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

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.

Utilizing micropellets as building blocks in cartilage tissue engineering

Osteoarthritis is the most common cause of pain and disability in Australia. This project describes a method where hundreds of cartilage microtissues are generated as tiny building blocks for assembly into larger tissues suitable for cartilage defect repair. Tissue engineering applications has the potential to overcome natural barriers and effectively repair damaged cartilage tissue. However, engineering few-millimeter thick cartilage, similar to human cartilage in the knee, remains a challenge. Utilizing micropellets as building blocks has the potential to overcome some of the challenges in cartilage tissue engineering.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Growth of confined cancer spheroids : a combined experimental marker, critical modelling approach

A critical step in the dissemination of ovarian cancer is the formation of multicellular spheroids from cells shed from the primary tumour. The objectives of this study were to apply bioengineered three-dimensional (3D) microenvironments for culturing ovarian cancer spheroids in vitro and simultaneously to build on a mathematical model describing the growth of multicellular spheroids in these biomimetic matrices. Cancer cells derived from human epithelial ovarian carcinoma were embedded within biomimetic hydrogels of varying stiffness and grown for up to 4 weeks. Immunohistochemistry, imaging and growth analyses were used to quantify the dependence of cell proliferation and apoptosis on matrix stiffness, long-term culture and treatment with the anti-cancer drug paclitaxel. The mathematical model was formulated as a free boundary problem in which each spheroid was treated as an incompressible porous medium. The functional forms used to describe the rates of cell proliferation and apoptosis were motivated by the experimental work and predictions of the mathematical model compared with the experimental output. This work aimed to establish whether it is possible to simulate solid tumour growth on the basis of data on spheroid size, cell proliferation and cell death within these spheroids. The mathematical model predictions were in agreement with the experimental data set and simulated how the growth of cancer spheroids was influenced by mechanical and biochemical stimuli including matrix stiffness, culture duration and administration of a chemotherapeutic drug. Our computational model provides new perspectives on experimental results and has informed the design of new 3D studies of chemoresistance of multicellular cancer spheroids.

A multiscale road map of cancer spheroids : incorporating experimental and mathematical modelling to understand cancer progression

Computational models represent a highly suitable framework, not only for testing biological hypotheses and generating new ones but also for optimising experimental strategies. As one surveys the literature devoted to cancer modelling, it is obvious that immense progress has been made in applying simulation techniques to the study of cancer biology, although the full impact has yet to be realised. For example, there are excellent models to describe cancer incidence rates or factors for early disease detection, but these predictions are unable to explain the functional and molecular changes that are associated with tumour progression. In addition, it is crucial that interactions between mechanical effects, and intracellular and intercellular signalling are incorporated in order to understand cancer growth, its interaction with the extracellular microenvironment and invasion of secondary sites. There is a compelling need to tailor new, physiologically relevant in silico models that are specialised for particular types of cancer, such as ovarian cancer owing to its unique route of metastasis, which are capable of investigating anti-cancer therapies, and generating both qualitative and quantitative predictions. This Commentary will focus on how computational simulation approaches can advance our understanding of ovarian cancer progression and treatment, in particular, with the help of multicellular cancer spheroids, and thus, can inform biological hypothesis and experimental design.

Gelatine methacrylamide-based hydrogels : an alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

Epithelial-mesenchymal transition and mesenchymal-epithelial transition are essential for the acquisition of stem cell properties in hTERT-immortalised oral epithelial cells

