Mouse Cell Types Research Articles

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Distinct gene programs underpinning disease tolerance and resistance in influenza virus infection.

When challenged with an invading pathogen, the host-defense response is engaged to eliminate the pathogen (resistance) and to maintain health in the presence of the pathogen (disease tolerance). However, the identification of distinct molecular programs underpinning disease tolerance and resistance remained obscure. We exploited transcriptional and physiological monitoring across 33 mouse strains, during invivo influenza virus infection, to identify two host-defense gene programs-one is associated with hallmarks of disease tolerance and the other with hallmarks of resistance. Both programs constitute generic responses in multiple mouse and human cell types. Our study describes the organizational principles of these programs and validates Arhgdia as a regulator of disease-tolerance states in epithelial cells. We further reveal that the baseline disease-tolerance state in peritoneal macrophages is associated with the pathophysiological response to injury and infection. Our framework provides a paradigm for the understanding of disease tolerance and resistance at the molecular level.

Whole-embryonic identification of maternal microchimeric cell types in mouse using single-cell RNA sequencing

Even though the mother and the fetus of placental mammals are immunologically non-self with respect to one other, mutual exchange of small numbers of cells between them is known to occur. Maternal cells entering the fetus, called maternal microchimeric cells (MMc cells), are thought to be involved in different physiological phenomena, such as establishing immune tolerance, tissue repair, and the pathogenesis or deterioration of some inflammatory diseases and congenital malformations. While specific MMc cell types have been reported as associated with these phenomena, the contribution of MMc cells to these different outcomes remains unknown. As one possibility, we hypothesized that different embryos have differing repertoires of MMc cell types, leading to or biasing embryos toward different fates. To date, no studies have succeeded in identifying the MMc cell type repertoire of a single embryo. Accordingly, here, we isolated MMc cells from whole mouse embryos, determined their types, and analyzed their MMc cell type variability. By combining our previously established, whole-embryonic MMc isolation method with single-cell RNA sequencing, we successfully estimated the cell type repertoires of MMc cells isolated from 26 mouse embryos. The majority of MMc cells were immune-related cells, such as myeloid cells and granulocytes. We also detected stem cell-like MMc cells expressing proliferation marker genes and terminally differentiated cells. As hypothesized, we noted statistically significant inter-individual variation in the proportion of immune-related cells in the different embryos. We here successfully estimated MMc cell types in individual whole mouse embryos. The proportion of immune-related cells significantly differed among the individual embryos, suggesting that the variations are one of the potential mechanisms underlying the differing MMc-related physiological phenomena in offspring. These findings provide insight into cell-level epigenetics by maternal cells.

51. Discerning cell types and states in spatial transcriptomics data using topic modelling

Glioblastoma multiforme (GBM) is the most common adult brain tumour. GBM cells include a diverse collection of normal and tumour cells with distinct molecular capabilities and with differential levels of sensitivity to treatment that led to treatment resistance. Spatial transcriptomics technologies (e.g. 10X Visium) have enabled analyzing tumour-normal cells interactions in a physical tissue context. Using Visium, a well-annotated cohort of adult GBM patient samples grown as patient-derived xenograft (PDX) models were profiled at different time points of the disease. However, the identification of cell types or states of Visium spots is a challenging problem as each spot contains a mixture of cells. Current tools for cell-type deconvolution are limited in discovering new novel cell types because they require a well-annotated reference dataset. Moreover, the PDX data has a mixture of human and mouse cell types at the same spot. In this work, we have applied topic modelling, a well-known probabilistic approach in natural language processing to discover the distribution of topics in language documents. Using the analogy between documents and spatial transcriptomic data, we were able to identify the main cell types in spatial PDX data without gene expression references dataset of tumour states, normal mouse, and immune cell types. Finally, we compared various probabilistic topic modelling algorithms to fit the PDX data, and to understand the cell-to-cell interactions between tumour and normal cells.

