Project description:Pancreatic ductal adenocarcinoma (PDAC) remains resistant to most treatments and demonstrates a complex pathobiology. Here, we deconvolute regional heterogeneity in the human PDAC tumor microenvironment (TME), a long-standing obstacle, to define precise stromal contributions to PDAC progression. Large scale integration of histology-guided multiOMICs profiling with clinical data sets and functional in vitro models uncovered two microenvironmental programs in PDAC that were anchored in fibroblast differentiation states. These sub-tumor microenvironments (subTMEs) co-occurred intratumorally and were spatially confined, producing patient-specific cellular and molecular heterogeneity associated with shortened patient survival. Each subTME was uniquely structured to support discrete aspects of tumor biology: reactive regions rich in activated fibroblast communities were immune-hot and promoted aggressive tumor progression while deserted regions enriched in extracellular matrix supported tumor differentiation yet were markedly chemoprotective. In conclusion, PDAC regional heterogeneity derives from biologically distinct reactive and protective TME elements with a defined, active role in PDAC progression.
Project description:OCT-embedded PDAC tissues were assessed for stromal and tumour epithelial regions which were both laser-capture microdissected from 33 patients. Integration of these proteomic profiles with transcriptomic data lead to the identification of two spatially confined tumour microenvironment programs: deserted and reactive.
Project description:We aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture. Three different culture conditions of EB culture were analyzed: suspension (standard condition), confinement in microwells of width/depth ratio 1:1 and 1:2. EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture.
Project description:Our work offers a novel concept of spatially confined microbiota, advancing sustainable biomanufacturing and function-robust microbiota design.
Project description:We aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture.
Project description:The spatial organization of cells within tissues is tightly linked to their biological function. Yet, methods to probe the entire transcriptome of multiple native tissue microenvironments at single cell resolution are lacking. Here, we introduce fragment-sequencing, a method that enables the transcriptomic characterization of single cells within spatially distinct tissue niches. Fragment-sequencing of the mouse metastatic liver revealed previously uncharacterized zonated genes and ligand-receptor interactions enriched in the different hepatic microenvironments and the metastatic niche.
Project description:Chronic allodynia stemming from peripheral stump neuromas can persist for extended periods, significantly compromising patients’ quality of life. Conventional managements for nerve stumps have demonstrated limited effectiveness in ensuring their orderly termination. In this study, we present a spatially confined conduit strategy, designed to enhance the self-organization of regenerating nerves after truncation. This innovative approach elegantly enables the autonomous slowing of axonal outgrowth in response to the gradually constricting space, concurrently suppressing neuroinflammation through YAP-mediated mechanotransduction activation. Meanwhile, the decelerating axons exhibit excellent alignment and remyelination, thereby helping to prevent failure modes in nerve self-organization, such as axonal twisting in congested regions and overgrowth beyond the conduit's capacity.
Project description:Molecular mechanisms underlying dorsolateral prefrontal cortex (dlPFC) dysfunction in schizophrenia (SCZ) are poorly understood. dlPFC cell types are spatially organized across the six layers into functional microcircuits, which regulate cognitive and emotional processes that are implicated in SCZ. While regional specificity across cortical layers and cell types has been demonstrated for some SCZ-linked genes, spatially-resolved transcriptomics (SRT) can more definitively map molecular associations of disease. We investigated spatial gene expression changes in the human dlPFC from neurotypical control (n=31) and SCZ (n=32) brain donors using the Visium platform with incorporation of immunostaining to label perineuronal nets, neurons and vasculature. SCZ-associated DEGs were then mapped across these labeled cellular microenvironments and cortical layers. Major transcriptional alterations were identified in synaptic and neuroimmune pathways, which we localized to neuropil and glia-enriched domains. Integrative analysis with bulk and single-cell RNA studies highlighted distinct roles for spatially-localized glial cell populations, and identified enrichment of novel DEGs in endothelial cells and microglia. These findings were supported by enrichment of SCZ genetic risk across similar domains, and association of laminae-specific transcription factors to SCZ risk variants. Cellular resolution SRT using Xenium extended laminar disease associations to spatially-organized cell types. The findings highlight unique advantages of SRT to identify novel SCZ-related biology, particularly for cellular populations and compartments that may be missed with alternative technologies. We provide data resources to enable sample-level visualization of the spatial transcriptomics data (Samui) and to explore SCZ-associated DEGs across spatial domains (iSEE-layer adjusted) and cellular microenvironments (iSEE-Neuropil, iSEE-Neuron, iSEE-vasculature, iSEE-PNNs).