Helpathon #4 - can you help Raissa?
Can you help Raissa find a more complex organoid-like brain and immune model based on rhesus microglia to study aging in relation to neuroinflammation and neurodegenerative diseases? Join Helpathon #4, look at www.tpihelpathon.nl/coming-up ! Raissa Timmerman is a PhD student at the alternative unit at the Biomedical Primate Research Center. A better understanding of aging of the brain is key to studying neuroinflammation and neurodegenerative diseases. We believe there is a potential for breakthrough in using our existing live macaque data obtained from past aging experiments to develop more complex in vitro rhesus brain-like models and then to correlate all this data with data from human in vitro models and human live data.
Helpathon #4 - can you help Anne-Marie?
Can you help Anne-Marie develop a more organ-like Rhesus 3D liver model in which she can study the dormancy and the waking up of malaria parasites? Join Helpathon #4, look at www.tpihelpathon.nl/coming-up ! Anne- Marie Zeeman is a researcher at the Biomedical Primate Research Center (BPRC). Anne-Marie studies recurrent malaria ( P. vivax). She successfully developed a single cell layer in vitro model to study compounds affecting dormant and active malaria parasites in the liver of Rhesus monkeys. We believe that the cross correlation between in vitro Rhesus and in vitro human models will provide the missing link required to improve the drug development process and aid transition. A more refined Rhesus in vitro model can reduce the number of monkeys currently used for testing drugs. The data from in vivo monkeys combined with new in vitro models could help validate and develop reliable human in vitro models making testing on monkeys unnecessary detours.
Helpathon #4 - can you help Frank?
Can you help Frank with integrating an immune system into a macaque lung organoid to address local immunity to tuberculosis with his vaccination strategy? Join Helpathon #4, look at www.tpihelpathon.nl/coming-up ! Frank Verreck does research on tuberculosis at the Biomedical Primate Research Center (BPRC). Tuberculosis is the most deadly infectious disease worldwide! For the past hundred years, BCG (Bacillus Calmette Guérin) vaccinations take place through the skin. Research shows that macaques can be better protected from this infection by vaccination through their lungs. Frank really wants to further study the potential of this alternative vaccination strategy. He wants to understand how this BCG vaccination works in macaques lungs.
Sign in for Helpathon #3: Saskia van Mil
Saskia van Mil calls for a Helpathon! She invites you to help her develop a human model for studying liver metabolism? You can sign in for this Helpathon here: tpihelpathon.nl. Online, 18th - 19th of June 2020.
Sign in for Helpathon #3: Daniela Salvatori
Daniela Salvatori calls for a Helpathon! She invites you to help her and TPI Utrecht to create a unique master course for animal free innovation. You can sign in for this Helpathon here: tpihelpathon.nl. Online, 18th - 19th of June 2020.
Scientific solutions for the gap in translational medicine: skin model platform with melanoma (3D melanoma)
The developing process of a new drug, from first testing to regulatory approval and ultimately to market is a long, costly, and risky path. Noteworthy is the fact that almost 95% of the drugs that go into human trials fail. According to the National Institutes of Health (NIH), 80 to 90% of drug research projects fail before they ever get tested in humans. The value of preclinical research, mainly conducted in animal model experiments for predicting the effectiveness of therapies and treatment strategies in human trials, has remained controversial. Only 6% of the animal studies are successfully translated into the human response. Breaking down failure rates by therapeutic area, oncology disorders account for 30% of all failures. The absence of human-relevant models with receptors, proteins, and drug interactions in the in situ microenvironment leaves a gap in the scientific discovery process of new therapies. In this context, the present work presents the development of a sophisticated in vitro skin model platform focus on boosting melanoma treatment. The results showed a physiological microenvironment of human skin with epidermal differentiation and development of stratified layers (basement membrane, stratum spinosum, stratum granulosum, and stratum corneum). Furthermore, it was observed the pathophysiological microenvironment of the melanoma with invasion or migration through the basement membrane into the dermis and no epidermal differentiation. Vemurafenib treatment, the gold standard which targets BRAF mutations, showed a decrease in proliferation and invasion of melanoma tumors, with an increase in epidermis keratinization. Melanoma incidence continues to increase year-on-year and is currently responsible for >80% of skin cancer deaths. It is the most common cutaneous form and is known to have the highest mutational load of all cancers. Nowadays, patients with advanced melanoma BRAFV600E mutation can benefit from monotherapies or targeted therapies. Although the initial response rate is effective, disease progression and tumor chemoresistance rapidly occur in the majority of patients. Therefore, the treatment of melanoma remains a challenge, and despite the advances, there is still an urgent need to identify new therapeutic strategies. 3D Model Melanoma is considered one important tool for studying the evolution of the pathology, as well as evaluating the effectiveness of new therapeutic approaches.
