Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition ...and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2
macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer.
Full text
Available for:
GEOZS, IJS, IMTLJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Recent spatial gene expression technologies enable comprehensive measurement of transcriptomic profiles while retaining spatial context. However, existing analysis methods do not address the limited ...resolution of the technology or use the spatial information efficiently. Here, we introduce BayesSpace, a fully Bayesian statistical method that uses the information from spatial neighborhoods for resolution enhancement of spatial transcriptomic data and for clustering analysis. We benchmark BayesSpace against current methods for spatial and non-spatial clustering and show that it improves identification of distinct intra-tissue transcriptional profiles from samples of the brain, melanoma, invasive ductal carcinoma and ovarian adenocarcinoma. Using immunohistochemistry and an in silico dataset constructed from scRNA-seq data, we show that BayesSpace resolves tissue structure that is not detectable at the original resolution and identifies transcriptional heterogeneity inaccessible to histological analysis. Our results illustrate BayesSpace's utility in facilitating the discovery of biological insights from spatial transcriptomic datasets.
Full text
Available for:
GEOZS, IJS, IMTLJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Ciliopathies are a class of inherited pleiotropic genetic disorders in which alterations in cilia assembly, maintenance, and/or function exhibit penetrance in the multiple organ systems. Olfactory ...dysfunction is one such clinical manifestation that has been shown in both patients and model organisms. Existing therapies for ciliopathies are limited to the treatment or management of symptoms. The last decade has seen an increase in potential curative therapeutic options including small molecules and biologics. Recent work in multiciliated olfactory sensory neurons has demonstrated the capacity of targeted gene therapy to restore ciliation in terminally differentiated cells and rescue olfactory function. This review will discuss the current understanding of the penetrance of ciliopathies in the olfactory system. Importantly, it will highlight both pharmacological and biological approaches, and their potential therapeutic value in the olfactory system and other ciliated tissues.
We undertook a structured and comprehensive search of peer-reviewed research literature encompassing in vitro, in vivo, model organism, and clinical studies. From these publications, we describe the olfactory system, and discuss the penetrance of ciliopathies and impact of cilia loss on olfactory function. In addition, we outlined the developing therapies for ciliopathies across different organ and cell culture systems, and discussed their potential therapeutic application to the mammalian olfactory system.
One-hundred sixty-one manuscripts were included in the review, centering on the understanding of olfactory penetrance of ciliopathies, and discussing the potential therapeutic options for ciliopathies in the context of the mammalian olfactory system. Forty-four manuscripts were used to generate a table listing the known congenital causes of olfactory dysfunction, with the first ten listed are linked to ciliopathies. Twenty-three manuscripts were used to outline the potential of small molecules for the olfactory system. Emphasis was placed on HDAC6 inhibitors and lithium, both of which were shown to stabilize microtubule structures, contributing to ciliogenesis and cilia lengthening. Seventy-five manuscripts were used to describe gene therapy and gene therapeutic strategies. Included were the implementation of adenoviral, adeno-associated virus (AAV), and lentiviral vectors to treat ciliopathies across different organ systems and application toward the olfactory system. Thus far, adenoviral and AAVmeditated ciliary restoration demonstrated successful proof-of-principle preclinical studies. In addition, gene editing, ex vivo gene therapy, and transplantation could serve as alternative therapeutic and long-term approaches. But for all approaches, additional assessment of vector immunogenicity, specificity, and efficacy need further investigation. Currently, ciliopathy treatments are limited to symptomatic management with no curative options. However, the accessibility and amenability of the olfactory system to treatment would facilitate development and advancement of a viable therapy.
The findings of this review highlight the contribution of ciliopathies to a growing list of congenial olfactory dysfunctions. Promising results from other organ systems imply the feasibility of biologics, with results from gene therapies proving to be a viable therapeutic option for ciliopathies and olfactory dysfunction.
Cilia of olfactory sensory neurons (OSNs) are the primary site of odor binding; hence, their loss results in anosmia, a clinical manifestation of pleiotropic ciliopathies for which there are no ...curative therapies. We used OSN-specific Ift88 knock-out mice (
) of both sexes to examine the mechanisms of ciliopathy-induced olfactory dysfunction and the potential for gene replacement to rescue odorant detection, restore olfactory circuitry, and restore odor-guided behaviors. Loss of OSN cilia in
mice resulted in substantially reduced odor detection and odor-driven synaptic activity in the olfactory bulb (OB). Defects in OSN axon targeting to the OB were also observed in parallel with aberrant odor-guided behavior. Intranasal gene delivery of wild-type IFT88 to
mice rescued OSN ciliation and peripheral olfactory function. Importantly, this recovery of sensory input in a limited number of mature OSNs was sufficient to restore axonal targeting in the OB of juvenile mice, and with delayed onset in adult mice. In addition, restoration of sensory input re-established course odor-guided behaviors. These findings highlight the spare capacity of the olfactory epithelium and the plasticity of primary synaptic input into the central olfactory system. The restoration of peripheral and central neuronal function supports the potential for treatment of ciliopathy-related anosmia using gene therapy.
