Unique Transcriptional Signatures Research Articles

BRAF Modulates Lipid Use and Accumulation.

Simple SummaryBRAF is a serine/threonine kinase that is commonly mutated across cancers. The BRAF V600E mutation is targetable with kinase inhibitors; however, many patients eventually develop resistance. Recent evidence suggests that tumors harboring BRAF mutations may oxidize fatty acids for energy rather than utilizing aerobic glycolysis (the Warburg effect). Understanding the metabolism of cells harboring BRAF mutations may uncover targets to improve therapy response. We studied the effects of BRAF mutation and expression on metabolism. We found that cell expressing BRAF V600E were enriched with immunomodulatory lipids and have a metabolism that is distinct from cells expressing wild type BRAF. We also found that patients with melanoma who did not respond to BRAF-targeted therapy had plasma lipid profiles that were different from patients who responded to this therapy. Overall, our findings indicate that targeting lipid metabolism may be a potential alternative strategy to improve patient responses to BRAF-targeted therapies.There is increasing evidence that oxidative metabolism and fatty acids play an important role in BRAF-driven tumorigenesis, yet the effect of BRAF mutation and expression on metabolism is poorly understood. We examined how BRAF mutation and expression modulates metabolite abundance. Using the non-transformed NIH3T3 cell line, we generated cells that stably overexpressed BRAF V600E or BRAF WT. We found that cells expressing BRAF V600E were enriched with immunomodulatory lipids. Further, we found a unique transcriptional signature that was exclusive to BRAF V600E expression. We also report that BRAF V600E mutation promoted accumulation of long chain polyunsaturated fatty acids (PUFAs) and rewired metabolic flux for non-Warburg behavior. This cancer promoting mutation further induced the formation of tunneling nanotube (TNT)-like protrusions in NIH3T3 cells that preferentially accumulated lipid droplets. In the plasma of melanoma patients harboring the BRAF V600E mutation, levels of lysophosphatidic acid, sphingomyelin, and long chain fatty acids were significantly increased in the cohort of patients that did not respond to BRAF inhibitor therapy. Our findings show BRAF V600 status plays an important role in regulating immunomodulatory lipid profiles and lipid trafficking, which may inform future therapy across cancers.

Distinct Adipogenic and Fibrogenic Differentiation Capacities of Mesenchymal Stromal Cells from Pancreas and White Adipose Tissue.

Pancreatic steatosis associates with β-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals.

PGC1α/β Expression Predicts Therapeutic Response to Oxidative Phosphorylation Inhibition in Ovarian Cancer.

OXPHOS inhibition in ovarian cancer can exploit the metabolic vulnerabilities conferred by high PGC1α/β expression and offers an effective approach to manage patients on the basis of PGC1α/β expression.

Diversity of satellite glia in sympathetic and sensory ganglia.

Satellite glia are the major glial type found in sympathetic and sensory ganglia in the peripheral nervous system, and specifically, contact neuronal cell bodies. Sympathetic and sensory neurons differ in morphological, molecular, and electrophysiological properties. However, the molecular diversity of the associated satellite glial cells remains unclear. Here, using single-cell RNA sequencing analysis, we identify five different populations of satellite glia from sympathetic and sensory ganglia. We define three shared populations of satellite glia enriched in immune-response genes, immediate-early genes, and ion channels/ECM-interactors, respectively. Sensory- and sympathetic-specific satellite glia are differentially enriched for modulators of lipid synthesis and metabolism. Sensory glia are also specifically enriched for genes involved in glutamate turnover. Furthermore, satellite glia and Schwann cells can be distinguished by unique transcriptional signatures. This study reveals the remarkable heterogeneity of satellite glia in the peripheral nervous system.

Uncovering the Connectivity Logic of the Ventral Tegmental Area.

