Evaluation of Three-Dimensional in Vitro Models to Study Tumor

May 3, 2017 - Leibniz Institute of Polymer Research Dresden e.V., Max Bergmann Center of Biomaterials Dresden, Hohe Straße 6, 01069 Dresden, Saxony, ...
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Evaluation of Three-Dimensional in Vitro Models to Study Tumor Angiogenesis Laura J. Bray†,‡ and Carsten Werner*,§,∥

ACS Biomater. Sci. Eng. 2018.4:337-346. Downloaded from pubs.acs.org by NEWCASTLE UNIV on 10/03/18. For personal use only.

†

Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove 4059 Queensland Australia ‡ Mater Research Institute - University of Queensland (MRI-UQ), Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD Australia § Leibniz Institute of Polymer Research Dresden e.V., Max Bergmann Center of Biomaterials Dresden, Hohe Straße 6, 01069 Dresden, Saxony, Germany ∥ Center for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstraße 105, 01307 Dresden, Saxony, Germany ABSTRACT: The tumor microenvironment is a key factor in the progression and metastasis of cancer, the complexity of which is not reproducible using two-dimensional (2D) culture models. While three-dimensional (3D) models have become more well-known and utilized over the past decade, only recently have these models been exploited in the advancement of methods to directly study the mechanisms of tumor development, such as angiogenesis. Here, we review the current “state-of-the-art” in 3D culture models developed for the study of tumor angiogenesis. We assess the potential for 3D tissue engineered models to fill the gap between 2D models and clinical application. We also discuss recent advances in combining materials science, engineering, and cell biology to highlight the most promising scaffolds for tumor engineering. KEYWORDS: biomaterials, hydrogel, microenvironment, cancer, angiogenesis

1. INTRODUCTION Cancer, in all of its forms, carries a major disease burden globally, and advancements in the field of cancer research require the development of sophisticated models mimicking the tumor microenvironment. Traditional two-dimensional (2D) culture methods cannot replicate the complexity and heterogeneity of the tumor microenvironment and remove the native context of tumor development. Moreover, 2D cultures disrupt natural cell−cell signaling mechanisms and display altered gene expression and phenotypic profiles.1−5 The development of novel three-dimensional (3D) materials that can support and provide appropriate mechanical and biological cues to imitate the surrounding tissue and extracellular matrix (ECM), as well as maintain multicellular applications, is a current challenge for bioengineers. The goal is to close the gap between 2D cultures and in vivo models to create modern and humanized culture technologies that can mimic the complexity of the disease seen in patients. Epithelial carcinomas represent the most common form of cancer.6 The normal epithelium consists of organized layers of cells with an underlying basement membrane, predominantly comprising collagen type IV and laminin, acting as a divide between the epithelial and stromal layers. This structure and its homeostasis is maintained through particular cell−cell and cell−matrix contacts. Many factors, both cellular and environ© 2017 American Chemical Society

mental, influence cancer progression. When tissue homeostasis has been disrupted by cancer cells, the tissue microenvironment instead begins to support the developing tumor.7−9 Therefore, the architecture of the tumor microenvironment depends on the type and stage of the cancer. Cancer tissue types should be taken into consideration when designing an appropriate model, as the native architecture should be mimicked using a detailed 3D cell and matrix arrangement encouraging cell−cell and cell− ECM interactions.

2. TUMOR MICROENVIRONMENT AND METASTASIS The tumor microenvironment consists of a neoplasm surrounded by a stromal environment containing acellular and cellular components. The ECM has a high quantity of proteoglycans and glycosaminoglycans, collagen, fibrin, and fibronectin.10 Angiogenesis (including lymphangiogenesis), and the greater vascular system, is a key factor in the growth, progression, and metastasis of cancer. In fact, angiogenesis is considered a hallmark of cancer,11 making angiogenesis a key Special Issue: Tissue Engineering and Biomaterials Approaches to Tumor Modeling Received: March 6, 2017 Accepted: May 1, 2017 Published: May 3, 2017 337

