Conventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely... Show moreConventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti-apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G2 cell-cycle arrest; Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy. Show less
Baranski, Z.; Booij, T.H.; Cleton-Jansen, A.M.; Price, L.S.; Water, B. van de; Bovee, J.V.M.G.; ... ; Danen, E.H.J. 2015
Traditional drug discovery approaches have been hampered by (in vitro) cell-culture models that poorly represent the situation in the human body. Principally, cells grow in the body in a three... Show moreTraditional drug discovery approaches have been hampered by (in vitro) cell-culture models that poorly represent the situation in the human body. Principally, cells grow in the body in a three-dimensional (3D) environment that cannot generally be captured using cell culture methods. For this reason, cell-culture models have been developed where cells grow in a 3D-environment, which allows them to form structures that are more comparable to tissue in the body. However, the full complexity of these advanced cell-culture models can only be fully used for routine drug testing if the cell culture model can be used on a large scale (also termed high-throughput screening or HTS), and if the readout can capture all of the biological complexity reflected by the 3D-cultured cells (high-content screening or HCS). Due to these technological limitations, 3D cellular models are not yet routinely applied in drug and drug-target discovery. This thesis describes the development of fully-scalable 3D cell-culture screening platforms in the context of cancer and polycystic kidney disease. Show less
Duinen, V. van; Zhu, D.; Ramakers, C.; Zonneveld, A.J. van; Vulto, P.; Hankemeier, T. 2019
Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have... Show morePre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure. Show less
A fundamental process in the development and progression of heart failure is fibrotic remodeling, characterized by excessive deposition of extracellular matrix proteins in response to injury.... Show moreA fundamental process in the development and progression of heart failure is fibrotic remodeling, characterized by excessive deposition of extracellular matrix proteins in response to injury. Currently, therapies that effectively target and reverse cardiac fibrosis are lacking, warranting novel therapeutic strategies and reliable methods to study their effect. Using a gelatin methacryloyl hydrogel, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and human fetal cardiac fibroblasts (hfCF), we developed a multi-cellular mechanically tunable 3D in vitro model of human cardiac fibrosis. This model was used to evaluate the effects of a promising anti-fibrotic drug-pirfenidone-and yields proof-of-concept of the drug testing potential of this platform. Our study demonstrates that pirfenidone has anti-fibrotic effects but does not reverse all TGF-beta 1 induced pro-fibrotic changes, which provides new insights into its mechanism of action. Show less
Gorte, J.; Beyreuther, E.; Danen, E.H.J.; Cordes, N. 2020
Pancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to... Show morePancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to surgery and chemotherapy. Owing to radiosensitive abdominal organs, high-precision proton beam radiotherapy has been regarded as superior to photon radiotherapy. To further elucidate the potential of combination therapies, we employed a more physiological 3D, matrix-based cell culture model to assess tumoroid formation capacity after photon and proton irradiation. Additionally, we investigated proton- and photon-irradiation-induced phosphoproteomic changes for identifying clinically exploitable targets. Here, we show that proton irradiation elicits a higher efficacy to reduce 3D PDAC tumoroid formation and a greater extent of phosphoproteome alterations compared with photon irradiation. The targeting of proteins identified in the phosphoproteome that were uniquely altered by protons or photons failed to cause radiation-type-specific radiosensitization. Targeting DNA repair proteins associated with non-homologous endjoining, however, revealed a strong radiosensitizing potential independent of the radiation type. In conclusion, our findings suggest proton irradiation to be potentially more effective in PDAC than photons without additional efficacy when combined with DNA repair inhibitors. Show less
Liu, T.; Berk, L. van den; Wondergem, J.A.J.; Tong, C.; Kwakernaak, M.C.; Braak, B. ter; ... ; Kieltyka, R.E. 2021