Decades of research have revealed the remarkable complexity of the midbrain dopamine (DA) system, which comprises cells principally located in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Neither homogenous nor serving a singular function, the midbrain DA system is instead composed of distinct cell populations that (1) receive different sets of inputs, (2) project to separate forebrain sites, and (3) are characterized by unique transcriptional and physiological signatures. To appreciate how these differences relate to circuit function, we first need to understand the anatomical connectivity of unique DA pathways and how this connectivity relates to DA-dependent motivated behavior. We and others have provided detailed maps of the input-output relationships of several subpopulations of midbrain DA cells and explored the roles of these different cell populations in directing behavioral output. In this study, we analyze VTA inputs and outputs as a high dimensional dataset (10 outputs, 22 inputs), deploying computational techniques well-suited to finding interpretable patterns in such data. In addition to reinforcing our previous conclusion that the connectivity in the VTA is dependent on spatial organization, our analysis also uncovered a set of inputs elevated onto each projection-defined VTADA cell type. For example, VTADA→NAcLat cells receive preferential innervation from inputs in the basal ganglia, while VTADA→Amygdala cells preferentially receive inputs from populations sending a distributed input across the VTA, which happen to be regions associated with the brain’s stress circuitry. In addition, VTADA→NAcMed cells receive ventromedially biased inputs including from the preoptic area, ventral pallidum, and laterodorsal tegmentum, while VTADA→mPFC cells are defined by dominant inputs from the habenula and dorsal raphe. We also go on to show that the biased input logic to the VTADA cells can be recapitulated using projection architecture in the ventral midbrain, reinforcing our finding that most input differences identified using rabies-based (RABV) circuit mapping reflect projection archetypes within the VTA.

Transcriptome-scale spatial gene expression in rat arcuate nucleus during puberty

BackgroundA variety of neurons in hypothalamus undergo a complicated regulation on transcription activity of multiple genes for hypothalamic–pituitary–gonadal axis activation during pubertal development. Identification of puberty-associated cell composition and characterization of the unique transcriptional signatures across different cells are beneficial to isolation of specific neurons and advanced understanding of their functions.MethodsThe hypothalamus of female Sprague–Dawley rats in postnatal day-25, 35 and 45 were used to define the dynamic spatial atlas of gene expression in the arcuate nucleus (ARC) by 10× Genomics Visium platform. A surface protein expressed selectively by kisspeptin neurons was used to sort neurons by flow cytometric assay in vitro. The transcriptome of the isolated cells was examined using Smart sequencing.ResultsFour subclusters of neurons with similar gene expression signatures in ARC were identified. Only one subcluster showed the robust expression of Kiss1, which could be isolated by a unique membrane surface biomarker Solute carrier family 18 member A3 (SLC18A3). Moreover, genes in different subclusters presenting three expression modules distinctly functioned in each pubertal stage. Different types of cells representing distinct functions on glial or neuron differentiation, hormone secretion as well as estradiol response precisely affect and coordinate with each other, resulting in a complicated regulatory network for hypothalamic–pituitary–gonadal axis initiation and modulation.ConclusionOur data revealed a comprehensive transcriptomic overview of ARC within different pubertal stages, which could serve as a valuable resource for the study of puberty and sexual development disorders.

CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity.

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

Neurovascular disease: 2022 update.

In this update we present a series of papers focused on topics that have emerged in vascular disease over the prior year. The first two papers focus on the pathogenesis of vascular malformations, the first on brain arteriovenous malformations, and the second on cerebral cavernous malformations. These disorders can lead to significant brain injuries from intracerebral hemorrhage (if they rupture) or other neurological complications, including seizures. The next set of papers reflects work that has advanced our understanding of how the brain and the immune system "communicate" after brain injury, including stroke (papers 3-6). The first of these shows that T cells are involved in white matter repair after ischemic injury, an effect dependent on microglia, demonstrating the important cross-talk between innate and adaptive immunity. The next two papers focus on B cells, which have been relatively understudied in the context of brain injury. The contribution of antigen-experienced B cells from the meninges and skull bone marrow, rather than blood-derived B cells in neuroinflammation opens up a very novel area of investigation. The possibility that antibody secreting B cells may contribute to vascular dementia will certainly be an active area for future investigations. Similarly, in paper 6, investigators found that CNS-infiltrating myeloid cells can originate from brain borders tissues. These cells have unique transcriptional signatures that are distinct from their blood-derived counterparts, and likely contribute to myeloid cell infiltration from bone-marrow niches in close proximity to the brain. The contribution of microglia, the primary innate immune cell of the brain, to amyloid deposition and propagation is then discussed, followed by work on how perivascular Aβ is potentially cleared along the cerebral vessels in patients with cerebral amyloid angiopathy. The final two papers focus on the contribution of senescent endothelial cells and pericytes. The first used a model of accelerated senescence (Hutchinson-Gilford progeria syndrome; HGPS) and shows the translational potential of an approach to reduce telomere shortening to slow aging. The final paper demonstrates how capillary pericytes contribute to basal blood flow resistance and slow modulation of blood flow throughout the brain. Interestingly, several of the papers identified therapeutic strategies that could be potentially translated into clinical populations.

