3D Cell Models Research Articles

Tumor Ablation Enhancement by Combining Radiofrequency Ablation and Irreversible Electroporation: An In Vitro 3D Tumor Study.

We hypothesized and demonstrated for the first time that significant tumor ablation enhancement can be achieved by combining radiofrequency ablation (RFA) and irreversible electroporation (IRE) using a 3D cervical cancer cell model. Three RFA (43, 50, and 60°C for 2min) and IRE protocols (350, 700, and 1050V/cm) were used to study the combining effect in the 3D tumor cell model. The in vitro experiment showed that both RFA enhanced IRE and IRE enhanced RFA can lead to a significant increase in the size of the ablation zone compared to IRE and RFA alone. It was also noted that the sequence of applying ablation energy (RFA→RE or IRE→RFA) affected the efficacy of tumor ablation enhancement. The electrical conductivity of 3D tumor was found to be increased after preliminary RFA or IRE treatment. This increase in tumor conductivity may explain the enhancement of tumor ablation. Another explanation might be that there is repeat injury to the transitional zone of the first treatment by the second one. The promising results achieved in the study can provide us useful clues about the treatment of large tumors abutting large vessels or bile ducts.

Angiopoietin like-4 as a novel vascular mediator in capillary cerebral amyloid angiopathy.

Increasing evidence suggests that vascular dysfunction in the brain is associated with early stages of Alzheimer's disease. Amyloid-β deposition in the microvasculature of the brain, a process referred to as capillary cerebral amyloid angiopathy (capillary CAA), propagates vascular remodelling, which results in impaired function of the blood-brain barrier, reduced cerebral perfusion and increased hypoxia. While improving vascular function may be an attractive new way to fight capillary CAA, the underlying factors that mediate vascular alterations in Alzheimer's disease and capillary CAA pathogenesis remain largely unknown. Here we provide first evidence that angiopoietin like-4 (ANGPTL4), a hypoxia-induced factor, is highly expressed by reactive astrocytes in well characterized post-mortem tissues of patients with capillary CAA. Our in vitro studies reveal that ANGPTL4 is upregulated and secreted by human cortical astrocytes under hypoxic conditions and in turn stimulates endothelial cell migration and sprouting in a 3D spheroid model of human brain endothelial cells. Interestingly, plasma levels of ANGPTL4 are significantly increased in patients with vascular dementia compared to patients with subjective memory complaints. Overall, our data suggest that ANGPTL4 contributes to pathological vascular remodelling in capillary CAA and that detection of ANGPTL4 levels may improve current diagnostics. Ways of counteracting the detrimental effects of ANGPTL4 and thus promoting cerebral vascular function may provide novel treatment regimens to halt the progression of Alzheimer's disease.

In vitro and in vivo models for studying Zika virus biology.

The emergence and rapid spread of Zika virus (ZIKV) in the Americas has prompted the development of in vitro and in vivo models to understand several aspects of ZIKV biology and boost the development of vaccines and antivirals. In vitro model studies include reverse genetics systems, two-dimensional (2D) cell models, such as primary cells and cell lines, and ex vivo three-dimensional (3D) models derived from skin, brain and placenta. While these models are cost-effective and allow rigorous control of experimental variables, they do not always recapitulate in vivo scenarios. Thus, a number of in vivo models have been developed, including mosquitoes (Aedes sp. and Culex sp.), embryonated chicken eggs, immunocompetent and immunodeficient mice strains, hamsters, guinea pigs, conventional swine and non-human primates. In this review, we summarize the main research systems that have been developed in recent years and discuss their advantages, limitations and main applications.

A Novel 3D in Vitro Tumor Model Based on Silk Fibroin/Chitosan Scaffolds To Mimic the Tumor Microenvironment.

