Specific Gene Expression Profiles Research Articles

Isolation of ascorbate peroxidase (APX) gene in lentil (Lens culinaris Medik.) and expression analysis under drought stress conditions

Objective: The objective of this study was to isolate partial cDNA that belongs to the ascorbate peroxidase (APX) gene of lentil (Lens culinaris Medik.) and to express LcAPX gene in lentil seedlings under drought stress conditions. Material and Methods: To identify the relationships between drought stress and LcAPX gene expression, lentil seedlings grown for 2 weeks were subjected to drought stress through not irrigating for 6, 13, and 20 days. Effects of drought stress were determined by measuring the stem relative water content (RWC). Gene expression changes in lentil seedlings were determined with real-time RT-qPCR. Results: The LcAPX gene expression levels of both drought-tolerant Firat-87 and drought-sensitive Ozbek cultivars varied with the severity of drought stress. The gene expression of LcAPX reached the highest level in Firat-87 cultivar on the 6th day, whereas a significant increase was observed only on the 20th day of the Ozbek cultivar, and this increase was relatively low as compared to the Fırat-87 cultivar. Conclusion: From the study conducted, it was concluded that time-dependent changes of the expression of LcAPX gene indicates that LcAPX gene had a highly specific gene expression profile and complex regulation in lentil drought response.

Abstract IA025: Using genetically engineered mouse models to study sarcoma metastasis

Abstract Despite aggressive conventional therapy, many patients with high-risk soft-tissue sarcoma develop metastatic disease. To investigate mechanism(s) of sarcoma metastasis, our lab has utilized genetically engineered mouse models. For example, we injected an adenovirus expressing Cre recombinase (adeno-Cre) into the gastrocnemius muscle of LSL-KrasG12D; p53Flox/Flox (KP) mice to initiate high grade undifferentiated pleomorphic sarcomas, and after amputation approximately 40% of the mice develop lung metastasis. Using a genetic approach, we found that miR-182, NEAT-1, and HIF-1a regulate metastasis to the lung. We also performed lineage tracing with complementary fluorescent proteins and CRISPR-generated bar codes to find that lung metastases from KP sarcomas arise from clones with specific gene expression profiles. Although the KP sarcoma model is useful for studying metastasis, one limitation of this model is that there are few non-synonymous mutations to engage the immune system. Therefore, we generated a high mutational load primary mouse model of soft tissue sarcoma by injecting adeno-Cre into the gastrocnemius muscle of p53Flox/Flox mice to delete p53 and also injected 3-methylcholanthrene (MCA) to generate primary p53/MCA undifferentiated pleomorphic sarcomas. In this p53/MCA model, the overall rate of lung metastasis after amputation was surprisingly low (~12%). We hypothesized that the immune system suppressed lung metastasis in this model. However, when we generated p53/MCA sarcomas in Rag2 −/− mice that lack mature B and T cells, we still observed a low rate of lung metastasis after amputation. These data suggest that mutations caused by MCA may have disabled tumor intrinsic factors needed to drive sarcoma metastasis. We are currently performing genome-wide screens in the p53/MCA model to search for genes required for sarcoma metastasis. Citation Format: David G. Kirsch, Rutulkumar Patel, Sophie R. Finkelstein, Joy Ban, Yuning J Tang, Jianguo Huang, Benjamin A. Alman, Yvonne M. Mowery. Using genetically engineered mouse models to study sarcoma metastasis [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA025.

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

Transcriptomic changes in the large organs in lethal meningococcal shock are reflected in a porcine shock model.