ODP443 Targeting NAD+ availability modulates 11β- Hydroxysteroid dehydrogenase 1 activity in mouse and human cell lines and primary cells

Abstract Four nicotinamide adenine dinucleotide coenzymes – NAD+, NADH, NADP+, and NADPH – are the central catalysts of metabolism. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is NADPH-dependent reductase that catalyses the reduction of the inactive glucocorticoid cortisone) to active cortisol (11-dehydrocorticosterone to corticosterone in rodents). 11β-HSD1 is in the endoplasmic reticulum (ER) lumen, and the level of NADPH in the ER regulates enzyme activity. Little is known about the biochemical mechanisms controlling cytosolic/ER NAD(P)(H) crosstalk. Here we examined how perturbed cellular NAD+ availability modulates 11β-HSD1 enzyme activity in the ER across a range of mouse and human cell types. 11β-HSD1 activity was measured in a series of transformed and primary mouse and human liver (HepG2), muscle, and Human Dermal Fibroblast cells (HDFn) depleted for NAD+ using FK866 to inhibit nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. To replete NAD+ we supplemented cells with 0.5mM of NAD+ precursors nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). FK866 (100nM, 48h) was effective at depleting NAD+ by approximately 90% in C2C12 myotubes and 50% in HepG2 liver cells. In each cell line, 11β-HSD1 reductase activity was decreased by FK866 compared to untreated controls (80% in C2C12 P<0. 0001, 55% in HepG2, P=0. 03 and 65% in HDFn P=0. 002). Restoring NAD+ level with NR (0.5mM, 6h) in C2C12 and (0.5mM, 24h) in HepG2 and HDFn rescued 11β-HSD1 enzyme activity compared with no precursor treatment (60% in C2C12 P=0. 0007, 52% in HepG2 P=0. 03 and 68% in HDFn P=0. 0006). Human dermal fibroblasts treated with TNFa (10nM, 24h) boosted 11β-HSD1 expression 4-fold, however this was not able to overcome the FK866 induced drop-in reductase activity. These data show that depletion of NAD+ and impaired 11β-HSD1 reductase activity in the ER is a generalised phenomenon across cell types. This suggests a crosstalk between cytosolic and ER NAD(P)(H) pools that can respond rapidly to changes in NAD+ precursor availability. This could provide new insight into the regulation of cellular glucocorticoid metabolism and ER NAD(P)(H) homeostasis. Presentation: No date and time listed

Complete deletion of Ectromelia virus p28 impairs virus genome replication in a mouse strain, cell type, and multiplicity of infection-dependent manner

p28 is a poxvirus-encoded E3 ubiquitin ligase that possesses an N-terminal KilA-N domain and a C-terminal RING domain. In Ectromelia virus (ECTV), disruption of the p28 RING domain severely attenuated virulence in A strain mice, which normally succumb to ECTV infection. Moreover, this mutant virus exhibited dramatically reduced genome replication and impaired factory formation in A strain mice peritoneal macrophages (PMs) infected at high multiplicity of infection (MOI) These defects were not observed in PMs isolated from C57BL/6 mice which survive ECTV infection, demonstrating that p28 functions in a context-specific manner. To further investigate p28 function, we completely deleted the p28 gene from ECTV (ECTV-Δp28). In contrast to previous findings, we found that the ECTV-Δp28 virus exhibited severely compromised virus production and genome replication in PMs isolated from A strain mice only when infected at low MOI. This defect was minimal in bone marrow-derived macrophages and two cell lines derived from A strain mice. Furthermore, this low MOI defect in virus production was also observed in PMs isolated from the susceptible BALB/c mouse strain, but not PMs isolated from C57BL/6 mice. Taken together, our data demonstrate that the requirement for ECTV p28 to establish a productive infection depends on the MOI, the cell type, as well as the mouse strain.

WhichTF is functionally important in your open chromatin data?

We present WhichTF, a computational method to identify functionally important transcription factors (TFs) from chromatin accessibility measurements. To rank TFs, WhichTF applies an ontology-guided functional approach to compute novel enrichment by integrating accessibility measurements, high-confidence pre-computed conservation-aware TF binding sites, and putative gene-regulatory models. Comparison with prior sheer abundance-based methods reveals the unique ability of WhichTF to identify context-specific TFs with functional relevance, including NF-κB family members in lymphocytes and GATA factors in cardiac cells. To distinguish the transcriptional regulatory landscape in closely related samples, we apply differential analysis and demonstrate its utility in lymphocyte, mesoderm developmental, and disease cells. We find suggestive, under-characterized TFs, such as RUNX3 in mesoderm development and GLI1 in systemic lupus erythematosus. We also find TFs known for stress response, suggesting routine experimental caveats that warrant careful consideration. WhichTF yields biological insight into known and novel molecular mechanisms of TF-mediated transcriptional regulation in diverse contexts, including human and mouse cell types, cell fate trajectories, and disease-associated cells.

