Deciphering Organoids: High-Dimensional Analysis of Biomimetic Cultures

Organoids are self-organising stem cell-derived ex vivo cultures widely adopted as biomimetic models of healthy and diseased tissues. Traditional low-dimensional experimental methods such as microscopy and bulk molecular analysis have generated remarkable biological insights from organoids. However, as complex heterocellular systems, organoids are especially well-positioned to take advantage of emerging high-dimensional technologies. In particular, single-cell methods offer considerable opportunities to analyse organoids at unprecedented scale and depth, enabling comprehensive characterisation of cellular processes and spatial organisation underpinning organoid heterogeneity. This review evaluates state-of-the-art analytical methods applied to organoids, discusses the latest advances in single-cell technologies, and speculates on the integration of these two rapidly developing fields

The Heterocellular Emergence of Colorectal Cancer.

Tissues contain multiple different cell types and can be considered to be heterocellular systems. Signaling between different cells allows tissues to achieve phenotypes that no cell type can achieve in isolation. Such emergent tissue-level phenotypes can be said to 'supervene upon' heterocellular signaling. It is proposed here that cancer is also an emergent phenotype that supervenes upon heterocellular signaling. Using colorectal cancer (CRC) as an example, I review how heterotypic cells differentially communicate to support emergent malignancy. Studying tumors as integrated heterocellular systems - rather than as solitary expansions of mutated cells - may reveal novel ways to treat cancer

Cell-type-specific signaling networks in heterocellular organoids

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids

Multiplexed single-cell analysis of organoid signaling networks

Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type–specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type–specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run

Eurasian Arctic greening reveals teleconnections and the potential for novel ecosystems

Arctic warming has been linked to observed increases in tundra shrub cover and growth in recent decades on the basis of significant relationships between deciduous shrub growth/biomass and temperature. These vegetation trends have been linked to Arctic sea ice decline and thus to the sea ice/albedo feedback known as Arctic amplification. However, the interactions between climate, sea ice and tundra vegetation remain poorly understood. Here we reveal a 50- year growth response over a >100,000 km2 area to a rise in summer temperature for alder (Alnus) and willow (Salix), the most abundant shrub genera respectively at and north of the continental treeline. We demonstrate that whereas plant productivity is related to sea ice in late spring, the growing season peak responds to persistent synoptic-scale air masses over West Siberia associated with Fennoscandian weather systems through the Rossby wave train. Substrate is important for biomass accumulation, yet a strong correlation between growth and temperature encompasses all observed soil types. Vegetation is especially responsive to temperature in early summer. These results have significant implications for modelling present and future Low Arctic vegetation responses to climate change, and emphasize the potential for structurally novel ecosystems to emerge fromwithin the tundra zone.Vertaisarviointia edeltävä käsikirjoitu

Summer warming explains widespread but not uniform greening in the Arctic tundra biome

Arctic warming can influence tundra ecosystem function with consequences for climate feedbacks, wildlife and human communities. Yet ecological change across the Arctic tundra biome remains poorly quantified due to field measurement limitations and reliance on coarse-resolution satellite data. Here, we assess decadal changes in Arctic tundra greenness using time series from the 30 m resolution Landsat satellites. From 1985 to 2016 tundra greenness increased (greening) at ~37.3% of sampling sites and decreased (browning) at ~4.7% of sampling sites. Greening occurred most often at warm sampling sites with increased summer air temperature, soil temperature, and soil moisture, while browning occurred most often at cold sampling sites that cooled and dried. Tundra greenness was positively correlated with graminoid, shrub, and ecosystem productivity measured at field sites. Our results support the hypothesis that summer warming stimulated plant productivity across much, but not all, of the Arctic tundra biome during recent decades

Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion.

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-β and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue

Comparative genetic, proteomic and phosphoproteomic analysis of C. <i>elegans </i>embryos with a focus on <i>ham</i>-1/STOX and <i>pig</i>-1/MELK in dopaminergic neuron development

Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) - an open access, searchable online database