BackgroundFulminant meningococcal sepsis with shock and multiple organ failure is associated with a massive systemic inflammatory response involving solid organs. We have previously established a porcine model of the disease to study pathophysiologic and possible therapeutic strategies.ObjectiveThis study examined whether the organ specific gene expression profile in such a large animal model reflects the profile seen in patients with fulminant meningococcal sepsis.Patients and methodsData from gene expression profiles induced in organs from patients (n=5) and the porcine model (n=8) were imported into the Ingenuity pathway analysis (IPA) software for comparison analysis. The number of meningococci in the organs were quantified by real time-PCR.ResultsThe all-over transcriptional activation between different organs revealed a striking concordance between the patients and the pigs regarding the pattern of transcriptional activation and activated pathways. Comparison analysis demonstrated similar pattern of upregulation of genes being associated with a large range of inflammatory biofunctions in the patients and the porcine model. Genes associated with biofunctions such as organismal death, morbidity and mortality were similarly downregulated in the patients and the porcine model. Comparison analysis of main predicted canonical pathways also demonstrated a high degree of similarity regarding up- and downregulation in both groups. Core analysis revealed different top-upstream regulators in the different organs in the patients. In the patients pro-inflammatory regulators were most activated in the lungs. In the other organs up-stream factors that regulate signaling pathways involved in development, growth, repair and homeostasis and triglyceride synthesis were most activated. In the porcine model, the top-upstream regulators were pro-inflammatory in all organs. The difference may reflect the shorter duration of the porcine experiment than the duration of the patient’s infection before death.ConclusionThe inflammatory responses measured on the transcriptomic level in organs in patients with fulminant meningococcal sepsis is reproduced in the porcine model of the disease, although some differences may exist regarding the top-upregulated factors in individual organs. Thus, this large animal model reproduces important immunological features of meningococcal sepsis and can be a valuable tool in further investigations of inflammatory aspects and possible treatment options

IL-18/IL-18R Signaling Is Dispensable for ILC Development But Constrains the Growth of ILCP/ILCs.

Innate lymphoid cells (ILCs) develop from ILC progenitors in the bone marrow. Various ILC precursors (ILCPs) with different ILC subset lineage potentials have been identified based on the expression of cell surface markers and ILC-associated key transcription factor reporter genes. This study characterized an interleukin (IL)-7Rα+IL-18Rα+ ILC progenitor population in the mouse bone marrow with multi-ILC lineage potential on the clonal level. Single-cell gene expression analysis revealed the heterogeneity of this population and identified several subpopulations with specific ILC subset-biased gene expression profiles. The role of IL-18 signaling in the regulation of IL-18Rα+ ILC progenitors and ILC development was further investigated using Il18- and Il18r1-deficient mice, in vitro differentiation assay, and adoptive transfer model. IL-18/IL-18R-mediated signal was found to not be required for early stages of ILC development. While Il18r1-/- lymphoid progenitors were able to generate all ILC subsets in vitro and in vivo like the wild-type counterpart, increased IL-18 level, as often occurred during infection or under stress, suppressed the growth of ILCP/ILC in an IL-18Ra-dependent manner via inhibiting proliferation and inducing apoptosis.

Abstract 2476: Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy

Abstract Glioblastomas multiforme (GBMs) are the most lethal tumors of the brain. Tumoral mesenchymal transformation is a hallmark of GBMs associated with alterations in cellular morphology and dynamic organization. However, little is known about the mechanisms that control this pathological process. Here, we report a comprehensive spatiotemporal study integrating novel intra-tumoral histopathological structures, ‘oncostreams’, with tumor dynamic properties, microenvironment assets and spatial molecular features. Cellular analyses of genetic engineered mouse models of glioma identified that oncostreams are heterogenous structures formed by elongated and aligned neoplastic cells enriched in non-neoplastic cells such as ACTA2+ mesenchymal like cells and CD68+ tumor associated microglia/macrophages (TAM). Deep learning analysis of H&E glioma histological samples from mouse and human gliomas identified that oncostream density correlates with tumor aggressiveness. To determine whether oncostreams fascicles are characterized by a specific gene expression profile, we performed transcriptomic analysis using laser capture microdissection coupled to RNA-sequencing. We found that oncostreams are defined by a transcriptomic signature enriched in mesenchymal genes. Network analyses identified that COL1A1 is a critical gene that regulates oncostream organization and function. Correspondingly, human and mouse high-grade gliomas with high oncostream densities showed prominent alignment of collagen fibers along these fascicles and higher COL1A1 expression compared to low-grade gliomas. To evaluate the functional role of COL1A1 in oncostream formation we generated a COL1A1-deficient GEMM of glioma. We observed that COL1A1 inhibition decreased oncostream formation, impaired tumor cell proliferation and remodeled the tumor microenvironment by diminishing CD68+ TAM cells, CD31+ endothelial vascular proliferation and ACTA2+ perivascular mesenchymal cells, thus increasing animal survival. Further studies, using time lapse confocal imaging in ex vivo glioma explants, and intravital imaging in vivo demonstrated that oncostreams are organized collective dynamic structures present at the tumor core and the invasive tumor border. Oncostreams dynamics increased the intra-tumoral spread of cells within the tumor and foster glioma aggressiveness through collective invasion of the normal brain parenchyma. The analysis of glioma invasion in COL1A1 knockdown tumors exhibited a reduction in collective migration patterns, strongly supporting its importance in tumor progression. We propose that oncostreams represent a novel pathological marker of potential value for diagnosis and COL1A1 depletion within oncostreams is a promising approach and reprogram mesenchymal transformation to reduce the tumor malignancy. Citation Format: Andrea Comba, Syed Faisal, Patrick J. Dunn, Anna E. Argento, Todd C. Hollon, Wajd N. Al-Holou, Maria L. Varela, Daniel B. Zamler, Gunnar L. Quass, Pierre F. Apostolides, Christine E. Brown, Phillip E. E. Kish, Alon Kahana, Celina G. Kleer, Sebastien Motsch, Maria G. Castro, Pedro R. Lowenstein. Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy [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 2476.

