Biology subjects

Brandenberg, N.

Publications and source records attributed to Brandenberg, N..

5 recordsLinked to original sources

bioRxiv2023

Dissection and reconstruction of the colorectal cancer tumor microenvironment

Patient-derived organoids (PDOs) are the reference in vitro human disease models. However, the utility of colorectal cancer (CRC) PDOs is hindered by the lack of a tumor microenvironment (TME). To address this limitation, we built a living biobank of CRC PDOs with autologous stromal and immune TME. We characterized the original tumors and traditional monocultures using single-cell RNA-seq (scRNA-seq) and whole exome sequencing (WES) to obtain insights into cell type selection and phenotypic drift in culture. Subsequently, we developed culture conditions supporting all cell types to recapitulate the CRC-TME around PDOs. From the transcriptomes of >180k cells obtained from 260 such co-cultures, we illuminated the mutual influence of cells within CRC tumors. Based on original tumor data, atlases of predicted interactions and transcriptional networks elucidated why monocultures were altered and suggested that TME reconstruction more accurately reflected original tumor behavior. We found that inflammatory signals were absent in vitro and recovered upon co-culture with tumor-infiltrating lymphocytes (TILs). We also functionally confirmed that stromal, not cancer cells, mediated immune evasion. Additionally, stroma induced an invasive phenotype in cancer cells. From this deep dive into CRC-TME interactions, we built the human CRC-TME atlas (https://crc-tme.com/), an online portal for interactive exploration of gene expression data, prediction of cell-cell interactions at the pathway and receptor/ligand levels, transcriptional networks, and more. We anticipate PDO cultures with reconstructed TMEs will be valuable for discovery efforts, preclinical studies, and personalized medicine, with the atlas as a framework and inspiration for future CRC-TME studies.

cancer biology↗

bioRxiv2021

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such a vascular compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays.

bioengineering↗

bioRxiv2021

Probing the killing potency of tumor-infiltrating lymphocytes on microarrayed autologous tumoroids

Immunotherapy has shown promise as an approach to fight cancer by harnessing the immune system. However, due to the lack of biomarkers to guide treatment regimens and predict response rates, there is an unmet need for more robust ex vivo and in vitro systems that recapitulate patient-specific tumor biology and enable response prediction for immune therapies in an autologous setting. To address this issue, we developed a high-content screening-compatible assay based on microcavity arrays to study tumor-infiltrating lymphocyte (TIL) functionality on 3D tumoroid models. We validated our system using the pmel-1 activated T cell mouse model to assess both cancer immunogenicity and T cell functionality. To demonstrate the translational potential of the platform, we used it to evaluate the response of patient-derived TILs to autologous human colorectal cancer (CRC) tumoroids. Using a combination of imaging and flow cytometry, we determined several features of the antitumor activity of TILs, including the extent of tumoroid killing and secretion of cytokines. We then used the approach to identify responders to immunotherapy, such as the immune checkpoint blockade (ICB) agent Nivolumab (PD-1 inhibitor) and Ipilimumab (CTLA-4 inhibitor). Our system allows not only the identification of immunogenic tumors, but also the testing of patients for response to immunomodulators, enabling personalized immuno-oncology.

cancer biology↗

bioRxiv2021

Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays

BackgroundThe pathways that control protein transport across the Blood-Brain Barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. MethodsWe applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. ResultsBBB organoid arrays allowed the simultaneous growth of more than 5000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. ConclusionsHuman BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.

cell biology↗

bioRxiv2021

Bioengineered embryoids mimic post-implantation development in vitro

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are not yet optimal for modeling post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. A high-throughput cell aggregation approach was employed to simultaneously coax mouse embryonic stem cells (ESCs) into hundreds of uniform epiblast-like (EPI) aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell (TSC) aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

developmental biology↗