Transcriptomic Analysis of Mycobacterium leprae-Stimulated Response in Peripheral Blood Mononuclear Cells Reveal Potential Biomarkers for Early Diagnosis of Leprosy.

We aimed to identify an unique host transcriptional signature in peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium leprae antigens to distinguish between patients with leprosy and non-leprosy controls for early diagnosis of the disease. Sixteen individuals were enrolled in the discovery cohort [eight patients with leprosy, comprising four multibacillary (MB) and four paucibacillary (PB); and eight non-leprosy controls, comprising four healthy house contacts (HHCs) and four endemic controls (ECs)]. The differences in the transcriptome response of PBMCs to M. leprae sonicate antigen were evaluated between leprosy patients and non-leprosy controls, and 12 differentially expressed genes (CCL2/MCP-1, IL-8, JAKM, ATP, ND1, SERP, FLJ10489, LINC00659, LOC34487, LOC101928143, MIR22, and NCF1C) were identified. The accuracy of the 12 differentially expressed genes was further validated for the diagnosis of leprosy using real-time quantitative PCR in 82 individuals (13 MB, 10 PB, 37 HHCs, and 22 ECs) in the validation cohort. We found that a 5 gene signature set IL-8, CCL2/MCP-1, SERP, LINC00659 and FLJ10489 had a suitable performance in discriminating leprosy from ECs. In addition, elevated expression of IL-8, CCL2/MCP-1, SERP and LINC00659 was associated with MB diagnosis compared with ECs, whereas increased expression of IL-8, CCL2/MCP-1, SERP and FLJ10489 was found to be useful biomarkers for PB diagnosis from ECs. Moreover, we found decreased expression of NCF1C among leprosy patients could distinguish leprosy from HHCs, whereas higher expression of CCL2 among MB than PB could distinguish different leprosy patients. In conclusion, among the 12 candidate host genes identified, a three gene signature IL-8, CCL2/MCP-1, and SERP showed the best performance in distinguishing leprosy patients from healthy controls. These findings may have implications for developing a rapid blood-based test for early diagnosis of leprosy.

Evaluation of Human Bone Marrow Mesenchymal Stromal Cell (MSC) Functions on a Biomorphic Rattan-Wood-Derived Scaffold: A Comparison between Cultured and Uncultured MSCs.

The reconstruction of large bone defects requires the use of biocompatible osteoconductive scaffolds. These scaffolds are often loaded with the patient’s own bone marrow (BM) cells to facilitate osteoinductivity and biological potency. Scaffolds that are naturally sourced and fabricated through biomorphic transitions of rattan wood (B-HA scaffolds) offer a unique advantage of higher mechanical strength and bioactivity. In this study, we investigated the ability of a biomorphic B-HA scaffold (B-HA) to support the attachment, survival and gene expression profile of human uncultured BM-derived mesenchymal stromal cells (BMSCs, n = 6) and culture expanded MSCs (cMSCs, n = 7) in comparison to a sintered, porous HA scaffold (S-HA). B-HA scaffolds supported BMSC attachment (average 98%) and their survival up to 4 weeks in culture. Flow cytometry confirmed the phenotype of cMSCs on the scaffolds. Gene expression indicated clear segregation between cMSCs and BMSCs with MSC osteogenesis- and adipogenesis-related genes including RUNX2, PPARγ, ALP and FABP4 being higher expressed in BMSCs. These data indicated a unique transcriptional signature of BMSCs that was distinct from that of cMSCs regardless of the type of scaffold or time in culture. There was no statistical difference in the expression of osteogenic genes in BMSCs or cMSCs in B-HA compared to S-HA. VEGF release from cMSCs co-cultured with human endothelial cells (n = 4) on B-HA scaffolds suggested significantly higher supernatant concentration with endothelial cells on day 14. This indicated a potential mechanism for providing vasculature to the repair area when such scaffolds are used for treating large bone defects.

Transcriptional modification of host cells harboring Toxoplasma gondii bradyzoites prevents IFN gamma-mediated cell death

Toxoplasma gondii develops a latent infection in the muscle and central nervous system that acts as a reservoir for acute-stage reactivation in vulnerable patients. Little is understood about how parasites manipulate host cells during latent infection and the impact this has on survival. We show that bradyzoites impart a unique transcriptional signature on infected host cells. Many of these transcriptional changes rely on protein export and result in the suppression of type I interferon (IFN) and IFNγ signaling more so than in acute stages. Loss of the protein export component, MYR1, abrogates transcriptional remodeling and prevents suppression of IFN signaling. Among the exported proteins, the inhibitor of STAT1 transcription (IST) plays a key role in limiting IFNγ signaling in bradyzoites. Furthermore, bradyzoite protein export protects host cells from IFNγ-mediated cell death, even when export is restricted to latent stages. These findings highlight the functional importance of host manipulation in Toxoplasma's bradyzoite stages.