Predicting Algorithm of Tissue Cell Ratio Based on Deep Learning Using Single-Cell RNA Sequencing

Background: Understanding the proportion of cell types in heterogeneous tissue samples is important in bioinformatics. It is a challenge to infer the proportion of tissues using bulk RNA sequencing data in bioinformatics because most traditional algorithms for predicting tissue cell ratios heavily rely on standardized specific cell-type gene expression profiles, and do not consider tissue heterogeneity. The prediction accuracy of algorithms is limited, and robustness is lacking. This means that new approaches are needed urgently. Methods: In this study, we introduced an algorithm that automatically predicts tissue cell ratios named Autoptcr. The algorithm uses the data simulated by single-cell RNA sequencing (ScRNA-Seq) for model training, using convolutional neural networks (CNNs) to extract intrinsic relationships between genes and predict the cell proportions of tissues. Results: We trained the algorithm using simulated bulk samples and made predictions using real bulk PBMC data. Comparing Autoptcr with existing advanced algorithms, the Pearson correlation coefficient between the actual value of Autoptcr and the predicted value was the highest, reaching 0.903. Tested on a bulk sample, the correlation coefficient of Lin was 41% higher than that of CSx. The algorithm can infer tissue cell proportions directly from tissue gene expression data. Conclusions: The Autoptcr algorithm uses simulated ScRNA-Seq data for training to solve the problem of specific cell-type gene expression profiles. It also has high prediction accuracy and strong noise resistance for the tissue cell ratio. This work is expected to provide new research ideas for the prediction of tissue cell proportions.

Single-Cell Transcriptomic Analysis of the Mouse Pancreas: Characteristic Features of Pancreatic Ductal Cells in Chronic Pancreatitis.

Chronic pancreatitis (CP) is a fibroinflammatory disorder of the pancreas. Our understanding of CP pathogenesis is partly limited by the incomplete characterization of pancreatic cell types. Here, we performed single-cell RNA sequencing on 3825 cells from the pancreas of one control mouse and mice with caerulein-induced CP. An analysis of the single-cell transcriptomes revealed 16 unique clusters and cell type-specific gene expression patterns in the mouse pancreas. Sub-clustering of the pancreatic mesenchymal cells from the control mouse revealed four clusters of cells with specific gene expression profiles (combinatorial expressions of Smoc2, Cxcl14, Tnfaip6, and Fn1). We observed that immune cells in the pancreas of the CP mice were abundant and diverse in cellular type. Compared to the control, 547 upregulated genes (including Mmp7, Ttr, Rgs5, Adh1, and Cldn2) and 257 downregulated genes were identified in ductal cells from the CP group. The elevated expression levels of MMP7 and TTR were further verified in the pancreatic ducts of CP patients. This study provides a preliminary description of the single-cell transcriptome profiles of mouse pancreata and accurately demonstrates the characteristics of pancreatic ductal cells in CP. The findings provide insight into novel disease-specific biomarkers and potential therapeutic targets of CP.