BACKGROUND INFORMATION: Evidence has shown that mesenchymal-epithelial transition (MET) and epithelial-mesenchymal transition (EMT) are linked to stem cell properties. We currently lack a model showing how the occurrence of MET and EMT in immortalised cells influences the maintenance of stem cell properties. Thus, we established a project aiming to investigate the roles of EMT and MET in the acquisition of stem cell properties in immortalised oral epithelial cells. RESULTS: In this study, a retroviral transfection vector (pLXSN-hTERT) was used to immortalise oral epithelial cells by insertion of the hTERT gene (hTERT(+)-oral mucosal epithelial cell line [OME]). The protein and RNA expression of EMT transcriptional factors (Snail, Slug and Twist), their downstream markers (E-cadherin and N-cadherin) and embryonic stem cell markers (OCT4, Nanog and Sox2) were studied by reverse transcription PCR and Western blots in these cells. Some EMT markers were detected at both mRNA and protein levels. Adipocytes and bone cells were noted in the multi-differentiation assay, showing that the immortal cells underwent EMT. The differentiation assay for hTERT(+)-OME cells revealed the recovery of epithelial phenotypes, implicating the presence of MET. The stem cell properties were confirmed by the detection of appropriate markers. Altered expression of alpha-tubulin and gamma-tubulin in both two-dimensional-cultured (without serum) and three-dimensional-cultured hTERT(+)-OME spheroids indicated the re-programming of cytoskeleton proteins which is attributed to MET processes in hTERT(+)-OME cells. CONCLUSIONS: EMT and MET are essential for hTERT-immortalised cells to maintain their epithelial stem cell properties.

Developing a three-dimensional in-vitro cell culture model for studying breast and prostate cancer using starPEG-heparin hydrogels

Introduction Hydrogels prepared from poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs) (1). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSC). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyse the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via Alamar Blue assays, light microscopy, and immunofluorescence staining for cytokeratin 8/18, Ki67 and E-Cadherin. Cancer cell lines were then pre-grown in hydrogels for 5-7 days and then re-seeded into starPEG-heparin hydrogels functionalised with RGD, SDF-1, bFGF and VEGF as spheroids with HUVECs and MSC and grown for 14 days as a tri-culture in Endothelial Cell Growth Medium (ECGM; Promocell). Cell lines were also seeded as a single cell suspension into the functionalised tri-culture system. Cultures were fixed in 4% paraformaldehyde and analysed via immunostaining for Von Willebrand Factor and CD31, as well as the above mentioned markers, and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualised in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. HUVEC tube formation and cancer line spheroid formation occured after 3-4 days. Interaction was visualised between tumours and HUVECs via confocal microscopy. Slightly increased interaction was seen between cancer tumours and micro-vascular tubes when seeded as single cells compared with the pre-formed spheroid approach. Further studies intend to utilise cytokine gradients to further optimise the ECM environment of in situ tumour angiogenesis. Discussion and Conclusions Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVECs and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer. References 1. Tsurkan MV, Chwalek K, Prokoph S, Zieris A, Levental KR, Freudenberg U, Werner C. Advanced Materials. 25, 2606-10, 2013. Disclosures The authors declare no conflicts of interest

Single molecule microscopy in 3D cell cultures and tissues

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments

Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14 days, cancer spheroids of 100-200µm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process.

Effective Cell-Centred Time-Domain Maxwell's Equations Numerical Solvers

This research work analyses techniques for implementing a cell-centred finite-volume time-domain (ccFV-TD) computational methodology for the purpose of studying microwave heating. Various state-of-the-art spatial and temporal discretisation methods employed to solve Maxwell's equations on multidimensional structured grid networks are investigated, and the dispersive and dissipative errors inherent in those techniques examined. Both staggered and unstaggered grid approaches are considered. Upwind schemes using a Riemann solver and intensity vector splitting are studied and evaluated. Staggered and unstaggered Leapfrog and Runge-Kutta time integration methods are analysed in terms of phase and amplitude error to identify which method is the most accurate and efficient for simulating microwave heating processes. The implementation and migration of typical electromagnetic boundary conditions. from staggered in space to cell-centred approaches also is deliberated. In particular, an existing perfectly matched layer absorbing boundary methodology is adapted to formulate a new cell-centred boundary implementation for the ccFV-TD solvers. Finally for microwave heating purposes, a comparison of analytical and numerical results for standard case studies in rectangular waveguides allows the accuracy of the developed methods to be assessed.