Single-cell analysis identifies dynamic gene expression networks that govern B cell development and transformation

Integration of external signals and B-lymphoid transcription factor activities organise B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells. We use single-cell transcriptomics/proteomics to identify differentially expressed gene networks across B cell development and correlate these networks with subtypes of B cell leukemia. Here we show unique transcriptional signatures that refine the pre-B cell expansion stages into pre-BCR-dependent and pre-BCR-independent proliferative phases. These changes correlate with reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. Our findings demonstrate the underlying heterogeneity of developing B cells and characterise developmental nodes linked to B cell transformation.

Vascular Smooth Muscle Cell Subpopulations and Neointimal Formation in Mouse Models of Elastin Insufficiency.

Using a mouse model of Eln (elastin) insufficiency that spontaneously develops neointima in the ascending aorta, we sought to understand the origin and phenotypic heterogeneity of smooth muscle cells (SMCs) contributing to intimal hyperplasia. We were also interested in exploring how vascular cells adapt to the absence of Eln. Approach and Results: We used single-cell sequencing together with lineage-specific cell labeling to identify neointimal cell populations in a noninjury, genetic model of neointimal formation. Inactivating Eln production in vascular SMCs results in rapid intimal hyperplasia around breaks in the ascending aorta's internal elastic lamina. Using lineage-specific Cre drivers to both lineage mark and inactivate Eln expression in the secondary heart field and neural crest aortic SMCs, we found that cells with a secondary heart field lineage are significant contributors to neointima formation. We also identified a small population of secondary heart field-derived SMCs underneath and adjacent to the internal elastic lamina. Within the neointima of SMC-Eln knockout mice, 2 unique SMC populations were identified that are transcriptionally different from other SMCs. While these cells had a distinct gene signature, they expressed several genes identified in other studies of neointimal lesions, suggesting that some mechanisms underlying neointima formation in Eln insufficiency are shared with adult vessel injury models. These results highlight the unique developmental origin and transcriptional signature of cells contributing to neointima in the ascending aorta. Our findings also show that the absence of Eln, or changes in elastic fiber integrity, influences the SMC biological niche in ways that lead to altered cell phenotypes.

Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation.

Endothelial tip cells guiding tissue vascularization are primary targets for angiogenic therapies. Whether tip cells require differential signals to develop their complex branching patterns remained unknown. Here, we show that diving tip cells invading the mouse neuroretina (D-tip cells) are distinct from tip cells guiding the superficial retinal vascular plexus (S-tip cells). D-tip cells have a unique transcriptional signature, including high TGF-β signaling, and they begin to acquire blood-retina barrier properties. Endothelial deletion of TGF-β receptor I (Alk5) inhibits D-tip cell identity acquisition and deep vascular plexus formation. Loss of endothelial ALK5, but not of the canonical SMAD effectors, leads to aberrant contractile pericyte differentiation and hemorrhagic vascular malformations. Oxygen-induced retinopathy vasculature exhibits S-like tip cells, and Alk5 deletion impedes retina revascularization. Our data reveal stage-specific tip cell heterogeneity as a requirement for retinal vascular development and suggest that non-canonical-TGF-β signaling could improve retinal revascularization and neural function in ischemic retinopathy.

The p53 transcriptional response across tumor types reveals core and senescence-specific signatures modulated by long noncoding RNAs