Drug development involves various evaluation processes to ascertain drug effects and rigorous analysis of biological indicators during in vitro preclinical studies. Two-dimensional (2D) cell cultures are commonly used in numerous in vitro studies, which are poor facsimiles of the in vivo conditions. Recently, three-dimensional (3D) tumor models mimicking the tumor microenvironment and reducing the use of experimental animals have been developed generating great interest to appraise tumor response to treatment strategies in cancer therapy. In this study, silk fibroin (SF) protein and chitosan (CS), two natural biomaterials, were chosen to construct the scaffolds of 3D cell models. Human non-small cell lung cancer A549 cells in the SF/CS scaffolds were found to have a great tendency to gather and form tumor spheres. A549 cell spheres in the 3D scaffolds showed biological and morphological characteristics much closer to the in vivo tumors. Besides, the cells in 3D models displayed better invasion ability and drug resistance than 2D models. Additionally, differences in drug-resistant and immune-related protein levels were found, which indicated that 3D models might resemble the real-life situation. These findings suggested that these 3D tumor models composed of SF/CS are promising to provide a valuable biomaterial platform in the evaluation of anticancer drugs.

P3-224 - Photodynamic therapy induced apoptosis and autophagy in 2D and 3D nasopharyngeal carcinoma cell models

P3-224 - Photodynamic therapy induced apoptosis and autophagy in 2D and 3D nasopharyngeal carcinoma cell models

Finite Element Formulation of Multiphasic Shell Elements for Cell Mechanics Analyses in FEBio.

With the recent implementation of multiphasic materials in the open-source finite element (FE) software FEBio (febio.org), 3D models of cells embedded within the tissue may now be analyzed, accounting for porous solid matrix deformation, transport of interstitial fluid and solutes, membrane potential, and reactions. The cell membrane is a critical component in cell models, which selectively regulates the transport of fluid and solutes in the presence of large concentration and electric potential gradients, while also facilitating the transport of various proteins. The cell membrane is much thinner than the cell; therefore, in an FE environment, shell elements formulated as 2D surfaces in 3D space would be preferred for modeling the cell membrane, for the convenience of mesh generation from image-based data, especially for convoluted membranes. However, multiphasic shell elements are yet to be developed in the FE literature and commercial FE software. This study presents a novel formulation of multiphasic shell elements and its implementation in FEBio. The shell model includes front- and back-face nodal degrees of freedom for the solid displacement, effective fluid pressure and effective solute concentrations, and a linear interpolation of these variables across the shell thickness. This formulation was verified against classical models of cell physiology and validated against reported experimental measurements in chondrocytes. This implementation of passive transport of fluid and solutes across multiphasic membranes makes it possible to model the biomechanics of isolated cells or cells embedded in their extracellular matrix, accounting for solvent and solute transport.

Leveraging proteomics to compare submerged versus air-liquid interface carbon nanotube exposure to a 3D lung cell model

With the emerging concern over the potential toxicity associated with carbon nanotube inhalation exposure, several in vitro methods have been developed to evaluate cellular responses. Since the major concern for adverse effects by carbon nanotubes is inhalation, various lung cell culture models have been established for toxicity testing, thus creating a wide variation of methodology. Limited studies have conducted side-by-side comparisons of common methods used for carbon nanotube hazard testing. The aim of this work was to use proteomics to evaluate global cellular response, including pro-inflammatory and pro-fibrotic mediators, of a 3D lung model composed of macrophages, epithelial cells, and fibroblasts which mimics the human alveolar epithelial tissue barrier. The cells were exposed to Mitsui 7 (M-7) multi-walled carbon nanotubes (MWCNT) under submerged and air-liquid interface (ALI) conditions and discovery proteomics identified 3500 proteins. The M-7 ALI exposure compared to control was found to increase expression in proteins related to oxidative stress that were not found to be enriched in submerged exposure. Comparison of MWCNT exposure methods, M-7 ALI exposure versus M-7 submerged exposure, yielded protein enrichment in pathways known to be associated with carbon nanotube exposure stress response, such as acute phase response signaling and NRF2-mediated oxidative stress response. This study demonstrates a comparison of commonly deployed carbon nanotube exposure methods. These data should be considered by the nanotoxicology community when interpreting or cross comparing in vitro exposure results.

