Biology subjects

Broguiere, N.

Publications and source records attributed to Broguiere, N..

6 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↗

bioRxiv2023

Bioengineered human colon organoids with in vivo-like complexity and function

Organoids and microphysiological systems, such as organs-on-a-chip, have emerged as powerful tools for modeling human gut physiology and disease in vitro. However, although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell-type diversity, and maturity necessary for mimicking adult human intestinal mucosa. To instead generate models closely resembling the in vivo cell-type composition and spatial compartmentalization, we herein integrated organoid and organ-on-a-chip technology to develop a primary human stem-cell-derived organoid model, called mini-colons. The luminal access and flow in human mini-colons removes shed cells to greatly enhance tissue longevity and differentiation over physically inaccessible human intestinal organoids that accumulate trapped cellular debris and waste. By establishing a gradient of growth factors, we replicated and sustained in vivo-like cell fate patterning and concurrent differentiation to secretory cell types and colonocytes. These long-lived human mini-colons contain abundant mucus-producing Goblet cells that lubricate the colonic epithelial lining. The stem and proliferative progenitor cells are also realistically confined to the crypts, facilitating stable homeostatic tissue turnover and preserving tissue integrity for several weeks. Also signifying mini-colon in vivo-like maturation, single-cell RNA sequencing showed emerging mature colonocytes and absorptive BEST4+ colonocytes. This methodology could be expanded to generate microtissues derived from the small intestine and incorporate additional microenvironmental components, thus emulating the intricate complexity of the native gut in an in vitro setting. Our bioengineered human organoids provide a highly accurate, long-lived, functional platform to systematically study human gut physiology and pathology, and for the development of novel therapeutic strategies.

bioengineering↗

bioRxiv2021

3D confinement regulates stem cell fate

Biophysical properties of the cellular microenvironment, including stiffness and geometry, influence cell fate. Recent findings have implicated geometric confinement as an important regulator of cell fate determination. Our understanding of how mechanical signals direct cell fate is based primarily on two-dimensional (2D) studies. To investigate the role of confinement on stem cell fate in three-dimensional (3D) culture, we fabricated a single cell microwell culture platform and used it to investigate how niche volume and stiffness affect human mesenchymal stem cell (hMSC) fate. The viability and proliferation of hMSCs in confined 3D microniches were compared with the fate of unconfined cells in 2D culture. Physical confinement biased hMSC fate, and this influence was modulated by the niche volume and stiffness. The rate of cell death increased, and proliferation markedly decreased upon 3D confinement. We correlated the observed differences in hMSC fate to YES-associated protein (YAP) localization. In 3D microniches, hMSCs displayed primarily cytoplasmic YAP localization, indicating reduced mechanical activation upon confinement. These results demonstrate that 3D geometric confinement can be an important regulator of cell fate, and that confinement sensing is linked to canonical mechanotransduction pathways.

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

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↗

bioRxiv2019

Morphogenesis guided by 3D patterning of growth factors in biological matrices

Three-dimensional (3D) control over the placement of bioactive cues is fundamental to understand cell guidance and develop engineered tissues. Two-photon patterning (2PP) provides such placement at micro- to millimeter scale, but non-specific interactions between proteins and functionalized extracellular matrices (ECMs) restrict its use. Here we report a 2PP system based on non-fouling hydrophilic photocages and Sortase A-based enzymatic coupling offering unprecedented orthogonality and signal-to-noise ratio in both inert hydrogels and complex mammalian matrices. Improved photocaged peptide synthesis, and protein functionalization protocols with broad applicability are introduced. Importantly, the method enables 2PP in a single step and in the presence of fragile biomolecules and cells. As a corollary, we demonstrate the guidance of axons through 3D-patterned nerve growth factor (NGF) within brain-mimetic ECMs. Our approach allows for the interrogation of the role of complex signaling molecules in 3D matrices, thus helping to better understand biological guidance in tissue development and regeneration.

bioengineering↗