Evaluation of Three-Dimensional in Vitro Models to Study Tumor

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Evaluation of three dimensional in vitro models to study tumour angiogenesis Laura Bray, and Carsten Werner ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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ACS Biomaterials Science & Engineering

Evaluation of three dimensional in vitro models to study tumour angiogenesis Laura J. Bray1, 2 and Carsten Werner3, 4, *

1

Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT),

60 Musk Avenue, Kelvin Grove 4059, Queensland, Australia 2

Mater Research Institute - University of Queensland (MRI-UQ), Translational Research

Institute, 37 Kent Street, Woolloongabba 4102, QLD, Australia 3

Leibniz Institute of Polymer Research Dresden e.V., Max Bergmann Center of Biomaterials

Dresden, Hohe Str. 6, 01069 Dresden, Saxony, Germany 4

Center for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstr.

105, 01307 Dresden, Saxony, Germany

*

Corresponding author: [email protected], Leibniz Institute of Polymer Research Dresden e.V., Hohe Str. 6, 01069 Dresden, Germany, Tel. +49 (0)351 4658 531 / Fax +49 (0)351 4658 533 ACS Paragon Plus Environment

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Abstract The tumour 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 utilised over the past decade, only recently have these models been exploited in the advancement of methods to directly study the mechanisms of tumour development, such as angiogenesis. Here, we review the current ‘state-of-the-art’ in 3D culture models developed for the study of tumour 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 tumour engineering.

Keywords: biomaterials, hydrogel, microenvironment, cancer, angiogenesis

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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 tumour microenvironment. Traditional two dimensional (2D) culture methods cannot replicate the complexity and heterogeneity of the tumour microenvironment, and remove the native context of tumour development. Moreover, 2D cultures disrupt natural cell-cell signalling 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 multi-cellular 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 humanised culture technologies that can mimic the complexity of the disease seen in patients.

Epithelial carcinomas represent the most common form of cancer.6 Normal epithelium consists of organised 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 environmental, influence cancer progression. When tissue homeostasis has been disrupted by cancer cells, the tissue microenvironment instead begins to support the developing tumour.7-9 Therefore, the architecture of the tumour 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 three-dimensional cell and matrix arrangement encouraging cell-cell and cell-ECM interactions.

2. The tumour microenvironment and metastasis ACS Paragon Plus Environment

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The tumour 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 target for anticancer treatments.12 Even so, tumour vascularisation does not occur by endothelial cells alone, but also through endothelial cell interactions with pericytes and bone marrow-derived precursor cells.13-15 Outside of the immediate vascular structures, supporting cells from the tumour microenvironment provide signalling cascades contributing to angiogenesis. These cells include immune cells (such as tumour associated macrophages), mesenchymal stromal cells (MSCs) and fibroblasts (such as cancer associated fibroblasts). The mechanisms of tumour angiogenesis occur during expansion due to the hypoxic and nutrient poor tumour core. Furthermore, it is known that tumours beyond 1-2 mm3 necessitate contact with the vascular system to reach a sufficient level of oxygen and nutrients delivered to the tumour, 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 tumour site. VEGF is a key regulator of vasculogenesis and angiogenesis.20,

21

This factor is comprised of 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 tumour with the oxygen and ACS Paragon Plus Environment

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nutrients.28 The subsequently formed tumour 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 tumour 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 tumours 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 tumour cells, intravasation, dissemination via lymphatic or blood vessels, extravasation and colonization. First, the tumour cells have to detach from the primary tumour 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 Secondly, tumour 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 the mass migration. Single cells can either migrate via a proteasedependent mesenchymal movement (undergo phenotypic changes: epithelial-to-mesenchymal transition) or a protease-independent amoeboid movement.34 In the vascular system, tumour cells have to escape from the immune system and withstand the 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


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As described, tumour 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 tumour angiogenesis to study this process requires novel models that can account for the aforementioned complexity of the tumour microenvironment (Figure 1).

Figure 1. Schematic overview of key biomaterials suitable for the engineering of tumour 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 tumour 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 ACS Paragon Plus Environment

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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 vascularised cancer tissue for research purposes.

There have been few attempts to engineer 3D in vitro systems, which would mimic the complex tumour microenvironment and compromise between 2D cultures and animal studies.41 Of importance, 3D in vitro culture systems have been recently recognised for higher degree of reflecting the in vivo response to the therapeutic agents, and thus have a high potential to become useful in preclinical analysis. Hence, what remains the present task for multidisciplinary teams is to develop a platform for 3D culture to study the mechanisms of tumour angiogenesis and recapitulate the interactions between tumours 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 tumour angiogenesis, focussing on the development of models for drug discovery, including suitable biomaterials, limitations and challenges. This review highlighted the use of static and dynamic cultures for modelling solid tumour angiogenesis. Here, we review the current ‘state-of-the-art’ 3D tissue engineered models that have been developed for the study of tumour angiogenesis, including blood cancer. We compare and contrast natural, semi-synthetic and synthetic biomaterials that have promise for tumour 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.