Eomes function is conserved between zebrafish and mouse and controls left-right organiser progenitor gene expression via interlocking feedforward loops.

The T-box family transcription factor Eomesodermin (Eomes) is present in all vertebrates, with many key roles in the developing mammalian embryo and immune system. Homozygous Eomes mutant mouse embryos exhibit early lethality due to defects in both the embryonic mesendoderm and the extraembryonic trophoblast cell lineage. In contrast, zebrafish lacking the predominant Eomes homologue A (Eomesa) do not suffer complete lethality and can be maintained. This suggests fundamental differences in either the molecular function of Eomes orthologues or the molecular configuration of processes in which they participate. To explore these hypotheses we initially analysed the expression of distinct Eomes isoforms in various mouse cell types. Next we compared the functional capabilities of these murine isoforms to zebrafish Eomesa. These experiments provided no evidence for functional divergence. Next we examined the functions of zebrafish Eomesa and other T-box family members expressed in early development, as well as its paralogue Eomesb. Though Eomes is a member of the Tbr1 subfamily we found evidence for functional redundancy with the Tbx6 subfamily member Tbx16, known to be absent from eutherians. However, Tbx16 does not appear to synergise with Eomesa cofactors Mixl1 and Gata5. Finally, we analysed the ability of Eomesa and other T-box factors to induce zebrafish left-right organiser progenitors (known as dorsal forerunner cells) known to be positively regulated by vgll4l, a gene we had previously shown to be repressed by Eomesa. Here we demonstrate that Eomesa indirectly upregulates vgll4l expression via interlocking feedforward loops, suggesting a role in establishment of left-right asymmetry. Conversely, other T-box factors could not similarly induce left-right organiser progenitors. Overall these findings demonstrate conservation of Eomes molecular function and participation in similar processes, but differential requirements across evolution due to additional co-expressed T-box factors in teleosts, albeit with markedly different molecular capabilities. Our analyses also provide insights into the role of Eomesa in left-right organiser formation in zebrafish.

BSCI-14 THE ROLE OF ICAM1 IN GLIOBLASTOMA TUMOUR ASSOCIATED MACROPHAGES UNDER HYPOXIC CONDITIONS

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive and highly fatal brain cancer in adults. Existing treatment methods are ineffective and we are in need of new treatments that extend the overall survival and improve quality-of-life. Cell adhesion molecules (CAMs) are proteins that enable cells to communicate with one another and the surrounding environment. Intracellular adhesion molecule 1 (ICAM1) is a CAM expressed by TAMs in GBM. Tumour associated macrophages (TAMs) are thought to enhance tumour growth and proliferation, particularly within the characteristic hypoxic tumour microenvironment (TME) of GBM. I hypothesize that the expression of ICAM1 on the surface of TAMs contributes to GBM cell invasiveness, especially in the hypoxic TME, by enhancing the interaction between tumour cells and macrophages, thereby facilitating the migration and invasion of the tumour cells. METHODOLOGY Assess the expression levels of ICAM1 in primary and immortalized human and mouse macrophages under hypoxic conditions. Analyze the effect of ICAM1 deficiency on macrophage behaviour including migration, proliferation, and adhesion to tumour cells. Intracranially inject GL261 glioma cells in ICAM1 deficient and wild type mice. RESULTS ICAM1 is highly expressed in different cell types within the GBM microenvironment, including TAMs. The expression is particularly enhanced when primary or immortalized macrophages are treated with tumour cell-conditioned medium and is further exacerbated upon incubation of these cells in hypoxic conditions. The migration levels of bone marrow derived macrophage mouse cell type is higher in wild type cells than in ICAM1 deficient cells and higher when co-cultured with tumour cell condition media. ICAM1 deficient mice succumbed to GBM more quickly compared with wild type. CONCLUSIONS It is evident that the hypoxic tumour microenvironment increases the expression of ICAM1 in macrophages. The tumour microenvironment increases migration levels of macrophages. The expression of ICAM1 in TAMs in hypoxic TME promotes GBM cell invasiveness, proliferation, aggressiveness and migration.