Pathway enrichment analysis of microarray data from human penis of diabetic and Peyronie's patients, in comparison with diabetic rat erectile dysfunction models

ABSTRACT Introduction Erectile dysfunction (ED) is a debilitating medical condition in which current treatments are minimally effective in diabetic patients due to neuropathy of the cavernous nerve, a peripheral nerve that innervates the penis. Loss of innervation causes apoptosis of penile smooth muscle, remodeling of corpora cavernosa (penile erectile tissue) morphology, and ED. Objective In this study, microarray and pathway analysis were used to obtain a global understanding of how signaling mechanisms are altered in diabetic patients and animal models as ED develops, in order to identify novel targets for disease management, and points of intervention for clinical therapy development. Methods Human corpora cavernosal tissue was obtained from diabetic (n=4) and Peyronie's (control, n=3) patients that were undergoing prosthesis implant to treat ED. Rat tissue was obtained from BB/WOR diabetic (n=5) and diabetes resistant (n=5) rats. RNA was extracted using TRIzol, DNase treated, and purified by Qiagen mini kit. Microarray was performed using the Human Gene 2.0 ST Array. 1) Alterations in patient and diabetic rat pathway signaling were examined using several analytical tools (ShinyGO, Metascape, WebGestalt, STRING) and databases, 2) Strengths/weaknesses of the different pathway analysis tools were compared, and 3) Comparison of human and rat (BB/WOR and Streptozotocin) pathway analysis was performed. Two technical replicates were performed. P value (FDR) < 0.15 was used as threshold for differential expression. FDR<0.05 was considered significant. Results Microarray identified 182 differentially expressed protein-coding genes. Pathway analysis revealed similar enrichments with different analytical tools. Down regulated pathways include development, tubular structure, sprouting, cell death, ischemia, angiogenesis, transcription, second messengers, and stem cell differentiation. ED patients, who have diabetes, incur significant loss of normal regulatory processes required for repair and replacement of injured corpora cavernosal tissue. Combined with loss of apoptotic regulatory mechanisms, this results in significant architectural remodeling of the corpora cavernosa, and loss of regenerative capacity in the penis. Conclusions Penis from diabetic ED patients lacks capacity for maintenance of corpora cavernosal architecture and regeneration, which are critical points for intervention for therapy development. The analysis of tissue specific gene expression profiles provides a means of understanding drivers of disease and identifying novel pathways for clinical intervention. This first report of microarray and pathway analysis in human corpora cavernosa, is critical for identification of novel pathways pertinent to ED and for validating animal models. Disclosure Work supported by industry: no.

DNA Methyltransferase 3b Accelerates the Process of Atherosclerosis.

Background DNA methylation plays a key role in establishing cell type–specific gene expression profiles and patterns in atherosclerosis. The underlying mechanism remains unclear. Previous studies have shown that DNA methyltransferase 3b (DNMT3b) may play an important role in atherosclerosis. This study aimed to establish the regulatory role of DNMT3b in the development of atherosclerosis. Methods We constructed a viral vector carrying Dnmt3b shRNA to transduce ApoE−/− mice. Meanwhile, healthy human peripheral blood Treg cells were treated with inhibitor of DNMT3b (AZA and EGCG) or transduced with DNMT3b shRNA. Results It showed that Dnmt3b silencing attenuated atherosclerosis, including decreased lesion size and macrophage content and increased collagen and smooth muscle cells content in ApoE−/− mice. To further investigate the possible mechanisms, combined with previous studies by our group, we showed that Foxp3-TSDR methylation level was significantly reduced Foxp3 expression and peripheral blood Treg levels were significantly increased by Dnmt3b shRNA vector transduction in animals committed to western diet for 12 and 18 weeks. Consistently, inhibition of DNMT3b (AZA and EGCG) decreased the expression levels of DNMT3b, which can increase the expression levels of FOXP3, and increase the levels of TGF-β and IL-10 and decrease the levels of IL-β and IFN-γ. After transduction with DNMT3b shRNA, the effect was more obvious. Conclusions DNMT3b accelerated atherosclerosis, and may be associated with FOXP3 hypermethylation status in regulatory T cells.

Pathway Enrichment Analysis of Microarray Data from Human Penis of Diabetic and Peyronie's Patients, in Comparison with Diabetic Rat Erectile Dysfunction Models