3D human brain cell models: New frontiers in disease understanding and drug discovery for neurodegenerative diseases

Neurodegenerative disorders have an enormous impact on society and healthcare budgets. There has been a high degree of failure in many recent clinical trials for disease-modifying therapeutics. A major factor in this failure is the difficulty of translating findings from animal-based cell models to human patients. The majority of non-animal neurodegenerative disease research has been conducted in 2 dimensional models of rodent neonatal neurons and glia. While these systems have provided valuable insights into neural cell function and dysfunction, they have largely reached the end of their useful life, as human stem cell technologies combined with major advances in microfluidic technologies have opened the door to development of patient-derived 3D brain cell models. These have major advantages in providing a micro-physiological system more closely reflecting the in vivo brain environment, and promote the interaction between different patient-derived brain cell-types. However, major challenges remain before these model systems will replace the 2D rodent models as the workhorse for neurodegenerative disease studies. Despite these challenges, we are likely to experience a rapid transition of research from old models to new patient derived 3D brain cell systems, which will likely improve translational outcomes for disease therapeutics.

Abstract 2548: An image-based method to detect and quantify T cell mediated cytotoxicity of 2D and 3D target cell models

Abstract T cell-mediated cytotoxicity plays an important role in a suite of new methods being developed with the goal of incorporating a patient's immune system to combat cancer. The purpose of this study was to develop a sensitive, image-based in vitro method to evaluate and optimize adoptive T cell immunotherapies. Experiments were performed using MDA-MB-231 and fibroblast target cells co-cultured in 2D and 3D models, along with T cell to target cell ratios of 20:1, 10:1 and 5:1. T cell activation procedures were also tested where T cells were activated in the presence of anti-CD3 and anti-CD28 antibodies plus an IL-2 superkine only, or in a directed fashion with antibodies, superkine, and varying concentrations of target breast cancer cells. Experimental data show that directing the activation of T cells to recognize surface antigens on specific target cancer cells enhances the killing ability of the cells when compared to results from nondirected cells. In addition, increased T cell to target cell ratios induce higher levels of target cell cytotoxicity. Finally, it was evident that assay performance using 2D cultured target cells could be reliably carried out through a 72-hour incubation period. However, it exhibited limited utility beyond this point due to increasing numbers of uninduced necrotic cells within wells, negating the effect of test conditions. This phenomenon was unseen from 3D cultured target cells, indicating their ability to maintain a higher level of viability when cultured in this manner. Necrotic induction can then be reliably attributed to cell treatments using multiday incubations. The combination confirms the ability of the image-based method to deliver dependable results to assess T cell-mediated cytotoxicity using 2D and 3D target cell models. Citation Format: Brad Larson, Diane Kambach, Wini Luty, Glauco Souza. An image-based method to detect and quantify T cell mediated cytotoxicity of 2D and 3D target cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2548.

Abstract 4099: Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis

Abstract For patients with colorectal cancer (CRC) peritoneal metastases (PM) represent a terminal tumor stage with limited therapeutic options. To increase therapeutic efficacy and overall survival, availability of appropriate patient-derived 3D cell culture (PD3D) and patient-derived xenografts (PDX) models of PM could help improving the predictability of drug response for a specific tumor, but also foster the identification of novel biomarkers and therapeutic targets for CRC-PM patients. In this context, we generated the first scaffold-based PD3D and matching PDX models of CRC-PM as platform to test for chemotherapy response and to identify novel biomarkers. For model establishment, surgical specimens were processed either for PD3D cell culture or transplanted subcutaneously (s.c.) onto immunocompromized NOD scid gamma (NSG) mice. For 3D cell culture models, tissue was dissected, digested and filtered before embedding into a scaffold matrix. For PDX models engrafted tumors were transferred to NMRI nu/nu mice for further passaging. They were characterized by histopathology, immunohistochemistry and gene expression analyses using real-time RT-PCR. Chemosensitivity of both sibling models was evaluated on a panel of conventional chemotherapeutic and of targeted drugs. The same panel of drugs was used in 15 matched PD3D/PDX models and revealed individual response patterns both in PD3D and PDX. Most interestingly, different drug response pattern was observed in models derived from tumor tissue of the omentum vs. tissue from the peritoneum of the same patient. Our results demonstrate, that matched models maintain basic characteristics such as the morphology of the patient tumor in early passages, reflect heterogeneous response rates, and can be used as preclinical platform for translational studies of potential clinical use. Citation Format: Christian RA Regenbrecht, Guido Gambara, Eva Pachmayr, Alessandra Silvestri, Mathias Dahlmann, Bernadette Brzezicha, Britta Buettner, Beate Rau, Ulrich Keilholz, Urike Stein, Wolfgang Walther. Novel patient-derived 3D (PD3D) cell models and matched patient-derived xenografts (PDX) from peritoneal metastasis of colorectal cancer for drug testing and biomarker analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4099.