Systematic identification of cell-fate regulatory programs using a single-cell atlas of mouse development.

Waddington's epigenetic landscape is a metaphor frequently used to illustrate cell differentiation. Recent advances in single-cell genomics are altering our understanding of the Waddington landscape, yet the molecular mechanisms of cell-fate decisions remain poorly understood. We constructed a cell landscape of mouse lineage differentiation during development at the single-cell level and described both lineage-common and lineage-specific regulatory programs during cell-type maturation. We also found lineage-common regulatory programs that are broadly active during the development of invertebrates and vertebrates. In particular, we identified Xbp1 as an evolutionarily conserved regulator of cell-fate determinations across different species. We demonstrated that Xbp1 transcriptional regulation is important for the stabilization of the gene-regulatory networks for a wide range of mouse cell types. Our results offer genetic and molecular insights into cellular gene-regulatory programs and will serve as a basis for further advancing the understanding of cell-fate decisions.

An intein-split transactivator for intersectional neural imaging and optogenetic manipulation

The cell-type-specific recording and manipulation is instrumental to disentangle causal neural mechanisms in physiology and behavior and increasingly requires intersectional control; however, current approaches are largely limited by the number of intersectional features, incompatibility of common effectors and insufficient gene expression. Here, we utilized the protein-splicing technique mediated by intervening sequences (intein) and devised an intein-based intersectional synthesis of transactivator (IBIST) to selectively control gene expression of common effectors in multiple-feature defined cell types in mice. We validated the specificity and sufficiency of IBIST to control fluorophores, optogenetic opsins and Ca2+ indicators in various intersectional conditions. The IBIST-based Ca2+ imaging showed that the IBIST can intersect five features and that hippocampal neurons tune differently to distinct emotional stimuli depending on the pattern of projection targets. Collectively, the IBIST multiplexes the capability to intersect cell-type features and controls common effectors to effectively regulate gene expression, monitor and manipulate neural activities.

Characterization of 2,2',4,4'-tetrabromodiphenyl ether (BDE47)-induced testicular toxicity via single-cell RNA-sequencing.

BackgroundThe growing male reproductive diseases have been linked to higher exposure to certain environmental compounds such as 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) that are widely distributed in the food chain. However, the specific underlying molecular mechanisms for BDE47-induced male reproductive toxicity are not completely understood.MethodsHere, for the first time, advanced single-cell RNA sequencing (ScRNA-seq) was employed to dissect BDE47-induced prepubertal testicular toxicity in mice from a pool of 76 859 cells.ResultsOur ScRNA-seq results revealed shared and heterogeneous information of differentially expressed genes, signaling pathways, transcription factors, and ligands-receptors in major testicular cell types in mice upon BDE47 treatment. Apart from disruption of hormone homeostasis, BDE47 was discovered to downregulate multiple previously unappreciated pathways such as double-strand break repair and cytokinesis pathways, indicative of their potential roles involved in BDE47-induced testicular injury. Interestingly, transcription factors analysis of ScRNA-seq results revealed that Kdm5b (lysine-specific demethylase 5B), a key transcription factor required for spermatogenesis, was downregulated in all germ cells as well as in Sertoli and telocyte cells in BDE47-treated testes of mice, suggesting its contribution to BDE47-induced impairment of spermatogenesis.ConclusionsOverall, for the first time, we established the molecular cell atlas of mice testes to define BDE47-induced prepubertal testicular toxicity using the ScRNA-seq approach, providing novel insight into our understanding of the underlying mechanisms and pathways involved in BDE47-associated testicular injury at a single-cell resolution. Our results can serve as an important resource to further dissect the potential roles of BDE47, and other relevant endocrine-disrupting chemicals, in inducing male reproductive toxicity.