ABSTRACT Introduction Erectile dysfunction (ED) is a debilitating medical condition in which current treatments are minimally effective in diabetic patients due to neuropathy of the cavernous nerve, a peripheral nerve that innervates the penis. Loss of innervation causes apoptosis of penile smooth muscle, remodeling of corpora cavernosa (penile erectile tissue) morphology, and ED. Objective In this study, microarray and pathway analysis were used to obtain a global understanding of how signaling mechanisms are altered in diabetic patients and animal models as ED develops, in order to identify novel targets for disease management, and points of intervention for clinical therapy development. Methods Human corpora cavernosal tissue was obtained from diabetic (n=4) and Peyronie's (control, n=3) patients that were undergoing prosthesis implant to treat ED. Rat tissue was obtained from BB/WOR diabetic (n=5) and diabetes resistant (n=5) rats. RNA was extracted using TRIzol, DNase treated, and purified by Qiagen mini kit. Microarray was performed using the Human Gene 2.0 ST Array. 1) Alterations in patient and diabetic rat pathway signaling were examined using several analytical tools (ShinyGO, Metascape, WebGestalt, STRING) and databases, 2) Strengths/weaknesses of the different pathway analysis tools were compared, and 3) Comparison of human and rat (BB/WOR and Streptozotocin) pathway analysis was performed. Two technical replicates were performed. P value (FDR) < 0.15 was used as threshold for differential expression. FDR<0.05 was considered significant. Results Microarray identified 182 differentially expressed protein-coding genes. Pathway analysis revealed similar enrichments with different analytical tools. Down regulated pathways include development, tubular structure, sprouting, cell death, ischemia, angiogenesis, transcription, second messengers, and stem cell differentiation. ED patients, who have diabetes, incur significant loss of normal regulatory processes required for repair and replacement of injured corpora cavernosal tissue. Combined with loss of apoptotic regulatory mechanisms, this results in significant architectural remodeling of the corpora cavernosa, and loss of regenerative capacity in the penis. Conclusions Penis from diabetic ED patients lacks capacity for maintenance of corpora cavernosal architecture and regeneration, which are critical points for intervention for therapy development. The analysis of tissue specific gene expression profiles provides a means of understanding drivers of disease and identifying novel pathways for clinical intervention. This first report of microarray and pathway analysis in human corpora cavernosa, is critical for identification of novel pathways pertinent to ED and for validating animal models. Disclosure No

CCR2+ Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling.

During mechanical force-induced alveolar bone remodeling, macrophage-mediated local inflammation plays a critical role. Yet, the detailed heterogeneity of macrophages is still unknown. Single-cell RNA sequencing was used to study the transcriptome heterogeneity of macrophages during alveolar bone remodeling. We identified macrophage subclusters with specific gene expression profiles and functions. CellChat and trajectory analysis revealed a central role of the Ccr2 cluster during development, with the CCL signaling pathway playing a crucial role. We further demonstrated that the Ccr2 cluster modulated bone remodeling associated inflammation through an NF-κB dependent pathway. Blocking CCR2 could significantly reduce the Orthodontic tooth movement (OTM) progression. In addition, we confirmed the variation of CCR2+ macrophages in human periodontal tissues. Our findings reveal that mechanical force-induced functional shift of the Ccr2 macrophages cluster mediated by NF-κB pathway, leading to a pro-inflammatory response and bone remodeling. This macrophage cluster may represent a potential target for the manipulation of OTM.

Identification of a specific immunophenotype associated with a consistent pattern of genetic mutations including SRFS2 and gene expression profile in MDS.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases classified by comprehensive diagnostics. Identification of homogeneous subgroups is desirable to understand differences in clinical course and to develop targeted treatment approaches. We identified a specific CD11b/CD16 expression pattern in granulocytes associated with reduced CD45 expression in myeloid progenitor cells (MPC) in MDS cases and assessed its genetic background by whole genome (WGS) and whole transcriptome sequencing (WTS). The cohort consisted of 32 MDS cases with the specific aberrant immunophenotype. Since all these 32 cases were found to be SRSF2 mutated additional 51 SRSF2 mutated MDS cases without this specific immunophenotype were selected as controls. For all cases WGS and WTS were performed. The immunophenotype newly identified in SRSF2 mutated MDS patients is characterized (1) by a specific maturation pattern, i.e. an increase of CD11b expression without CD16 expression followed by an increase in CD16 expression without further CD11b expression and (2) by only dim CD45 expression of MPC. STAG2 mutations were exclusively found in MDS cases with the specific immunophenotype (17/32, 53% vs. 0%, p< 0.001). Hence, >50% of cases with the specific immunophenotype were characterized by co-mutations in SRSF2 and STAG2. In addition, cluster analysis revealed a specific gene expression profile of such cases. We here for the first time describe a specific immunophenotype which defines MDS cases with SRSF2 mutations and a consistent and specific mutational and gene expression profile. This comprehensive data warrants analysis of further such cases to assess the feasibility of defining a new sub-entity of MDS.

