]. Various studies have attempted to illustrate a pattern between substrate stiffness of alginate hydrogels and cancer cell proliferation [30,31]. A substrate stiffness of 150–200 kPa allows a high cell proliferation rate of MCF-7 breast cancer cells [31]. Alginate scaffolds are often investigated to recapitulate the primary site of breast cancer and also evaluate drug efficacies under 3D conditions [32].

Alginate has various advantages over other polymeric materials, including low cost and ease in achieving tailored mechanical properties by varying the crosslinker concentration and crosslinking time. However, an increase in crosslinker concentration of alginate scaffolds also enhances cellular toxicity, limiting their application as tissue-engineered constructs. The other major limitation includes a lack of surface ligands on alginate scaffolds for cell attachment [29]. Thus, the alginate scaffold surface is commonly covalently modified with adhesion peptides to improve cell-scaffold interactions such as the incorporation of arginine-glycine-aspartic acid (RGD) peptide sequence on the alginate scaffold surface [30].

To further overcome these limitations, researchers have also investigated the various combination of alginate with other polymeric materials (e.g., matrigel, gelatin) [33,34]. These studies develop alginate-matrigel and alginate-gelatin based 3D in vitro models for a highly metastatic breast cancer cell line (MDA-MB-231). The results from these studies suggest that scaffolds comprised of a blend of 50% alginate and 50% matrigel provide high structural stability to scaffolds with increased biological activity [33,34]. In comparison, the hydrogel composition of 1% alginate with 7% or 9% gelatin concentrations provides enough surface ligands for cell adhesion, proliferation, and aggregation.

Chitosan:

Chitosan, a shrimp shell derived polymer, offers many benefits for tissue engineering applications, such as excellent biocompatibility and biodegradability [35]. However, chitosan exhibits weak mechanical properties; thus, its combination with various polymer combinations have been attempted to strengthen its mechanical properties. A recent study showed improved mechanical stability of the chitosan-silk-based scaffold at a blending ratio of 1:5 (chitosan: silk) [36]. Although significant studies have been conducted for the use of chitosan as drug delivery agents [37], recent studies also involve the use of chitosan-based scaffolds for the development of cancer models [38,39]. In vitro models have also been built using composite scaffold with silk fibroin, chitosan, and alginate with the 3D culture of colonic carcinoma [40]. A recent study showed alteration in the morphological response of prostate cancer cells by varying chitosan-alginate scaffold stiffness [38]. Since normal prostate tissue and prostate cancer contain chondroitin sulfate as a constituent of their extracellular matrix, this additive is also included in recent efforts to design in vitro chitosan-chondroitin sulfate-based scaffolds that promotes EMT of prostate cancer [39]. In a recent study, chitosan hydrogel was incorporated with hydroxyapatite mineral with the native bioactive factors enabled through seeding of human bone marrow mesenchymal stem cells (MSCs) to create an in vitro model mimicking many aspects of breast cancer bone metastasis [41]. Unique experiments enabled with this model with co-culture of MSCs and MDA-MB-231 breast cancer cells elucidate that MSCs caused upregulation in the expression of metastasis-associated gene metadherin within the breast cancer cells.

Silk:

Silk obtained from various sources such as Bombyx mori silkworm and Antheraea pernyi silkworm is a natural biodegradable material that consists of two major proteins, fibroin, and sericin. The 3D Silk fibroin scaffolds with co-cultures of cancer cells with osteoblasts provide a means to study interactions between cancer and bone environments [42]. The purification of silk to regenerated silk fibroin (RSF) involves removing the sericin layer that is believed to elicit prompt immune response during in vivo applications. RSF based scaffolds have been extensively investigated for tissue engineering applications due to their fast biodegradation, excellent water holding capacity, and good mechanical properties [43]. The mechanical properties of silk fibroin are mainly attributed to the large number of β-sheets domains, which influences the crystallinity of RSF. A molecular dynamics simulation study showed that nanoscale (2–4 nm) confinement of β-sheet provides silk fibers a greater stiffness, strength, and mechanical toughness [44]. Recently, an increased interest has been developed to tailor the mechanical properties of RSF to create improved 3D in vitro cancer models. In other studies, 3D silk scaffolds with bone-morphogenetic -protein-2 functionalization were used as a trap to capture metastasizing prostate cancer cells and evaluate their function [45–47]. The increased mechanical properties of chemically crosslinked silk scaffolds (eSF) significantly impacts the cancer migration rate [48]. The results showed enhanced migration of cancer cells on 2% eSF hydrogels over 3%, where no cell migration was observed. The primary cause of such variation in the migration rate is attributed to high matrix stiffness (1136 ± 94 Pa) of 3% eSF hydrogels compared to lower stiffness of the 2% eSF hydrogels (488 ± 72 Pa). A recent study also showed the application of silk fibroin scaffolds in drug studies. In this study, 3D in vitro silk fibroin scaffolds were created to evaluate the effect of doxorubicin-loaded folate conjugated fibroin nanoparticles on MDA-MB-231 breast cancer cells co-cultured with osteoblasts. Results showed decreased growth of cancer cells along with downregulation in invasiveness and angiogenesis markers [49].