Abstract 6397: Understanding the glioblastoma microenvironment with spatial resolution in PDX models

Abstract Glioblastoma multiforme (GBM) is the most common adult brain tumour, and despite aggressive treatment, it recurs fatally. GBM tumours include diverse populations of malignant and non-neoplastic cells with distinct molecular capabilities and with differential levels of sensitivity to treatment. Understanding the cell dynamics that occur during the development of GBM resistance to therapy could reveal key aspects of this process, including how resistance is acquired in time and how the diverse cell types of the tumour microenvironment (TME) contribute to this phenotype. In addition to the role of the TME, GBM exhibits significant tumour heterogeneity with diverse genetic clones coexisting in the same tumor, as well as cells with similar genetic backgrounds capable of adopting distinct transcriptional states and subtypes. The complex and dynamic interactions between tumor and TME remain to be fully studied. This work focuses on the in vivo spatial organization in GBM during disease progression. We generated spatial transcriptomic data from a set of adult GBM samples grown as patient-derived xenograft (PDX) models, profiled at different time points of the disease. Three PDX lines from one GBM patient (derived from tumor core, vascularized area, and infiltrating front) were used to recapitulate the genetic and phenotypic heterogeneity observed in the human disease. Two replicates from each of 8 PDX mice were collected from early, mid, and late time points of tumour growth and data was generated using the 10X Genomics Visium platform. We developed a robust computational pipeline capable of distinguishing admixture of human (tumour) and mouse cells (TME), using state-of-the-art tools. Human and mouse cell types and states were identified using pooled and separate single-cell references of human GBM states, and mouse brain cells from both normal and tumour conditions. With this approach we observe spatially distinct patterns of both (a) tumour infiltration patterns specific to the each PDX line that includes non-random distribution of GBM transcriptional states and genetic clones, and (b) spatially distinct infiltration of TME components including microglial and macrophage populations. Overall, our approach addresses the challenge of understanding the tumor-TME relationship by application of spatial profiling in PDX models, and provides a computational pipeline complex multi-species analysis in the spatial transcriptomic field. Citation Format: Aly O. Abdelkareem, Katalin Osz, Donna Senger, Jennifer A. Chan, Sorana Morrissy. Understanding the glioblastoma microenvironment with spatial resolution in PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6397.

A single-cell lung atlas of complement genes identifies the mesothelium and epithelium as prominent sources of extrahepatic complement proteins.

To understand functional duality of the complement system in host defense and lung injury, a more comprehensive view of its localized production in the lung, and the impact of age on complement production are essential. Here, we explored the expression of complement genes through computational analysis of preexisting single cell RNA sequencing data from lung transcriptomes of healthy young (3 months) and old C57BL/6 mice (24 months), and humans. We characterized the distribution of 48 complement genes. Across 28 distinct immune and non-immune cell types in mice, mesothelial cells expressed the greatest number of complement genes (e.g., C1ra, C2, C3), and regulators (e.g., Serping1, Cfh). C5 was abundant in type II alveolar epithelial cells and C1q in interstitial lung macrophages. There were only moderate differences in gene expression between young and old mice. Among 57 human lung cell types, mesothelial cells showed abundant complement expression. A few differences in gene expression (e.g., FCN1, CFI, C6, C7) were also evident between mice and human lung cells. Our findings present a novel perspective on the expression patterns of complement genes in normal lungs. These findings highlight the potential functions of complement in tissue-specific homeostasis and immunity and may foster a mechanistic understanding of its role in lung health and disease.

Quantitative relationship between cerebrovascular network and neuronal cell types in mice.

SUMMARYThe cerebrovasculature and its mural cells must meet brain regional energy demands, but how their spatial relationship with different neuronal cell types varies across the brain remains largely unknown. Here we apply brain-wide mapping methods to comprehensively define the quantitative relationships between the cerebrovasculature, capillary pericytes, and glutamatergic and GABAergic neurons, including neuronal nitric oxide synthase-positive (nNOS+) neurons and their subtypes in adult mice. Our results show high densities of vasculature with high fluid conductance and capillary pericytes in primary motor sensory cortices compared with association cortices that show significant positive and negative correlations with energy-demanding parvalbumin+ and vasomotor nNOS+ neurons, respectively. Thalamo-striatal areas that are connected to primary motor sensory cortices also show high densities of vasculature and pericytes, suggesting dense energy support for motor sensory processing areas. Our cellular-resolution resource offers opportunities to examine spatial relationships between the cerebrovascular network and neuronal cell composition in largely understudied subcortical areas.