Deconvolving Clinically Relevant Cellular Immune Cross-talk from Bulk Gene Expression Using CODEFACS and LIRICS Stratifies Patients with Melanoma to Anti-PD-1 Therapy.

This work presents two new computational methods that can deconvolve a large collection of bulk tumor gene expression profiles into their respective cell type-specific gene expression profiles and identify cell type-specific ligand-receptor interactions predictive of response to immune-checkpoint blockade therapy. This article is highlighted in the In This Issue feature, p. 873.

Single-Cell Transcriptomics Reveals the Molecular Anatomy of Sheep Hair Follicle Heterogeneity and Wool Curvature.

Wool is the critical textile raw material which is produced by the hair follicle of sheep. Therefore, it has important implications to investigate the molecular mechanism governing hair follicle development. Due to high cellular heterogeneity as well as the insufficient cellular, molecular, and spatial characterization of hair follicles on sheep, the molecular mechanisms involved in hair follicle development and wool curvature of sheep remains largely unknown. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to comprehensively dissect the cellular composition of complex skin tissues and unveil the differentiation and spatial signatures of epidermal and hair follicle development. However, such studies are lacking so far in sheep. Here, single-cell suspensions from the curly wool and straight wool lambskins were prepared for unbiased scRNA-seq. Based on UAMP dimension reduction analysis, we identified 19 distinct cell populations from 15,830 single-cell transcriptomes and characterized their cellular identity according to specific gene expression profiles. Furthermore, novel marker gene was applied in identifying dermal papilla cells isolated in vitro. By using pseudotime ordering analysis, we constructed the matrix cell lineage differentiation trajectory and revealed the dynamic gene expression profiles of matrix progenitors' commitment to the hair shaft and inner root sheath (IRS) cells. Meanwhile, intercellular communication between mesenchymal and epithelial cells was inferred based on CellChat and the prior knowledge of ligand–receptor pairs. As a result, strong intercellular communication and associated signaling pathways were revealed. Besides, to clarify the molecular mechanism of wool curvature, differentially expressed genes in specific cells between straight wool and curly wool were identified and analyzed. Our findings here provided an unbiased and systematic view of the molecular anatomy of sheep hair follicle comprising 19 clusters; revealed the differentiation, spatial signatures, and intercellular communication underlying sheep hair follicle development; and at the same time revealed the potential molecular mechanism of wool curvature, which will give important new insights into the biology of the sheep hair follicle and has implications for sheep breeding.

AtheroSpectrum Reveals Novel Macrophage Foam Cell Gene Signatures Associated With Atherosclerotic Cardiovascular Disease Risk.

Whereas several interventions can effectively lower lipid levels in people at risk for atherosclerotic cardiovascular disease (ASCVD), cardiovascular event risks remain, suggesting an unmet medical need to identify factors contributing to cardiovascular event risk. Monocytes and macrophages play central roles in atherosclerosis, but studies have yet to provide a detailed view of macrophage populations involved in increased ASCVD risk. A novel macrophage foaming analytics tool, AtheroSpectrum, was developed using 2 quantitative indices depicting lipid metabolism and the inflammatory status of macrophages. A machine learning algorithm was developed to analyze gene expression patterns in the peripheral monocyte transcriptome of MESA participants (Multi-Ethnic Study of Atherosclerosis; set 1; n=911). A list of 30 genes was generated and integrated with traditional risk factors to create an ASCVD risk prediction model (30-gene cardiovascular disease risk score [CR-30]), which was subsequently validated in the remaining MESA participants (set 2; n=228); performance of CR-30 was also tested in 2 independent human atherosclerotic tissue transcriptome data sets (GTEx [Genotype-Tissue Expression] and GSE43292). Using single-cell transcriptomic profiles (GSE97310, GSE116240, GSE97941, and FR-FCM-Z23S), AtheroSpectrum detected 2 distinct programs in plaque macrophages-homeostatic foaming and inflammatory pathogenic foaming-the latter of which was positively associated with severity of atherosclerosis in multiple studies. A pool of 2209 pathogenic foaming genes was extracted and screened to select a subset of 30 genes correlated with cardiovascular event in MESA set 1. A cardiovascular disease risk score model (CR-30) was then developed by incorporating this gene set with traditional variables sensitive to cardiovascular event in MESA set 1 after cross-validation generalizability analysis. The performance of CR-30 was then tested in MESA set 2 (P=2.60×10-4; area under the receiver operating characteristic curve, 0.742) and 2 independent data sets (GTEx: P=7.32×10-17; area under the receiver operating characteristic curve, 0.664; GSE43292: P=7.04×10-2; area under the receiver operating characteristic curve, 0.633). Model sensitivity tests confirmed the contribution of the 30-gene panel to the prediction model (likelihood ratio test; df=31, P=0.03). Our novel computational program (AtheroSpectrum) identified a specific gene expression profile associated with inflammatory macrophage foam cells. A subset of 30 genes expressed in circulating monocytes jointly contributed to prediction of symptomatic atherosclerotic vascular disease. Incorporating a pathogenic foaming gene set with known risk factors can significantly strengthen the power to predict ASCVD risk. Our programs may facilitate both mechanistic investigations and development of therapeutic and prognostic strategies for ASCVD risk.