Optimizing CAR-T-cell therapy using 3D tumor models and real-time cell imaging
Chimeric antigen receptor (CAR) T-cell therapy accounts for one of the most promising therapeutic advances in cancer immunotherapy. In this form of adoptive cell transfer, T-cells of a patient are engineered to express so-called ‘CARs’, in which the antigen-recognition capacity of antibodies is combined with T-cell activating domains. So far, CAR-T-cell therapy obtained its most impressive results in hematological malignancies resulting in the approval of five CAR-T cell products by the FDA for hematologic indications. However, CAR-T-cell therapy has not mirrored its success in solid tumors. The poor efficacy of CAR-T-cell therapy in solid tumors has, in part, been attributed to the lack of understanding in how CAR-T-cells function in a solid tumor microenvironment. Classical validation methods rely on the use of specificity and functionality assays in 2D models against adherent target cells or target cells in suspension. Yet, by using these models, observations made in vitro may differ greatly to an in vivo situation where tumors are engrafted in 3D structures. We developed a more relevant and translational 3D tumor model using eGFP+ target cells. This allows us to couple 3D tumor cell killing by CAR-T-cells to live-cell imaging, providing an efficient quantification of target cell death. As proof- of-concept, we used a 3D model of eGFP+ glioblastoma cells and CAR-T-cells targeting a pan-cancer antigen. This 3D glioblastoma model allowed us to show that classical scFv-based CAR-T-cell therapy of glioblastoma cells can be improved by nanoCAR-T-cells. Furthermore, combining nanoCAR-T-cell therapy with a genetic approach of nanobody-based anti-PD-L1 immune checkpoint blockade further increased the cytotoxicity of the nanoCAR-T-cell therapy.
Biotransformation of two proteratogenic anti-epileptics in the zebrafish (Danio rerio) embryo
The zebrafish (Danio rerio) embryo has gained interest as an alternative model for developmental toxicity testing, which still mainly relies on in vivo mammalian models (e.g., rat, rabbit). However, cytochrome P450 (CYP)-mediated drug metabolism, which is critical for the bioactivation of several proteratogens, is still under debate for this model. Therefore, we investigated the potential capacity of zebrafish embryos/larvae to bioactivate two known mammalian proteratogens, carbamazepine (CBZ) and phenytoin (PHE) into their mammalian active metabolites, carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. Zebrafish embryos were exposed to three concentrations (31.25, 85, and 250 μM) of CBZ and PHE from 51⁄4 to 120 hours post fertilization (hpf) at 28.5°C under a 14/10 hour light/dark cycle. For species comparison, also adult zebrafish, rat, rabbit and human liver microsomes (200 μg/ml) were exposed to 100 μM of CBZ or PHE for 240 minutes at 28.5°C. Potential formation of the mammalian metabolites was assessed in the embryo medium (48, 96, and 120 hpf); pooled (n=20) whole embryos/larvae extracts (24 and 120 hpf); and in the microsomal reaction mixtures (at 5 and 240 minutes) by targeted investigation using a UPLC–Triple Quadrupole MS system with lamotrigine (0.39 μM) as internal standard. Our study showed that zebrafish embryos metabolize CBZ to E-CBZ, but only at the end of organogenesis (from 96 hpf onwards), and no biotransformation of PHE to HPPH occurred. In contrast, our in vitro drug metabolism assay showed that adult zebrafish metabolize both compounds into their active mammalian metabolites. However, significant differences in metabolic rate were observed among the investigated species. These results highlight the importance of including the zebrafish in the in vitro drug metabolism testing battery for accurate species selection in toxicity studies.
Lung tumor spheroids for onco-immunological research
Lung cancer thrives in a complex multicellular tumor microenvironment that impacts tumor growth, metastasis, response, and resistance to therapy. While orthotopic murine lung cancer models can partly recapitulate this complexity, they do not resonate with high-throughput immunotherapeutic drug screening assays. To address the current need for relevant and easy-to-use lung tumor models, we established a protocol for fully histo-compatible murine and human lung tumor spheroids, generated by co-culturing lung fibroblasts with tumor cells in ultra-low adherence 96-well plates. Moreover, we describe their application potential to study tumor-stroma organization, T-cell motility, and infiltration as well as distinct macrophage subsets’ behavior using confocal microscopy. Finally, we report on a 3D target specific T-cell killing assay that allows spatio-temporal assessment using live cell imaging and flow cytometry. This lung tumor spheroid platform can serve as a blueprint for other solid cancer types to comply with the need for straightforward onco-immunology assays.