PCGA: a comprehensive web server for phenotype-cell-gene association analysis.

Most complex disease-associated loci mapped by genome-wide association studies (GWAS) are located in non-coding regions. It remains elusive which genes the associated loci regulate and in which tissues/cell types the regulation occurs. Here, we present PCGA (https://pmglab.top/pcga), a comprehensive web server for jointly estimating both associated tissues/cell types and susceptibility genes for complex phenotypes by GWAS summary statistics. The web server is built on our published method, DESE, which represents an effective method to mutually estimate driver tissues and genes by integrating GWAS summary statistics and transcriptome data. By collecting and processing extensive bulk and single-cell RNA sequencing datasets, PCGA has included expression profiles of 54 human tissues, 2,214 human cell types and 4,384 mouse cell types, which provide the basis for estimating associated tissues/cell types and genes for complex phenotypes. We develop a framework to sequentially estimate associated tissues and cell types of a complex phenotype according to their hierarchical relationships we curated. Meanwhile, we construct a phenotype-cell-gene association landscape by estimating the associated tissues/cell types and genes of 1,871 public GWASs. The association landscape is generally consistent with biological knowledge and can be searched and browsed at the PCGA website.

PD12-12 TUMOR ANTIGEN VACCINE ENHANCES ANTI-TUMOR EFFECTS OF IMMUNE CHECKPOINT INHIBITORS AGAINST REFRACTORY CANCERS

You have accessJournal of UrologyCME1 May 2022PD12-12 TUMOR ANTIGEN VACCINE ENHANCES ANTI-TUMOR EFFECTS OF IMMUNE CHECKPOINT INHIBITORS AGAINST REFRACTORY CANCERS Shohei Ueda, Miho Ushjima, Atsushi Irie, Yasuharu Nishimura, and Masatoshi Eto Shohei UedaShohei Ueda More articles by this author , Miho UshjimaMiho Ushjima More articles by this author , Atsushi IrieAtsushi Irie More articles by this author , Yasuharu NishimuraYasuharu Nishimura More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002538.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Progressive and chemoresistant bladder cancer is problematic because of the poor prognosis. immune checkpoint blockade(ICB) have exhibited drastic anti-tumor efficacy in some patients, but the response rates is limited. Therefore, to improve efficacy of immunotherapy for refractory cancer, we have tried to develop a combination immunotherapy of tumor antigen vaccine and ICB in tumor-bearing mouse models. METHODS: We identified genes highly expressed in tumors but not in normal tissues by comparing RNA-seq data of mouse bladder cancer MB49, cisplatin-resistant MB49-CR, and lymphoma RMA cell lines, all of which are refractory to ICB, with datasets of 26 normal mouse tissues. Subsequently tumor-associated antigens-derived peptides predicted to have high binding affinities to MHC-I and -II molecules were identified by prediction algorithms. We also selected immunogenic and cancer-specific mutated Neo-antigenic peptides. MB49, MB49-CR, or RMA bearing mice were treated with these peptides vaccine and ICB. Finally, we investigated tumor infiltrating immune cells by flow cytometry, TCR repertoire analysis, and RNA-seq of inoculated tumors. RESULTS: In 50-80% of mice pre-immunized with these peptides, a growth of MB49 and RMA inoculated s.c. was prevented and those immunized mice rejected re-transplanted tumors. Furthermore, combination therapy of these peptide vaccines, and ICB synergistically inhibited tumor-growth of MB49-CR, MB49 and RMA indicating that the combination immunotherapy described above enhances therapeutic efficacy to treat refractory cancers. We also observed increases of tumor infiltrating CD8+ T cells expressing limited TCR-alpha and beta genes, and drastic changes of various types of tumor-infiltrating immune cells in tumor-bearing mice treated with the combination therapy. CONCLUSIONS: These results suggest that the combination immunotherapy described above enhances therapeutic efficacy to treat refractory cancer. Source of Funding: This work was supported by KAKENHI © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e199 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shohei Ueda More articles by this author Miho Ushjima More articles by this author Atsushi Irie More articles by this author Yasuharu Nishimura More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement PDF DownloadLoading ...