DOI: 10.1021/acsbiomaterials.7b00139 ACS Biomater. Sci. Eng. 2018, 4, 337−346

Review

ACS Biomaterials Science & Engineering target for anticancer treatments.12 Even so, tumor vascularization does not occur by endothelial cells alone but also through endothelial cell interactions with pericytes and bone marrowderived precursor cells.13−15 Outside of the immediate vascular structures, supporting cells from the tumor microenvironment provide signaling cascades contributing to angiogenesis. These cells include immune cells (such as tumor associated macrophages), mesenchymal stromal cells (MSCs), and fibroblasts (such as cancer associated fibroblasts). The mechanisms of tumor angiogenesis occur during expansion due to the hypoxic and nutrient poor tumor core. Furthermore, it is known that tumors beyond 1−2 mm3 necessitate contact with the vascular system to reach a sufficient level of oxygen and nutrients delivered to the tumor, without which, they can become dormant.16−18 This leads to a process known as the “angiogenic switch”, which involves the release of pro-angiogenic growth factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF; FGF-2),17,19 for the purpose of attracting new endothelial cells to the tumor site. VEGF is a key regulator of vasculogenesis and angiogenesis.20,21 This factor comprises VEGF-A, including the isoforms VEGF121, VEGF165 and VEGF189,22 and VEGF-B.23 Another important angiogenesis factor, VEGF-C is a member of the VEGF receptor family that is expressed on adult lymphatic endothelium.24,25 Also modifiers of angiogenesis are the FGF-1 and FGF-2 (or bFGF) forms and their associated receptors.26,27 Along with endothelial cell migration and proliferation, this multifaceted course of events also involves ECM degradation. Once the endothelial cells arrive, they begin to form new capillaries to supply the tumor with the oxygen and nutrients.28 The subsequently formed tumor capillaries are distinct from normal vessels as they are irregularly shaped, tortuous, leaky, and immature. Nevertheless, the neovessels further provide a route for cancer cells to disseminate within the body. A high microvessel density of a primary tumor correlates with increased metastatic potential. This can be quantified using immunohistochemistry for CD34, CD31 (blood vessels), and D2−40 (lymphatic vessels).29−32 However, little is known about the types and patterns of angiogenesis of primary tumors and their corresponding metastasis.33 The importance of understanding the mechanisms of metastasis as a terminal stage of malignant growth has been widely recognized. This process includes the following sequential steps: invasion of tumor cells, intravasation, dissemination via lymphatic or blood vessels, extravasation, and colonization. First, the tumor cells have to detach from the primary tumor by altering their cell−cell-adhesions (e.g., cadherins) and cell−ECM contacts (e.g., integrins). Moreover, their invasion is preceded by the active degradation of the ECM by matrix metalloproteinases (MMP), the urokinase plasminogen activator (uPA) system, and heparanase.34 Second, tumor cells are able to migrate collectively or as single cells. When they migrate collectively, the cells are connected via intercellular junctions and circulate as emboli in the vessels. Cells at the edge will create tube-like microtracks by remodelling the ECM in order to ensure mass migration. Single cells can either migrate via a protease-dependent mesenchymal movement (undergo phenotypic changes: epithelial-to-mesenchymal transition) or a protease-independent ameboid movement.34 In the vascular system, tumor cells have to escape from the immune system and withstand mechanical stress. Eventually, the circulating cells will adhere to capillary beds within the target organ and extravasate. If

conditions permit cell survival, they will either proliferate within the organ parenchyma or become dormant.34 As described, tumor angiogenesis comprises many stages of development, and it is unknown whether these stages are similar in all tissue environments. Therefore, therapeutic interventions are reliant upon understanding the mechanisms involved in these complex processes within target tissues. Recapitulation of tumor angiogenesis to study this process requires novel models that can account for the aforementioned complexity of the tumor microenvironment (Figure 1).

Figure 1. Schematic overview of key biomaterials suitable for the engineering of tumor angiogenesis models and associated biological components required to mimic the complexity of this disease.

In vivo systems offer improved predictive value over 2D systems and are used routinely to understand the genetic origins of tumor development and progression, and for drug testing purposes. In this context, there have been in vivo systems developed to study angiogenesis and test drug efficacy,35 although detailed analysis of these studies remains challenging. Moreover, there are ethical considerations and increased pressure for the reduction of animals in research.36−38 In addition, distinct differences between rodent and human biology remain, resulting in altered physiological responses within the host animal when compared with the human body.17,39,40 Therefore, there remains a need for human 3D in vitro cultures that can reproduce the phenotype of vascularized cancer tissue for research purposes. There have been few attempts to engineer 3D in vitro systems, which would mimic the complex tumor microenvironment and compromise between 2D cultures and animal studies.41 Of importance, 3D in vitro culture systems have recently been recognized to mimic the in vivo response to therapeutic reagents to a higher degree, and thus, have potential to become useful in preclinical analyses. Hence, what remains the present task for multidisciplinary teams is to develop a platform for 3D culture to study the mechanisms of tumor angiogenesis and recapitulate the interactions between tumors and the surrounding vasculature. It is important that these platforms allow for precise control over the design of the tissue microenvironment including mechanical, biological, and biochemical properties as well as cell/matrix architecture for an individual tissue type. In a previous review,42 we described relevant cell culture technologies to study tumor angiogenesis, focusing on the development of models for drug discovery, including suitable biomaterials, limitations, and challenges. This 338

