RESUMO
Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Assuntos
Imageamento Tridimensional , Transcriptoma , Animais , Camundongos , Humanos , Transcriptoma/genética , Imageamento Tridimensional/métodos , Software , Perfilação da Expressão Gênica/métodos , Linfonodos/patologia , Linfonodos/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Metástase Linfática , FemininoRESUMO
Little is known about the contribution of 3D surface geometry to the development of multilayered tissues containing fibrous extracellular matrix components, such as those found in bone. In this study, we elucidate the role of curvature in the formation of chiral, twisted-plywood-like structures. Tissues consisting of murine preosteoblast cells (MC3T3-E1) were grown on 3D scaffolds with constant-mean curvature and negative Gaussian curvature for up to 32 days. Using 3D fluorescence microscopy, the influence of surface curvature on actin stress-fiber alignment and chirality was investigated. To gain mechanistic insights, we did experiments with MC3T3-E1 cells deficient in nuclear A-type lamins or treated with drugs targeting cytoskeleton proteins. We find that wild-type cells form a thick tissue with fibers predominantly aligned along directions of negative curvature, but exhibiting a twist in orientation with respect to older tissues. Fiber orientation is conserved below the tissue surface, thus creating a twisted-plywood-like material. We further show that this alignment pattern strongly depends on the structural components of the cells (A-type lamins, actin, and myosin), showing a role of mechanosensing on tissue organization. Our data indicate the importance of substrate curvature in the formation of 3D tissues and provide insights into the emergence of chirality.
RESUMO
Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.
Assuntos
Fenômenos Mecânicos , Membrana Celular , MorfogêneseRESUMO
We have studied the collective motion of polar active particles confined to ellipsoidal surfaces. The geometric constraints lead to the formation of vortices that encircle surface points of constant curvature (umbilics). We have found that collective motion patterns are particularly rich on ellipsoids with four umbilics where vortices tend to be located near pairs of umbilical points to minimize their interaction energy. Our results provide a perspective on the migration of living cells, which most likely use the information provided from the curved substrate geometry to guide their collective motion.
RESUMO
The complex arrangement of the extracellular matrix (ECM) produced by cells during tissue growth, healing and remodelling is fundamental to tissue function. In connective tissues, it is still unclear how both cells and the ECM become and remain organized over length scales much larger than the distance between neighbouring cells. While cytoskeletal forces are essential for assembly and organization of the early ECM, how these processes lead to a highly organized ECM in tissues such as osteoid is not clear. To clarify the role of cellular tension for the development of these ordered fibril architectures, we used an in vitro model system, where pre-osteoblastic cells produced ECM-rich tissue inside channels with millimetre-sized triangular cross sections in ceramic scaffolds. Our results suggest a mechanical handshake between actively contracting cells and ECM fibrils: the build-up of a long-range organization of cells and the ECM enables a gradual conversion of cell-generated tension to pre-straining the ECM fibrils, which reduces the work cells have to generate to keep mature tissue under tension.