Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.
In this work, a novel fiber optic sensor based on Fabry–Pérot interferometry is adopted in an optical coherence photoacoustic microscopy (OC-PAM) system to enable high-resolution in vivo imaging. The complete OC-PAM system is characterized using the fiber optic sensor for photoacoustic measurement. After characterization, the performance of the system is evaluated by imaging zebrafish larvae in vivo. With a lateral resolution of 3.4 μm and an axial resolution of 3.7 μm in air, the optical coherence microscopy subsystem visualizes the anatomy of the zebrafish larvae. The photoacoustic microscopy subsystem reveals the vasculature of the zebrafish larvae with a lateral resolution of 1.9 μm and an axial resolution of 37.3 μm. As the two modalities share the same sample arm, we obtain inherently co-registered morphological and vascular images. This OC-PAM system provides comprehensive information on the anatomy and vasculature of the zebrafish larvae. Featuring compactness, broad detection bandwidth, and wide detection angle, the fiber optic sensor enables a large field of view with a static sensor position. We verified the feasibility of the fiber optic sensor for dual-modality in vivo imaging. The OC-PAM system, as a non-invasive imaging method, demonstrates its superiority in the investigation of zebrafish larvae, an animal model with increasing significance in developmental biology and disease research. This technique can also be applied for functional as well as longitudinal studies in the future.
Osteosarcoma (OS) and Ewing sarcoma (ES) are the most common bone cancers in children. They are rare cancers and thus difficult to study due to scarcity of patient material, large genomic instability and a wide histological heterogeneity (in OS) or a lack of satisfactory transgenic animal model and availability of preclinical tests (in ES). There is a dire need for new models and novel therapeutic approaches. Although patient-derived xenografts (PDXs) may recapitulate human tumor biology and predict drug response, propagating PDXs in mice limits its use as a drug-testing platform. We have established and standardized ES and OS spheroid culture and developed a semi-automated drug-screening platform in tumor spheroids. We established several robust techniques for spheroid formation, with clear pathophysiological gradients, but without central necroses at the onset of drug treatment. We performed RNA-seq comparing spheroid transcription profiles to 2D culture and observed dramatic changes in overall expression patterns. We observed upregulation of genes shown to correlate with poor prognosis in OS patients. We saw upregulation of processes associated with regulation of cell migration, negative regulation of proliferation and modulation of the extracellular matrix (ECM). In addition to ES spheroid models, we created bioprinted 3D-models of ES cell lines and of cells obtained from ES PDXs, using extrusion bioprinting techniques (where cells are encapsulated within the cross-linked polymers, thus allowing homogeneous distribution and high cell density). PDX-derived cells were kept in liquid culture and as 3D-bioprinted constructs, while their transcription profiles were compared with the initial PDX. The mevalonate pathway was the most overrepresented in all ES 3D-models, consistent with predominant upregulation of this metabolic pathway integral to tumor growth and progression. After 15 days in 3D-bioprinted culture, we observed pronounced upregulation of genes involved in ECM signaling, suggesting that the construct promoted in vivo-like tumor-ECM interactions, without further promoting main proliferation and cell survival pathways, which was observed in liquid culture. Furthermore, we showed potential for combinatorial treatment with statins and confirmed feasibility of drug testing in patient-derived 3D models. Finally, as our spheroid models showed upregulation of many processes involved in metastasis (genes associated with invasion, migration, angiogenesis and hypoxia), we focused on lung as the most common site of metastasis in ES and OS patients. We are thus establishing mixed airway organoid/tumoroid cultures, to investigate further the lung metastatic niche, with a goal to provide proof of concept for patient-specific 3D-models of lung metastatic tumors to guide personalized drug selection for patients with advanced disease. Citation Format: Branka Radic Sarikas, Mathias Ilg, Marica Markovic, Caterina Sturtzel, Eva Scheuringer, Justine Zulini, Martin Metzelder, Florian Halbritter, Martin Distel, Didier Surdez, Olivier Delattre, Aleksandr Ovsianikov, Heinrich Kovar. 3D-models of pediatric bone sarcomas for personalized therapeutic screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6245.
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