Developmentally Regulated Modulation of Lumbar Motoneurons by Metabotropic Glutamate Receptors: A Cellular and Behavioral Analysis in Newborn Mice.

The present study explores the impact of metabotropic glutamate receptor (mGluR) activation on activity-dependent synaptic plasticity (ADSP) and the intrinsic membrane properties of lumbar motoneurons (MNs) using a combination of biochemical, pharmacological, electrophysiological and behavioral techniques. Using spinal cord slices from C57BL/6JRJ mice at two developmental stages, 1-3 and 8-12 postnatal days (P1-P3; P8-P12, respectively), we found that ADSP expressed at glutamatergic synapses between axons conveyed in the ventrolateral funiculus (VLF) and MNs, involved mGluR activation. Using specific agonists of the three groups of mGluRs, we observed that mGluR stimulation causes subtype-specific and developmentally regulated modulation of the ADSP and synaptic transmission at VLF-MN synapses as well as the intrinsic membrane properties of MNs. RT-qPCR analysis revealed a downregulation of mGluR gene expression with age in the ventral part of the lumbar spinal cord. Interestingly, the selective harvest by laser microdissection of MNs innervating the Gastrocnemius and Tibialis anterior muscles unraveled that the level of Grm2 expression is higher in Tibialis MNs compared to Gastrocnemius MNs suggesting a specific mGluR gene expression profile in these two MN pools. Finally, we assessed the functional impact of mGluR modulation on electrically induced bouts of fictive locomotion in the isolated spinal cord preparation of P1-P3 mice, and in vivo during spontaneous episodes of swimming activity in both P1-P3 and P8-P12 mouse pups. We observed that the mGluR agonists induced distinct and specific effects on the motor burst amplitudes and period of the locomotor rhythms tested and that their actions are function of the developmental stage of the animals. Altogether our data show that the metabotropic glutamatergic system exerts a complex neuromodulation in the developing spinal lumbar motor networks and provide new insights into the expression and modulation of ADSP in MNs.

Pathway Enrichment Analysis of Microarray Data Fom Human Penis of Diabetic and Peyronie's Patients, in Comparison With Diabetic Rat Erectile Dysfunction Models

