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
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
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
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
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
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
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