The p53 pathway is a universal tumor suppressor mechanism that limits tumor progression by triggering apoptosis or permanent cell cycle arrest, called senescence. In recent years, efforts to reactivate p53 function in cancer have proven to be a successful therapeutic strategy in murine models and have gained traction with the development of a range of small molecules targeting mutant p53. However, knowledge of the downstream mediators of p53 reactivation in different oncogenic contexts has been limited. Here, we utilized a panel of murine cancer cell lines from three distinct tumor types susceptible to alternative outcomes following p53 restoration to define unique and shared p53 transcriptional signatures. While we found that the majority of p53-bound sites and p53-responsive transcripts are tumor-type specific, analysis of shared targets identified a core signature of genes activated by p53 across all contexts. Furthermore, we identified repression of E2F and Myc target genes as a key feature of senescence. Characterization of p53-induced transcripts revealed core and senescence-specific long noncoding RNAs (lncRNAs) that are predominantly chromatin associated and whose production is coupled to cis-regulatory activities. Functional investigation of the contributions of p53-induced lncRNAs to p53-dependent outcomes highlighted Pvt1b, the p53-dependent isoform of Pvt1, as a mediator of p53-dependent senescence via Myc repression. Inhibition of Pvt1b led to decreased activation of senescence markers and increased levels of markers of proliferation. These findings shed light on the core and outcome-specific p53 restoration signatures across different oncogenic contexts and underscore the key role of the p53-Pvt1b-Myc regulatory axis in mediating proliferative arrest.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Linking Biophysical and Transcriptional Profiles of In Vivo-Treated Human Leukemias on a Single-Cell Level Uniquely Resolves Subpopulations of Response

Introduction: Assays are needed to define the susceptibility of heterogeneous primary human tumor cells. We previously showed that single-cell mass accumulation rate (MAR) - conceptually similar to growth rate - can accurately define drug sensitivity ex vivo (Stevens et al. Nature Biotechnology, 2016). This live-cell assay is performed using a suspended microchannel resonator (SMR), which maintains cell viability and permits the downstream application of single-cell whole transcriptome sequencing (scRNA-seq) to identify molecular correlates of drug response. By comparing leukemias exposed to antineoplastic therapy or vehicle in vivo , we sought to identify unique transcriptional signatures that, when coupled with single-cell biophysical properties, provide a more nuanced view of therapeutic susceptibility.Methods: We transplanted a patient-derived xenograft of BCR-ABL-rearranged, B-cell leukemia into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ recipients. This model was derived from a patient whose leukemia relapsed following combination chemotherapy and multiple tyrosine kinase inhibitors. Clinical sequencing revealed an ABL1 Y253H resistance mutation that was confirmed in the PDX. Upon engraftment, animals initiated therapy with ponatinib 40 mg/kg/day or vehicle via oral gavage. Splenocytes were harvested on treatment day 5, and immunomagnetically enriched leukemia cells were individually profiled for mass and MAR using the SMR, from which they were sequentially released directly into micro PCR tubes. Smart-Seq2 whole transcriptome amplification, library preparation, and 30-bp paired-end sequencing was then performed on these individual cells.Results: Leukemia cells treated with ponatinib in vivo had significantly lower mass and normalized MAR (MAR/mass) than vehicle-treated controls (p<2e-16 by Welch two-sample t-test, Figure 1A). Single-cell RNA-seq libraries generated from both treatment conditions were of appropriate quality and complexity, with 5,540 ± 81 and 3,826 ± 92 genes mapped per cell for vehicle- and ponatinib-treated cells, respectively. These figures are in line with our published experience (Kimmerling et al. Nature Communications , 2016). Library quality was temporally stable, with consistent average single-cell complexities maintained over the full course of collection experiments.Principal components analysis segregated cells by treatment status according to PC1 (positive enrichment for protein translation genes) and PC2 (negative enrichment for B-cell activation and B-cell receptor signaling genes) (Figure 1B). While single-cell measurements of biophysical properties or gene expression independently resolved treated and untreated cells at the population level (p<2e-16 by Welch two-sample t-test for both normalized MAR as well as PC1), layering single-cell mass and MAR on top of the transcriptomic data enabled us to identify and characterize subpopulations within treatment groups. For instance, biophysical measurements defined a subset of ponatinib-treated cells that maintained high mass and MAR despite continued therapeutic pressure. No subset of cells corresponding to this physically distinct subpopulation was identified by analysis of single-cell transcriptional profiles alone. Critically, linking biophysical and gene expression measurements from the same cells revealed gene sets with functional enrichment for cell cycle and translation - distinct from the gene sets identified from principal components analysis - that correlated with single-cell mass and mass-normalized MAR, respectively.Conclusions: Integrating the biophysical and molecular profiles of human leukemias on a single-cell level using the SMR platform coupled with scRNA-seq permits assessment of in vivo therapeutic susceptibility within hours of tissue sampling, with the potential to resolve and characterize intratumoral heterogeneity with greater precision. We are now focused on defining the clinical significance of such physical-molecular tumor subpopulations and interrogating mechanisms of therapeutic resistance illuminated by these methods in the context of phase II-like in vivo treatment studies. [Display omitted] DisclosuresStevens:Travera: Equity Ownership, Other: Co-founder. Kimmerling:Travera: Equity Ownership, Other: Co-founder. Manalis:Affinity Bio: Equity Ownership, Other: Co-founder; Travera: Consultancy, Equity Ownership, Other: Co-founder.

