Bioengineered 3D platform to explore cell–ECM interactions and drug resistance of epithelial ovarian cancer cells
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.
Section snippets
Abbreviations
CLSM, confocal laser scanning microscopy; EOC, epithelial ovarian cancer; FXIII, factor XIII; MMP, matrix metalloproteinase; mab, monoclonal antibody; PFA, paraformaldhyde; PBE, phosphate-buffered EDTA; PEG, polyethylene glycol; RGD, Arg-Gly-Asp; SEM, scanning electron microscopy; TEM, transmission electron microscopy.
Materials
Cell culture reagents, rhodamine415-conjugated phalloidin, Alexa488-conjugated anti-mouse IgG, DAPI, PCR reagents, CyQuant® and AlamarBlue® assays were from Invitrogen. Amino
Cell spheroid formation within biomimetic hydrogels
To replicate the in vivo behaviour of the EOC cell lines OV-MZ-6 and SKOV-3, we used synthetic hydrogels displaying key features of the natural ECM (e.g. cell-integrin binding sites and degradability by cell-secreted/activated proteases) as a 3D culture system and compared this to conventional monolayer cultures in 2D. EOC cells embedded within hydrogels formed cell spheroids, similar to those found in ascites fluid accumulated in the peritoneal cavity of patients diagnosed with advanced
Third dimension in cell cultures
Matrix-based 3D in vitro culture models are increasingly becoming essential tools in cancer research as they allow cell responses that more closely mimic events occurring in vivo during cancer formation and progression. They provide a pathophysiological context that more accurately replicates solid cancer microenvironments compared to monolayer cultures in 2D [1], [2], [9], [10]. In this context, we first examined the behaviour of two EOC cell types in 2D on conventional culture plastic, and in
Conclusions
We provide evidence that this 3D hydrogel system offers well-defined and reproducible extracellular microenvironments for studying pathophysiological processes in carcinogenesis. Both EOC cell lines embedded within hydrogels grew as multicellular spheroids from single cells, mimicking ovarian cancer metastases in vivo where cells are shed from the primary cancer and aggregate as spheroids within the ascites in the peritoneal cavity. Changes in extracellular microenvironment characteristics
Acknowledgements
The authors are grateful to Dr Deborah Stenzel, Dr Christina Theodoropoulos and Dr Luke Nothdurft from the Analytical Electron Microscopy Facility of the Queensland University of Technology (QUT) and Dr Leonore de Boer from the Cell Imaging Facility of the Institute of Health and Biomedical Innovation (IHBI) for their assistance in the microscopy techniques. This study was supported by the National Health and Medical Research Council (NHMRC) of Australia, a Smart State Fellowship of the
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