PO-270 3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment

IntroductionTumour initiation, progression and treatment are influenced by its microenvironment (TME). Chronic inflammation is linked to cancer in diverse pathologies and most aggressive solid tumours, including non-small cell lung carcinoma (NSCLC), have high levels of immune cell infiltration. Tumor-associated macrophages (TAM) are among the most abundant infiltrating cells, with tumour-promoting effects such as induction of proliferation, angiogenesis and evasion from adaptive immunity. Recapitulation of the interaction between the different cellular players, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression and for prediction of therapeutic response.Material and methodsIn this work, we established a 3D cell model, the 3D-3-culture, enclosing three cellular components: NSCLC tumour cell spheroids, cancer-associated fibroblasts and monocytes. The model is based on alginate microencapsulation, which allows direct interaction between different cell types and is compatible with continuous monitoring and functional assessment in long-term culture using stirred systems.Results and discussionsIn the 3D-3-culture, the invasive and immunosuppressive microenvironment of NSCLC was recreated, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen I, IV and fibronectin) and matrix metalloproteinases (MMP1/9). Our system supported cell migration and promoted cell-cell interactions within the alginate microcapsules, allowing the infiltration of monocytes into the tumour tissue and transpolarization into an M2-like macrophage phenotype. Upon challenge with chemo- and immunotherapeutic agents, the response of each cellular component to therapy was assessed. The macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages.ConclusionThe crosstalk between the ECM and tumour, stroma and immune cells in microencapsulated 3D-3-cultures promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. This 3D cell model constitutes a novel tool to study macrophage plasticity and repolarization in response to chemotherapeutic and immunomodulatory drugs.We acknowledge support from the IMI Joint Undertaking (grant agreement no.115188) and FCT (iNOVA4Health—UID/Multi/04462/2013, SFRH/BD/52202/2013, SFRH/BD/52208/2013).Utrecht University, Pharmaceutical Sciences, Utrecht, The Netherlands

Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening

Traditional high-throughput drug screening in oncology routinely relies on two-dimensional (2D) cell models, which inadequately recapitulate the physiologic context of cancer. Three-dimensional (3D) cell models are thought to better mimic the complexity of in vivo tumors. Numerous methods to culture 3D organoids have been described, but most are nonhomogeneous and expensive, and hence impractical for high-throughput screening (HTS) purposes. Here we describe an HTS-compatible method that enables the consistent production of organoids in standard flat-bottom 384- and 1536-well plates by combining the use of a cell-repellent surface with a bioprinting technology incorporating magnetic force. We validated this homogeneous process by evaluating the effects of well-characterized anticancer agents against four patient-derived pancreatic cancer KRAS mutant-associated primary cells, including cancer-associated fibroblasts. This technology was tested for its compatibility with HTS automation by completing a cytotoxicity pilot screen of ~3300 approved drugs. To highlight the benefits of the 3D format, we performed this pilot screen in parallel in both the 2D and 3D assays. These data indicate that this technique can be readily applied to support large-scale drug screening relying on clinically relevant, ex vivo 3D tumor models directly harvested from patients, an important milestone toward personalized medicine.