Dendritic cell metabolism regulates allergic sensitization in asthma

Abstract Allergic asthma is a respiratory disease characterized by airway inflammation driven by T-helper 2 (Th2) cytokines and antigen-specific IgE (Ag-specific IgE). Ag-specific IgE production requires T follicular helper 13 cells (Tfh13s), which are a population of IL-4/13+ Tfh cells. Tfh13 polarization is induced by type 2 conventional dendritic cells (cDC2s), however, the mechanisms by which cDC2s regulate Tfh13 polarization are unclear. We hypothesized that cDC2s would exhibit distinct transcriptional responses following in vivo exposure to the Tfh13 inducing stimuli, Alternaria, compared to non-Tfh13 polarizing stimuli, such as LPS or the protease papain. Thus, we treated mice intranasally with LPS, papain, or Alternaria plus fluorescently tagged OVA and sorted OVA+ migratory cDCs from the mediastinal lymph node (mLN) for RNA-seq analysis. Principal component analysis revealed that these stimuli induced unique transcriptional signatures in migratory cDCs. Gene set enrichment analysis demonstrated significant downregulation of oxidative phosphorylation (OXPHOS) genes in Alternaria stimulated cDCs compared to LPS or papain. We therefore hypothesized that Alternaria induces metabolic perturbations in cDC2s, whereby they upregulate glycolytic metabolism and decrease OXPHOS, and this metabolic profile was involved in Tfh13 polarization. To test this hypothesis, mice were either fed ad libitum or fasted for 12 hours prior to Alternaria exposure. Fasted mice exhibited a significant reduction in mLN Tfh13s and Th2 cells. Thus, we demonstrate that glucose metabolism may regulate allergic sensitization in asthma. Future experiments will aim to elucidate the role of cDC2-intrinsic metabolism in Tfh13 polarization.

Reconceptualising Resilience Within a Translational Framework is Supported by Unique and Brain-Region Specific Transcriptional Signatures in Mice

Chronic social defeat (CSD) in mice has been increasingly employed in experimental resilience research. Particularly, the degree of CSD-induced social avoidance is used to classify animals into resilient (socially non-avoidant) versus susceptible (avoidant). Inspired by human data pointing to threat-safety discrimination and responsiveness to extinction training of aversive memories as characteristics of resilient individuals, we here describe a translationally informed stratification which identified three phenotypic subgroups of mice following CSD

584 Transcriptomic analysis to identify protective dermal papilla signature in occipital scalp

Androgenetic alopecia (AGA) is defined by the miniaturization of hair follicles (HF) on the frontal (F) scalp, while occipital (O) HF are spared. Miniaturization is characterized by both thinning of the hair fiber and a decrease in cell number within the HF dermal papilla (DP). While previous research has looked at differences in HF stem cells between miniaturizing and growing HF in humans, to our knowledge no groups have looked at differences in the ex vivo DP between the F and O sites. Since the DP in FHF is affected by miniaturization, we hypothesized that a unique transcriptional signature in the ODP protects them from miniaturization. To investigate this, we carried out unbiased transcriptomic profiling of patient-matched DP and dermal sheath (DS) from FHF and OHF from 4 males undergoing hair transplantation surgery. By cross comparing, we identified 16 genes that are expressed at significantly higher levels in the ODP, compared to these 3 other dermal cell types. We believe this ODP signature protects DP from size reduction and cell loss during transition through the hair cycle, and thus protects OHF from miniaturization. Genes, previously identified as core DP genes, such as HHIP and SOSTDC1 were present in the unique ODP signature. Previous research using a murine patch assay to assess inductivity has shown that cultured FDP are significantly less inductive than cultured ODP. To interrogate the roles of ODP signature genes and evaluate their protective role against AGA, we developed an animal-free model of inductivity for high throughput analysis. When ODP cells are combined with epidermal keratinocytes (KC), cells self-organize into organoid structures with an ODP core, surrounded by KC. The KC in this model expresses a hair differentiation marker, K75 at significantly higher levels compared to organoids with a non-follicular fibroblast core, suggesting DP-induced KC reprogramming. We are now using this model to assess how ODP signature genes contribute to the KC reprogramming ability of DP organoids.