ProbC: joint modeling of epigenome and transcriptome effects in 3D genome

BackgroundHi-C and its high nucleosome resolution variant Micro-C provide a window into the spatial packing of a genome in 3D within the cell. Even though both techniques do not directly depend on the binding of specific antibodies, previous work has revealed enriched interactions and domain structures around multiple chromatin marks; epigenetic modifications and transcription factor binding sites. However, the joint impact of chromatin marks in Hi-C and Micro-C interactions have not been globally characterized, which limits our understanding of 3D genome characteristics. An emerging question is whether it is possible to deduce 3D genome characteristics and interactions by integrative analysis of multiple chromatin marks and associate interactions to functionality of the interacting loci.ResultWe come up with a probabilistic method ProbC to decompose Hi-C and Micro-C interactions by known chromatin marks. ProbC is based on convex likelihood optimization, which can directly take into account both interaction existence and nonexistence. Through ProbC, we discover histone modifications (H3K27ac, H3K9me3, H3K4me3, H3K4me1) and CTCF as particularly predictive of Hi-C and Micro-C contacts across cell types and species. Moreover, histone modifications are more effective than transcription factor binding sites in explaining the genome’s 3D shape through these interactions. ProbC can successfully predict Hi-C and Micro-C interactions in given species, while it is trained on different cell types or species. For instance, it can predict missing nucleosome resolution Micro-C interactions in human ES cells trained on mouse ES cells only from these 5 chromatin marks with above 0.75 AUC. Additionally, ProbC outperforms the existing methods in predicting interactions across almost all chromosomes.ConclusionVia our proposed method, we optimally decompose Hi-C interactions in terms of these chromatin marks at genome and chromosome levels. We find a subset of histone modifications and transcription factor binding sites to be predictive of both Hi-C and Micro-C interactions and TADs across human, mouse, and different cell types. Through learned models, we can predict interactions on species just from chromatin marks for which Hi-C data may be limited.

FindPC: An R package to automatically select the number of principal components in single-cell analysis.

Principal component analysis is widely used in analyzing single-cell genomic data. Selecting the optimal number of principal components (PCs) is a crucial step for downstream analyses. The elbow method is most commonly used for this task, but it requires one to visually inspect the elbow plot and manually choose the elbow point. To address this limitation, we developed six methods to automatically select the optimal number of PCs based on the elbow method. We evaluated the performance of these methods on real single-cell RNA-seq data from multiple human and mouse tissues and cell types. The perpendicular line method with 30 PCs has the best overall performance, and its results are highly consistent with the numbers of PCs identified manually. We implemented the six methods in an R package, findPC, that objectively selects the number of PCs and can be easily incorporated into any automatic analysis pipeline. findPC R package is freely available at https://github.com/haotian-zhuang/findPC. Supplementary data are available at Bioinformatics online.

Feature Detection by Retinal Ganglion Cells.

Retinal circuits transform the pixel representation of photoreceptors into the feature representations of ganglion cells, whose axons transmit these representations to the brain. Functional, morphological, and transcriptomic surveys have identified more than 40 retinal ganglion cell (RGC) types in mice. RGCs extract features of varying complexity; some simply signal local differences in brightness (i.e., luminance contrast), whereas others detect specific motion trajectories. To understand the retina, we need to know how retinal circuits give rise to the diverse RGC feature representations. A catalog of the RGC feature set, in turn, is fundamental to understanding visual processing in the brain. Anterograde tracing indicates that RGCs innervate more than 50 areas in the mouse brain. Current maps connecting RGC types to brain areas are rudimentary, as is our understanding of how retinal signals are transformed downstream to guide behavior. In this article, I review the feature selectivities of mouse RGCs, how they arise, and how they are utilized downstream. Not only is knowledge of the behavioral purpose of RGC signals critical for understanding the retinal contributions to vision; it can also guide us to the most relevant areas of visual feature space.