Decellularized biological scaffolds based in vitro models of bone metastasis

Decellularized extracellular matrix (dECM) based scaffolds show much promise in their use for tissue engineering. The process of decellularization involves complete elimination of cells (<50 ng dsDNA per mg ECM dry weight) from the harvested tissue/organ using various physical, chemical, and enzymatic methods while preserving the composition of the native extracellular matrix. The significant advantage of the dECM approach is to mimic the complex ECM niche of a specific tissue in a precise manner. The dECM technique has been widely used to construct bioengineered organs or tissues such as kidney [50], liver [51], blood vessels [52], and trachea [53]. However, designing an in vitro cancer model employing the dECM approach is very challenging due to significant inherent variation in native ECM; thus, intensive optimization is required to evade the batch to batch variation. The reported dECM based 3D in vitro cancer models are prepared from various normal and cancerous tissues depending upon their applications. However, it has been observed that cancer cells respond differently to different local ECM niche. A recent study with MCF-7 breast cancer cells cultured on dECM based scaffolds derived from normal and cancerous tissue of the breast indicates that MCF-7 cells cultured on cancerous dECM exhibited upregulation of migration factors such as MMP-9 and undergo EMT compared to cells grown on normal tissue-derived dECM [54]. Thus, it is expected that structural and compositional variations between normal and cancerous dECM could determine the fate of cancer cells [54]. The dECM approach has been explored to establish a connection between ECM niche and cancer progression. However some studies also have also shown the effect of ECM variation on drug response. Increased drug resistance is observed for MCF-7 cancer cells against 5-fluorouracil cultured on breast cancer tissue-derived dECM compared to 2D monolayer cells [55]. Further, the dECM approach is also used to screen drugs for highly metastatic breast cancer cells. Li and group have created a layered tissue matrix scaffold (TMS) for MDA-MB-231 breast cancer cells by coculturing MDA-MB-231 and GM637 (fibroblast cells). The major objective of the study was to create 3D in vitro cancer model that mimics layered tissue structures in vivo, to study migration behavior of breast cancer cells and to screen anti-cancer drugs. [56]. The schematic given in Figure 3 depicts steps of TMS formation. Briefly, decellularized breast tissue (DBT) of mice was utilized to fabricate porous DBT-TMS with a porosity of 100 μm. The MDA-MB-231 cells were seeded on this porous DBT-TMS followed by a coating of a blank TMS layer and finally coated with a second hydrogel layer seeded with GM637 cells to create a 3D in vitro model. Although various dECM based 3D in vitro cancer models have been successfully developed, employing the dECM technique is always relatively more expensive and needs strict adherence to procedure.

Figure 3.

Decellularized extracellular matrix (dECM) based multilayered hydrogel scaffolds (A) Fabrication steps of layered tissue matrix scaffold (TMS) system and culturing of breast cancer cells (MM231) and fibroblast cells (GM637) in separate layers of a hydrogel. (B) Hematoxylin and eosin (H&E) staining of MM231 and the first layer of hydrogel. (C) H&E staining of MM231 cells, a middle blank layer, and second GM637 cells loaded the hydrogel layer. (D) Cell distribution analyzed after 3 days of culture using DAPI staining. (E) Immunostaining of Ki-67 (green, MM231 cells) and HER2 (red, GM637 cells) representing the location of two cell types. (F to I) Cell migration between different hydrogel layers was analyzed by Live/Dead Cell staining. Scale bars, 100 μm. Adapted from [56] Copyright (2017) American Association for the Advancement of Science.