DOI: 10.1021/acsbiomaterials.7b00139 ACS Biomater. Sci. Eng. 2018, 4, 337−346

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339

59,71 lung cancer PEG

Synthetic biocompatible, high water content, tunable properties, and has biological functionalities that can be covalently incorporated

68−70

65−67

breast cancer, prostate cancer, hepatocarcinoma glioblastoma poly(ethylene glycol) (PEG)-heparin gelatin methacryloyl (GelMA)

Semisynthetic biocompatible, high water content, adjustable properties, can be functionalized with different biological molecules, cells can locally degrade the matrix with MMP sequences; glycosaminoglycan-based heparin matrix has affinity for a wide variety of biomolecules contains both integrin-binding and protease-cleavage sites and has reproducible and tunable qualities

64 peripheral nerve tumors

hydrogel that is cytocompatible, biocompatible, biodegradable, and is a blood coagulant; fibrin architecture mimics tumor ECM hydrogel is cytocompatible, biocompatible, biodegradable, and has chemical similarity to ECM

fibrin fibrinogen/gelatin/ alginate decellularized matrices

preserved blood vessel structures and preserved extracellular components

62 63

55−61 hydrogel microenvironment that is cytocompatible, has cell adhesion sites, and mechanical properties that can be adjusted in a limited range Matrigel

breast cancer, prostate cancer, colon cancer, oral cancer, glioblastoma, leukemia breast cancer, ovarian cancer, prostate cancer, glioblastoma breast cancer glioma Natural hydrogel microenvironment that is cytocompatible, biocompatible, biodegradable, cross-linkable, and is used widely for studies of angiogenesis

material

Table 1. Matrices for 3D Tumor Angiogenesis Engineering

characteristics

3. IN VITRO SCAFFOLD-BASED MODELS FOR ENGINEERING TUMOR ANGIOGENESIS There is a growing trend in the use of scaffold-based platforms to study the tumor microenvironment. The materials used to manufacture the scaffolds to model tumor angiogenesis can be natural, synthetic, or semisynthetic (Table 1).41 The goal of material selection is to develop a scaffold that is biodegradable, biocompatible, compatible with and can interact with cells, and can be tuned to obtain desirable mechanical and physical characteristics that mimic the ECM. Past research has revealed that gradients of nutrients, biomolecules, and oxygen significantly impact the development of cancer. Moreover, tissue organization will be determined by our ability to imitate their original ECM. In this way, the ECM is known to manipulate endothelial cell proliferation, migration, and sprouting.43 The development of 3D matrices that can mimic the complex process of tissue-specific endothelial cell recruitment and tumor angiogenesis development will provide researchers with a platform to study potential therapeutic intervention points. In order to achieve this, an engineered platform with appropriate properties must be developed. To recapitulate the complex tumor microenvironment, the scaffold must include mechanical stability, degradability, diffusion, appropriately presented adhesive ligands, and loading of growth factors and cytokines. Furthermore, the artificial microenvironment should allow for manipulation of the individual components of the platform to allow for flexibility in experimental design. 3.1. Natural Scaffolds. 3D approaches for tumor angiogenesis engineering are predominantly focused on using natural-based scaffolds. This includes the commonly used basement membrane extract, Matrigel, collagen type I, and fibrin glue. These scaffolds inherently convey biomolecules and therefore, cell signals, which direct cancer morphogenesis,72 as well as endothelial cell capillary formation and sprouting.73,74 While natural scaffolds for tumor engineering are the most utilized, predominantly due to their availability and ease of use, there are various disadvantages including limited stiffness range, fast degradability, instability, and natural batch-to-batch variability. 3.1.1. Collagen I. Collagen I is a frequently used scaffold for 3D cell culture, as it naturally contains the tripeptide RGD (Arg-Gly-Asp). Collagen I hydrogels are also amenable in that they can be modified synthetically to adjust their range of physiochemical properties. Collagen hydrogels are biocompatible, and their matrix architecture makes them suitable for mimicking the microenvironmental conditions of a solid tumor in culture. One of the first tumor angiogenesis models was developed by Janvier et al. in 1997, where a sandwich model was applied to study prostate cancer angiogenesis in vitro.46 The coculture model consisted of human foreskin fibroblasts grown within a collagen hydrogel and PC-3 prostate adenocarcinoma cells