A major challenge in the use of HepG2 cell culture models for drug toxicity screening is their lack of maturity in 2D culture. 3D culture in Matrigel promotes the formation of spheroids that... Show moreA major challenge in the use of HepG2 cell culture models for drug toxicity screening is their lack of maturity in 2D culture. 3D culture in Matrigel promotes the formation of spheroids that express liver-relevant markers, yet they still lack various primary hepatocyte functions. Therefore, alternative matrices where chemical composition and materials properties are controlled to steer maturation of HepG2 spheroids remain desired. Herein, a modular approach is taken based on a fully synthetic and minimalistic supramolecular matrix based on squaramide synthons outfitted with a cell-adhesive peptide, RGD for 3D HepG2 spheroid culture. Co-assemblies of RGD-functionalized squaramide-based and native monomers resulted in soft and self-recovering supramolecular hydrogels with a tunable RGD concentration. HepG2 spheroids are self-assembled and grown (≈150 µm) within the supramolecular hydrogels with high cell viability and differentiation over 21 days of culture. Importantly, significantly higher mRNA and protein expression levels of phase I and II metabolic enzymes, drug transporters, and liver markers are found for the squaramide hydrogels in comparison to Matrigel. Overall, the fully synthetic squaramide hydrogels are proven to be synthetically accessible and effective for HepG2 differentiation showcasing the potential of this supramolecular matrix to rival and replace naturally-derived materials classically used in high-throughput toxicity screening. Show less
Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative... Show morePurpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials. Show less
A new type of tripodal squaramide-based supramolecular hydrogels is developed and studied. The mechanical properties of hydrogel with a wide stiffness range that can be easily modulated and also... Show moreA new type of tripodal squaramide-based supramolecular hydrogels is developed and studied. The mechanical properties of hydrogel with a wide stiffness range that can be easily modulated and also special and temporal controlled either by decorating with the activated group to offer additional chemical crosslinks or using hybrid hydrogel by incorporating the second network through light irradiation to overcome the weakness of traditional self-assembled supramolecular materials. Moreover, the designed synthetic hydrogel systems are biocompatible with several cell lines and have the potential to use as a 3D culture substrate. Show less
There is an urgent need for more physiologically relevant cell culture methods to guide compound selection in pre-clinical stages of the drug development pipeline. This thesis describes the... Show moreThere is an urgent need for more physiologically relevant cell culture methods to guide compound selection in pre-clinical stages of the drug development pipeline. This thesis describes the development of the OrganoPlate, a microfluidic platform that enables enhanced physiology in cell culture models by combining 3D cell culture, co-culture and perfusion flow, whilst maintaining ease of use, compatibility and throughput. Phaseguides are capillary pressure barriers that enable microfluidic liquid routing and patterning without the use of membrane or other physical barriers. This technology was further developed to enable complex liquid routing using only a standard pipette Phaseguide technology was implemented for gel patterning in a dedicated 3D cell culture device embedded in a standard 384 wells plate. Each plate contains up to 96 microfluidic networks that enable perfusion culture of extracellular matrix embedded tissues and perfused epithelial or endothelial tubules. The standard dimensions and high quality optical readout allows interrogation of these tissues using high content readers as well as other standard readout equipment. The platform has been used for the culture of a variety of tissue types and disease models by the authors, but has also been adopted by expert and non-expert users across the field. Show less
Chondrosarcoma and giant cell tumour of bone (GCTB) are bone tumours characterized by recurrent mutations (IDH1/IDH2 and H3F3A, respectively) that induce remodelling of the epigenetic landscape.... Show moreChondrosarcoma and giant cell tumour of bone (GCTB) are bone tumours characterized by recurrent mutations (IDH1/IDH2 and H3F3A, respectively) that induce remodelling of the epigenetic landscape. The standard of care for both of these sarcoma subtypes is surgery and alternative treatment options for patients with inoperable disease are currently lacking (chondrosarcoma) or suboptimal (GCTB). Therefore, the aim of this thesis was to identify novel therapeutic targets for high-grade chondrosarcoma as well as GCTB, with a focus on potential therapies that could counteract the remodelling of the epigenome. PARP and HDAC inhibition, alone or in combination treatment strategies, were identified as promising therapeutic strategies for chondrosarcoma or both of these bone tumours, respectively. Additionally, this thesis describes the development and use of novel 3D cell culture models which can be used to improve the translation of preclinical findings to the clinic. Show less