Biological response of an in vitro human 3D lung cell model exposed to brake wear debris varies based on brake pad formulation

Wear particles from automotive friction brake pads of various sizes, morphology, and chemical composition are significant contributors towards particulate matter. Knowledge concerning the potential adverse effects following inhalation exposure to brake wear debris is limited. Our aim was, therefore, to generate brake wear particles released from commercial low-metallic and non-asbestos organic automotive brake pads used in mid-size passenger cars by a full-scale brake dynamometer with an environmental chamber simulating urban driving and to deduce their potential hazard in vitro. The collected fractions were analysed using scanning electron microscopy via energy-dispersive X-ray spectroscopy (SEM–EDS) and Raman microspectroscopy. The biological impact of the samples was investigated using a human 3D multicellular model consisting of human epithelial cells (A549) and human primary immune cells (macrophages and dendritic cells) mimicking the human epithelial tissue barrier. The viability, morphology, oxidative stress, and (pro-)inflammatory response of the cells were assessed following 24 h exposure to ~ 12, ~ 24, and ~ 48 µg/cm2 of non-airborne samples and to ~ 3.7 µg/cm2 of different brake wear size fractions (2–4, 1–2, and 0.25–1 µm) applying a pseudo-air–liquid interface approach. Brake wear debris with low-metallic formula does not induce any adverse biological effects to the in vitro lung multicellular model. Brake wear particles from non-asbestos organic formulated pads, however, induced increased (pro-)inflammatory mediator release from the same in vitro system. The latter finding can be attributed to the different particle compositions, specifically the presence of anatase.

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells.

Sulforaphane (SFN) exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane-N-acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs) and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H2O2 challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway and the induction of intracellular glutathione (GSH) played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

3D multicellular model of shock wave-cell interaction

Understanding the interaction between shock waves and tissue is critical for advancing the use of shock waves for medical applications, such as cancer therapy. This work aims to study shock wave-cell interaction in a more realistic environment, relevant to in vitro and in vivo studies, by using 3D computational models of healthy and cancerous cells. The results indicate that for a single cell embedded in an extracellular environment, the cellular geometry does not influence significantly the membrane strain but does influence the von Mises stress. On the contrary, the presence of neighbouring cells has a strong effect on the cell response, by increasing fourfold both quantities. The membrane strain response of a cell converges with more than three neighbouring cell layers, indicating that a cluster of four layers of cells is sufficient to model the membrane strain in a large domain of tissue. However, a full 3D tissue model is needed if the stress evaluation is of main interest. A tumour mimicking multicellular spheroid model is also proposed to study mutual interaction between healthy and cancer cells and shows that cancer cells can be specifically targeted in an early stage tumour-mimicking environment. Statement of SignificanceThis work presents 3D computational models of shock-wave/cell interaction in a biophysically realistic environment using real cell morphology in tissue-mimicking phantoms and multicellular spheroids. Results show that cell morphology does not strongly influence the membrane strain but influences the von Mises stress. While the presence of neighbouring cells significantly increases the cell response, four cell layers are enough to capture the membrane strain change in tissue. However, a full tissue model is necessary if accurate stress analysis is needed. The work also shows that cancer cells can be specifically targeted in early stage tumour mimicking environment. This work is a step towards realistic modelling of shock-wave/cell interactions in tissues and provides insight on the use of 3D models for different scenarios.