Nanoclay-based in vitro models of bone metastasis

Clay minerals have been extensively used in the biomedical industry for various applications, including wound healing, drug delivery, and tissue regeneration [57,58]. Nanoclays are nanoparticles of layered silicates with one octahedral alumina sheet sandwiched between two tetrahedral silica sheets [59]. Nanoclays have previously been used as filler materials to improve the mechanical properties of polymeric materials when added in small quantities. The altered phase model describes the mechanisms of property enhancement by nanoclays [60]. While commercial clays are investigated for biomedical applications[61], Katti & Katti group pioneered the use of engineered nanoclays with tailored clay modifications in tissue engineering scaffolds [62]. Further, nanoclay modified with amino acids was developed to mineralize hydroxyapatite (HAP), mimicking biomineralization in human bone [63]. The modified nanoclay was used to develop nanocomposite scaffolds with polymers, and hMSCs were cultured to investigate cellular response. Results indicated the formation of mineralized bone-like ECM via vesicular delivery by osteogenically differentiated MSCs on PCL/in situ HAPclay without the use of osteogenic supplements [64,65]. The sequential culture of prostate and breast cancer cells on the bone mimetic scaffolds indicate mesenchymal to the epithelial transition of prostate and breast cancer cells (Figure 4). Further, the impact of breast cancer cells mediated osteogenesis at the bone site was demonstrated through Wnt/β-catenin signaling [66]. Katti and coworkers showed that cancer-derived factors such as dickkopf-1 (DKK-1) and endothelin-1 (ET-1) are involved in modulating bone mineralization via Wnt/β-catenin pathway [66]. In another study, the prostate cancer phenotype was observed to influence bone mineralization at metastases [67]. As the evaluation of cancer at metastasis is significant, so is the impact of cancer cells on the bone site, as mortalities due to prostate and breast cancer result from skeletal failures due to metastasis. Further, the bone microenvironment is observed to confer drug resistance in breast cancer cells at metastases. Results showed bone-microenvironment secreted interleukin-6 (IL-6) activated signal transducer and activator of transcription 3 (STAT3) in breast cancer cells, which conferred chemoresistance by inhibiting apoptosis and promoting efflux of drugs [16].

Figure 4.

Nanoclay based scaffolds showing bone metastasis of prostate and breast cancer (A-B) Prostate cancer cells MDAPCa2b (left) and PC3 (right) grown on human mesenchymal stem cells (hMSCs) derived bone-mimetic nanoclay based scaffold. Reprinted from [15] with permission from Elsevier. (C-D) breast cancer cells MCF 7 (left) and MDAMB 231 (right) grown on hMSCs derived bone-mimetic nanoclay based scaffold. Scale bars, 10 μm. Adapted from [67] Copyright (2019) Wiley Periodicals, Inc.

Conclusion

In recent years, studies towards building realistic 3D in vitro models of cancer are extensively popular. Although the pharmaceutical and drug delivery space is enthused towards the design of 3D systems to evaluate drug penetration and efficacies, the development of 3D systems by itself is insufficient for recapitulating the metastasis environment. Hence, efforts towards the use of hypoxia chambers to study 3D cultures are also limiting. The limitation results from the fact that the true nature of hypoxia is an inherent characteristic of cancer tumors that can be captured accurately by the in vitro 3D models that are genuinely replicative of the biological environment of the bone site.

Here, we describe the efforts in the literature that use various novel bone tissue engineering approaches to develop bone-like environments using advanced biomaterials based on tissue engineering. While therapeutic strategies ranging from bone stabilizing drugs and tissue-engineered bone replacement are currently administered, recent advances in the evaluation of bone metastasis through in vitro models suggest transformative approaches in the future. One such study proposes inducing tumor dormancy in bone microenvironment at metastasis as a new therapeutic strategy for bone metastasis [68]. Indeed, the mortality of prostate cancer and breast cancer results from skeletal failures. The newly arrived cancer cells at the bone site dramatically affect osteogenesis [66], disrupt bone formation, and hence the skeletal failures, as also observed, are the concurrent effect of breast cancer at bone [69]. Thus, understanding both the role of the bone microenvironment in causing metastasis, and the role of cancer cells on bone remodeling, are essential. Also, patient-derived xenografts and organoid cultures prepared instead of the commercial cancer cell lines are being used [70] that further helps replicate the complexity of tumor-stroma interactions. Overall, the advanced manufacturing studies in tissue engineering and the use of advanced nanobiomaterials, are leading to the development of realistic 3D models of metastasis of cancer. It is expected that these studies will lead to new therapies resulting from advanced critical knowledge of the microenvironments at metastasis in the future and reduce the substantial cancer burden on humanity.