3. In vitro scaffold-based models for engineering tumour angiogenesis ACS Paragon Plus Environment

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There is a growing trend in the use of scaffold-based platforms to study the tumour microenvironment. The materials used to manufacture the scaffolds to model tumour angiogenesis can be either natural, synthetic or semi-synthetic (Table 1).41 The goal of material selection is to develop a scaffold that is biodegradable, biocompatible, are 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 organisation 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 tissuespecific endothelial cell recruitment and tumour 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 tumour 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.

Table 1. Matrices for 3D tumour angiogenesis engineering Material

Natural Collagen I

Characteristics

Tumour Angiogenesis Application

References

Hydrogel microenvironment that is cytocompatible, biocompatible, biodegradable, crosslinkable, and is used widely for studies of

Breast cancer, prostate cancer, Colon cancer, oral cancer, glioblastoma, leukaemia

44-54


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Matrigel

Fibrin

Fibrinogen/Gelatin/Alginate

Decellularised matrices

angiogenesis. Hydrogel microenvironment that is cytocompatible, has cell adhesion sites and mechanical properties can be adjusted in a limited range. Hydrogel that is cytocompatible, biocompatible, biodegradable, and is a blood coagulant. Fibrin architecture mimics tumour ECM Hydrogel is cytocompatible, biocompatible, biodegradable, chemical similarity to ECM Preserved blood vessel structures, preserved extracellular components

Semi-synthetic Poly(ethylene glycol) (PEG)- Biocompatible, high Heparin water content, adjustable properties, can be functionalised with different biological molecules, cells can locally degrade matrix with MMP sequences; glycosaminoglycan-based heparin matrix has affinity for a wide variety of biomolecules Gelatin methacryloyl Contains both integrin(GelMA) binding and proteasecleavage sites and has reproducible and tunable qualities Synthetic PEG Biocompatible, high water content, tunable properties, biological functionalities can be covalently incorporated

Breast cancer, ovarian cancer, prostate cancer, glioblastoma

55-61

Breast cancer

62

Glioma

63

Peripheral nerve tumours

64

Breast cancer, prostate cancer, hepatocarcinoma

65-67

Glioblastoma

68-70

Lung cancer

59, 71

3.1 Natural Scaffolds


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3D approaches for tumour 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 tumour engineering are the most utilised, 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 tumour in culture.

One of the first tumour 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 co-culture model consisted of human foreskin fibroblasts grown within a collagen hydrogel, and PC-3 prostate adenocarcinoma cells embedded in a fibrin hydrogel containing human umbilical vein endothelial cells (HUVECs). After 28 days, increased HUVEC tube formation was visualised when they were in tri-culture with fibroblasts and cancer cells, but not when in coculture with PC-3 cells alone. This result highlighted the influence of the supporting fibroblastic cells during tumour angiogenesis. The main limitations of this model were mechanical stability, in particular hydrogel degradation and shrinking throughout the


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experiments. Although, this was a pioneering step towards the utility of 3D engineered models for angiogenesis studies.

Similarly, Nyga et al. developed a 3D model of colon tumour 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 non-compressed collagen hydrogel containing HUVECs and CF56 colon fibroblasts. In this model, endothelial cell migration, but not tube formation, was visualised in the outer parts of the construct. Also, the collagen hydrogel shrank due to the fibroblast activity, stratifying the collagen layers.

Collagen hydrogels were utilised 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 towards 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 tumour angiogenesis. Collagen I hydrogels were utilised to culture preformed spheroids of endothelial cells, fibroblasts and the breast cancer tumour cell line MDA-MB-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 tumour angiogenesis. Another method utilised telomerase-immortalised human microvascular endothelial cells cultured on top of a collagen I hydrogel. Beneath that construct, MDA-MB231 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 ACS Paragon Plus Environment

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presence of MDA-MB-231 cells but not MCF-7 cells. This result is expected, as MDA-MB231 cells are described to be of a more invasive phenotype than the MCF-7 cell line.

Microfluidic devices have become more utilised in 3D engineering of tumour angiogenesis as the importance of shear forces and dynamic blood flow for the maintenance of vascular structures and tumour angiogenesis physiology is realised.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 tumour angiogenesis.48,

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In order to recapitulate a single vessel structure, a

microchannel was surrounded by collagen I hydrogel. The hydrogel was seeded MDA-MB231 cells before seeding endothelial cells through the microchannel. This study showed that, in the presence of endothelial cells, the tumour 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 co-culture combined 2D and 3D culture.49,

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In this

report, microchannels were set

Evaluation of Three-Dimensional in Vitro Models to Study Tumor

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