Matrix stiffness and tumor-associated macrophages modulate epithelial to mesenchymal transition of human adenocarcinoma cells

The tumor microenvironment (TME) is gaining increasing attention in oncology, as it is recognized to be functionally important during tumor development and progression. Tumors are heterogeneous tissues that, in addition to tumor cells, contain tumor-associated cell types such as immune cells, fibroblasts, and endothelial cells. These other cells, together with the specific extracellular matrix (ECM), create a permissive environment for tumor growth. While the influence of tumor-infiltrating cells and mechanical properties of the ECM in tumor invasion and progression have been studied separately, their interaction within the complex TME and the epithelial -to-mesenchymal transition (EMT) is still unclear. In this work, we develop a 3D co-culture model of lung adenocarcinoma cells and macrophages in an interpenetrating network hydrogel, to investigate the influence of the macrophage phenotype and ECM stiffness in the induction of EMT. Rising ECM stiffness increases both tumor cell proliferation and invasiveness. The presence of tumor-associated macrophages and the ECM stiffness jointly contribute to an invasive phenotype, and modulate the expression of key EMT-related markers. Overall, these findings support the utility of in vitro 3D cancer models that allow one to study interactions among key components of the TME.

The tyrosine-kinase inhibitor sunitinib targets vascular endothelial (VE)-cadherin: a marker of response to antitumoural treatment in metastatic renal cell carcinoma

BackgroundVascular endothelial (VE)-cadherin is an endothelial cell-specific protein responsible for endothelium integrity. Its adhesive properties are regulated by post-translational processing, such as tyrosine phosphorylation at site Y685 in its cytoplasmic domain, and cleavage of its extracellular domain (sVE). In hormone-refractory metastatic breast cancer, we recently demonstrated that sVE levels correlate to poor survival. In the present study, we determine whether kidney cancer therapies had an effect on VE-cadherin structural modifications and their clinical interest to monitor patient outcome.MethodsThe effects of kidney cancer biotherapies were tested on an endothelial monolayer model mimicking the endothelium lining blood vessels and on a homotypic and heterotypic 3D cell model mimicking tumour growth. sVE was quantified by ELISA in renal cell carcinoma patients initiating sunitinib (48 patients) or bevacizumab (83 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials).ResultsHuman VE-cadherin is a direct target for sunitinib which inhibits its VEGF-induced phosphorylation and cleavage on endothelial monolayer and endothelial cell migration in the 3D model. The tumour cell environment modulates VE-cadherin functions through MMPs and VEGF. We demonstrate the presence of soluble VE-cadherin in the sera of mRCC patients (n = 131) which level at baseline, is higher than in a healthy donor group (n = 96). Analysis of sVE level after 4 weeks of treatment showed that a decrease in sVE level discriminates the responders vs. non-responders to sunitinib, but not bevacizumab.ConclusionsThese data highlight the interest for the sVE bioassay in future follow-up of cancer patients treated with targeted therapies such as tyrosine-kinase inhibitors.

A physiologically relevant 3D collagen-based scaffold–neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models

3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. Statement of SignificanceTraditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

Ascitic fluid from advanced ovarian cancer patients compromises the activity of receptor tyrosine kinase inhibitors in 3D cell clusters of ovarian cancer cells

Ovarian cancer patients in the advanced stages of the disease show clinical ascites, which is associated with a poor prognosis. There is limited understanding of the effect of ascitic fluid on ovarian cancer cells and their response to anticancer drugs. We investigated the antitumour effects of EGFR/Her-2 (canertinib) and c-Met (PHA665752) inhibitors in a 3D cell model of three ovarian cancer lines. Single and combined inhibitor treatments affected cell growth of OVCAR-5 and SKOV-3 cell lines but not OV-90 cell line. Growth reduction was correlated with the down expression of PCNA, EGFR, HER-2, c-MET, ERK and AKT and their phosphorylation status in cells in growth factor supplemented media. However, these effects were not re-producible in OVCAR-5 and SKOV-3 cell lines when they were exposed to ascitic fluid obtained from three ovarian cancer patients. Serum albumin and protein components in the ascitic fluids may reduce the cellular uptake of the inhibitors.

Refining Radiation for the Next Century.

![][1] Wilhelm Roentgen's report more than a century ago describing X-ray imaging fostered in the application of ionizing radiation (IR) for cancer therapy. Today, about half of all the patients with cancer are treated with radiotherapy and it remains one of our most effective