collagen I

tumor angiogenesis application

refs

review highlighted the use of static and dynamic cultures for modeling solid tumor angiogenesis. Here, we review the current “state-of-the-art” 3D tissue engineered models that have been developed for the study of tumor angiogenesis, including blood cancer. We compare and contrast natural, semisynthetic, and synthetic biomaterials that have promise for tumor angiogenesis engineering. New approaches have been included that were developed over the past few years, and applications of these models for research, industry, and the clinic are discussed.

44−54

ACS Biomaterials Science & Engineering

DOI: 10.1021/acsbiomaterials.7b00139 ACS Biomater. Sci. Eng. 2018, 4, 337−346

Review

ACS Biomaterials Science & Engineering embedded in a fibrin hydrogel containing human umbilical vein endothelial cells (HUVECs). After 28 days, increased HUVEC tube formation was visualized when they were in triculture with fibroblasts and cancer cells but not when in coculture with PC3 cells alone. This result highlighted the influence of the supporting fibroblastic cells during tumor angiogenesis. The main limitations of this model were mechanical stability, in particular hydrogel degradation and shrinking throughout the experiments, although, this was a pioneering step toward the utility of 3D engineered models for angiogenesis studies. Similarly, Nyga et al. developed a 3D model of colon tumor angiogenesis using collagen hydrogels.50 In this model, the authors attempted to increase the core density of cancer tissue through partial plastic compression. The compressed collagen matrix contained HT29 human colon adenocarcinoma cells, which was surrounded by a noncompressed collagen hydrogel containing HUVECs and CF56 colon fibroblasts. In this model, endothelial cell migration, but not tube formation, was visualized in the outer parts of the construct. Also, the collagen hydrogel shrank due to the fibroblast activity, stratifying the collagen layers. Collagen hydrogels were utilized for the study of oral squamous carcinoma angiogenesis.54 In this setup, endothelial cells were seeded on the surface of the collagen hydrogel containing OSCC-3 cells as a monolayer. It was found that endothelial cells occupied the collagen hydrogel toward the cancer cells, suggesting cancer-induced chemoattraction. There were no stromal cells included in this model. Correa de Sampaio et al. created a model to recapitulate the early stages of breast tumor angiogenesis. Collagen I hydrogels were utilized to culture preformed spheroids of endothelial cells, fibroblasts, and the breast cancer tumor cell line MDAMB-231.45 Sprouting from the endothelial cells occurred and could be inhibited by the broad spectrum MMP inhibitor, GM6001. This study added to the evidence of stromal cell involvement in tumor angiogenesis. Another method utilized telomerase-immortalized human microvascular endothelial cells cultured on top of a collagen I hydrogel. Beneath that construct, MDA-MB-231 or MCF7 cells were cultured in an additional distinct collagen I hydrogel.53 In this coconstruct, endothelial cells invaded the collagen layer and formed a tubular network in the presence of MDA-MB-231 cells but not MCF-7 cells. This result is expected, as MDA-MB-231 cells are described to be of a more invasive phenotype than the MCF-7 cell line. Microfluidic devices have become more utilized in 3D engineering of tumor angiogenesis as the importance of shear forces and dynamic blood flow for the maintenance of vascular structures and tumor angiogenesis physiology is realized.48,75 Microfluidic channels can imitate blood flow, delivering oxygen and nutrients and therefore can mimic the complex in vivo microenvironment more so than traditional models.76 Recent research described the development of a microfluidic approach to study the effects of shear forces and blood flow on breast tumor angiogenesis.48,53 In order to recapitulate a single vessel structure, a microchannel was surrounded by collagen I hydrogel. The hydrogel was seeded MDA-MB-231 cells before seeding endothelial cells through the microchannel. This study showed that, in the presence of endothelial cells, the tumor cells had significantly increased expression of proangiogenic genes under flow conditions. Nevertheless, there was no endothelial cell invasion into the collagen hydrogel.

A second microfluidic approach for coculture combined 2D and 3D culture.49,51 In this report, microchannels were set