Bioengineered 3D platform to explore cell–ECM interactions and drug resistance of epithelial ovarian cancer cells

Abstract

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell–matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40–60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

Introduction

A growing body of evidence suggests that three-dimensional (3D) models, in contrast to two-dimensional (2D) flat, conventional culture plastic, replicate more accurately the actual microenvironment where cells reside in tissues; and therefore the behaviour of cells cultured in 3D will reflect more closely their in vivo responses [1], [2], [3], [4]. Consequently, 3D culture models are becoming increasingly crucial research tools, in particular in cancer cell biology [5], [6], [7], as they are a bridge between traditional culture on plastic and in vivo experiments [8], [9], [10].

Currently, in vitro 3D culture models for cancer research include multicellular spheroids grown in suspension [11], [12] and cells embedded within naturally derived extracellular matrices (ECM) (e.g. reconstituted ECM-protein-based matrices for Matrigel™ [13], [14], [15], [16], [17], collagen gels [18], [19] and cell-secreted ECMs [20]). Although these 3D culture systems have profoundly revolutionised fundamental cancer research [2], they have experimental and interactive limitations. Multicellular spheroids grown as independent cell agglomerates in the absence of an ECM do not interact with their extracellular milieu and do not have physical resistance provided by the ECM [21]. The gold standard 3D matrix Matrigel™ and other naturally derived hydrogels have ECM-like biological properties, but their inherent characteristics offer limited design flexibility in fine-tuning different matrix properties (physical and biological) independently [22]. In addition, their relatively poor handling characteristics and batch-to-batch composition differences affect the reproducibility of experimental outcomes and comparative studies [4], [9].

Emerging approaches in biomaterial sciences and regenerative medicine have focused on the development of synthetic hydrogels that mimic the key features of natural extracellular microenvironments due to flexible biochemical and biophysical characteristics [23], [24], [25], [26]. As provisional matrices and drug delivery platforms in biomaterial-based regenerative medicine, these biomimetic hydrogels have demonstrated that they can be equipped with specific biological functionalities that influence cellular performance in vitro and in vivo [27], [28], [29]. In the context of cancer biology, cell–ECM interactions are fundamental to carcinogenesis and related signalling events [30], [31], [32]. The possibility of re-creating controlled extracellular microenvironments, characterised by cell-integrin binding sites and susceptibility to proteolytic remodelling, are key features of synthetic hydrogels that can be exploited to study cancer cell–ECM interactions. Such synthetic and defined matrices can overcome the limitations of naturally derived matrices. Hence, these attributes make biomimetic hydrogels an exciting alternative to the currently used 3D systems [26], [33], [34].

Specifically, we used synthetic hydrogels that are formed from peptide-functionalised multiarm polyethylene glycol (PEG) macromolecules via the factor XIII (FXIII)-catalysed cross-linking mechanism, a reaction occurring during fibrin clot formation in natural wound healing. Desired biological functions can be conferred on these matrices through stable and specific incorporation of peptides (e.g. the Arg-Gly-Asp (RGD) integrin-binding motif), and other proteins (e.g. growth factors) by means of the same cross-linking reaction and simultaneously with the gel formation [35], [36]. The sensitivity of these matrices to degradation by cell-secreted/activated proteases (e.g. matrix metalloproteinases (MMP)), can be precisely controlled by designing MMP-substrates within the hydrogel network [28], [29]. The matrix stiffness can be regulated by changing the polymer dry mass of the hydrogel, without changing their biological and biochemical characteristics [29], [36].

The aim of our study was to provide a proof of concept that this synthetic 3D platform offers a versatile cell culture model to analyse interactions crucial in cancer progression and anti-cancer drug resistance. We have hypothesised that the malleability of the hydrogel characteristics, in contrast to natural gold standard gels (e.g. Matrigel™), may be exploited to dismantle complex cell–ECM interactions of carcinogenesis in vivo into more simple and defined questions trackable in an in vitro setting. We used two cell lines as cancer cell models – OV-MZ-6 [37] and SKOV-3 [38] – both were derived from peritoneal ascites (fluid that surrounds the cancer in the abdominal cavity) of human epithelial ovarian cancer (EOC), an aggressive form of ovarian cancer typically diagnosed at an advanced stage. At this late disease stage, chemotherapy resistance occurs, for reasons as yet unknown [39]. The ability of both cancer cell lines, embedded within these synthetic hydrogels, to grow multicellular spheroids from single cells and their resistance to anti-cancer drugs compared to cell monolayers grown on conventional 2D plastic surfaces [40], [41] were investigated. The influences of extracellular microenvironment characteristics, achieved by systematic and independent alteration of the physical, biological and biochemical properties of these synthetic matrices, on the behaviour of the EOC cells cultured in 3D were determined.