Ciliopathies, for which there are no curative therapies, are genetic disorders that alter cilia morphology and/or function in numerous tissue types, including the olfactory system, leading to sensory dysfunction. We show that
intranasal gene delivery restores peripheral olfactory function in a ciliopathy mouse model, including axonal targeting in the juvenile and adult olfactory bulb. Gene therapy also demonstrated restoration of olfactory perception by rescuing odor-guided behaviors. Understanding the therapeutic window and viability for gene therapy to restore odor detection and perception may facilitate translation of therapies to ciliopathy patients with olfactory dysfunctions.
The mammalian main olfactory system contains several subsystems that differ not only in the receptors they express and the glomerular targets they innervate within the main olfactory bulb (MOB), but ...also in the strategies they use to process odor information. The canonical main olfactory system employs a combinatorial coding strategy that represents odorant identity as a pattern of glomerular activity. By contrast, the "GC-D/necklace" olfactory subsystem-formed by olfactory sensory neurons expressing the receptor guanylyl cyclase GC-D and their target necklace glomeruli (NGs) encircling the caudal MOB-is critical for the detection of a small number of semiochemicals that promote the acquisition of food preferences. The formation of these socially-transmitted food preferences requires the animal to integrate information about two types of olfactory stimuli: these specialized social chemosignals and the food odors themselves. However, the neural mechanisms with which the GC-D/necklace subsystem processes this information are unclear. We used stimulus-induced increases in intrinsic fluorescence signals to map functional circuitry associated with NGs and canonical glomeruli (CGs) in the MOB. As expected, CG-associated activity spread laterally through both the glomerular and external plexiform layers associated with activated glomeruli. Activation of CGs or NGs resulted in activity spread between the two types of glomeruli; there was no evidence of preferential connectivity between individual necklace glomeruli. These results support previous anatomical findings that suggest the canonical and GC-D/necklace subsystems are functionally connected and may integrate general odor and semiochemical information in the MOB.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and ...spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-β signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-β signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-β signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.
Bardet–Biedl syndrome (BBS) is a hereditary genetic disorder that results in numerous clinical manifestations including olfactory dysfunction. Of at least 21 BBS‐related genes that can carry multiple ...mutations, a pathogenic mutation, BBS1M390R, is the single most common mutation of clinically diagnosed BBS outcomes. While the deletion of BBS‐related genes in mice can cause variable penetrance in different organ systems, the impact of the Bbs1M390R mutation in the olfactory system remains unclear. Using a clinically relevant knock‐in mouse model homozygous for Bbs1M390R, we investigated the impact of the mutation on the olfactory system and tested the potential of viral‐mediated, wildtype gene replacement therapy to rescue smell loss. The cilia of olfactory sensory neurons (OSNs) in Bbs1M390R/M390R mice were significantly shorter and fewer than those of wild‐type mice. Also, both peripheral cellular odor detection and synaptic‐dependent activity in the olfactory bulb were significantly decreased in the mutant mice. Furthermore, to gain insight into the degree to which perceptual features are impaired in the mutant mice, we used whole‐body plethysmography to quantitatively measure odor‐evoked sniffing. The Bbs1M390R/M390R mice showed significantly higher odor detection thresholds (reduced odor sensitivity) compared to wild‐type mice; however, their odor discrimination acuity was still well maintained. Importantly, adenoviral expression of Bbs1 in OSNs restored cilia length and re‐established both peripheral odorant detection and odor perception. Together, our findings further expand our understanding for the development of gene therapeutic treatment for congenital ciliopathies in the olfactory system.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Intranasal delivery of solutions is a straightforward methodology for viral vector transduction and gene transfer to the epithelia within the nasal cavity. Beyond the simplicity of the technique, ...intranasal delivery has demonstrated restricted transduction of the olfactory and respiratory epithelial tissues. Here we outline the procedure of viral vector intranasal delivery in early postnatal and adult mice, as well as adult rats. The procedure allows for robust transduction and ectopic gene delivery that can be used for the visualization of cellular structures, protein distribution, and assessment of viral vector-mediated therapies.
Neurons exhibit strong coupling of electrochemical and metabolic activity. Increases in intrinsic fluorescence from either oxidized flavoproteins or reduced nicotinamide adenine dinucleotide ...(phosphate) NAD(P)H in the mitochondria have been used as an indicator of neuronal activity for the functional mapping of neural circuits. However, this technique has not been used to investigate the flow of olfactory information within the circuitry of the main olfactory bulb (MOB). We found that intrinsic flavoprotein fluorescence signals induced by electrical stimulation of single glomeruli displayed biphasic responses within both the glomerular (GL) and external plexiform layers (EPL) of the MOB. Pharmacological blockers of mitochondrial activity, voltage-gated Na+ channels, or ionotropic glutamate receptors abolished stimulus-dependent flavoprotein responses. Blockade of GABAA receptors enhanced the amplitude and spatiotemporal spread of the flavoprotein signals, indicating an important role for inhibitory neurotransmission in shaping the spread of neural activity in the MOB. Stimulus-dependent spread of fluorescence across the GL and EPL displayed a spatial distribution consistent with that of individual glomerular microcircuits mapped by neuroanatomic tract tracing. These findings demonstrated the feasibility of intrinsic fluorescence imaging in the olfactory systems and provided a new tool to examine the functional circuitry of the MOB.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain ...unclear. Here, we report that
mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within
OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.
PDF