BackgroundErectile dysfunction (ED) is a debilitating medical condition in which current treatments are minimally effective in diabetic patients due to neuropathy of the cavernous nerve, a peripheral nerve that innervates the penis. Loss of innervation causes apoptosis of penile smooth muscle, remodeling of corpora cavernosa (penile erectile tissue) morphology, and ED. AimIn this study, microarray and pathway analysis were used to obtain a global understanding of how signaling mechanisms are altered in diabetic patients and animal models as ED develops, in order to identify novel targets for disease management, and points of intervention for clinical therapy development. Methods and OutcomesHuman corpora cavernosal tissue was obtained from diabetic (n = 4) and Peyronie's (control, n = 3) patients that were undergoing prosthesis implant to treat ED, and BB/WOR diabetic (n = 5) and resistant (n = 5) rats. RNA was extracted using TRIzol, DNase treated, and purified by Qiagen mini kit. Microarray was performed using the Human Gene 2.0 ST Array. (i) Alterations in patient and diabetic rat pathway signaling were examined using several analytical tools (ShinyGO, Metascape, WebGestalt, STRING) and databases, (ii) Strengths/weaknesses of the different pathway analysis tools were compared, and (iii) Comparison of human and rat (BB/WOR and Streptozotocin) pathway analysis was performed. Two technical replicates were performed. P value (FDR) < .15 was used as threshold for differential expression. FDR < 0.05 was considered significant. ResultsMicroarray identified 182 differentially expressed protein-coding genes. Pathway analysis revealed similar enrichments with different analytical tools. Down regulated pathways include development, tubular structure, sprouting, cell death, ischemia, angiogenesis, transcription, second messengers, and stem cell differentiation. ED patients, who have diabetes, incur significant loss of normal regulatory processes required for repair and replacement of injured corpora cavernosal tissue. Combined with loss of apoptotic regulatory mechanisms, this results in significant architectural remodeling of the corpora cavernosa, and loss of regenerative capacity in the penis. Clinical TranslationThis first report of microarray and pathway analysis in human corpora cavernosa, is critical for identification of novel pathways pertinent to ED and for validating animal models. Strengths and LimitationsThe analysis of tissue specific gene expression profiles provides a means of understanding drivers of disease and identifying novel pathways for clinical intervention. ConclusionPenis from diabetic ED patients lacks capacity for maintenance of corpora cavernosal architecture and regeneration, which are critical points for intervention for therapy development.Searl T, Ohlander S, McVary KT, et al., Pathway Enrichment Analysis of Microarray Data Fom Human Penis of Diabetic and Peyronie's Patients, in Comparison With Diabetic Rat Erectile Dysfunction Models. J Sex Med 2022;19:37–53.

Characterization of normal and cancer stem-like cell populations in murine lingual epithelial organoids using single-cell RNA sequencing

The advances in oral cancer research and therapies have not improved the prognosis of patients with tongue cancer. The poor treatment response of tongue cancer may be attributed to the presence of heterogeneous tumor cells exhibiting stem cell characteristics. Therefore, there is a need to develop effective molecular-targeted therapies based on the specific gene expression profiles of these cancer stem-like cell populations. In this study, the characteristics of normal and cancerous organoids, which are convenient tools for screening anti-cancer drugs, were analyzed comparatively. As organoids are generally generated by single progenitors, they enable the exclusion of normal cell contamination from the analyses. Single-cell RNA sequencing analysis revealed that p53 signaling activation and negative regulation of cell cycle were enriched characteristics in normal stem-like cells whereas hypoxia-related pathways, such as HIF-1 signaling and glycolysis, were upregulated in cancer stem-like cells. The findings of this study improved our understanding of the common features of heterogeneous cell populations with stem cell properties in tongue cancers, that are different from those of normal stem cell populations; this will enable the development of novel molecular-targeted therapies for tongue cancer.

P66.03 The Functional Role of TGF-β Signaling in SCLC Heterogeneity and Metastasis

Small cell lung cancer (SCLC) is the most aggressive lung cancer and highly heterogeneous phenotypes have been observed in human and mouse SCLC cells. Tumor heterogeneity has been proven to contribute to SCLC malignant progression and/or metastasis in RP-based models. And the unique therapeutic vulnerabilities across different SCLC subtypes have been linked with their specific gene expression profile, indicating the need of personal therapeutic strategy in clinic. The TGF-β signaling consists of secreted TGF-β family members, receptors and SMAD proteins, and is implicated in multiple cell biological processes, such as cell proliferation and survival, epithelial to mesenchymal transition (EMT), cell fate control, immune response, and tumorigenesis. Dysregulation of the TGF-β signaling pathway has been reported in different cancer types and dual role of this pathway in cancer development and progression has been documented. Activation of this pathway results in cell cycle arrest and apoptosis at the early-stage of cancer development, indicating a tumor suppressive function. On the other hand, TGF-β could promote EMT as well as tumor malignant progression at the late-stage disease. Up to date, the contribution of the TGF-β pathway in SCLC development and progression remains elusive. Using the Rb1L/L/Trp53 L/L (RP) mouse model, we find that mouse SCLC cells with adherent growth pattern are composed of mesenchymal-like (Mes) and epithelial-like (Epi) subpopulations. Take advantage of in vitro functional assay and in vivo allograft assay, we find that the Mes cells harbor higher malignant transformation and metastatic capabilities than Epi cells. Through the analysis of microarray data, we find that the TGF-β signaling is enriched in Mes cells. And the inhibition of TGF-β signaling in Mes cells significantly abrogates its metastatic ability in allograft assay. Moreover, genetic deletion of the key components of the TGF-β signaling pathway, dramatically attenuated SCLC metastasis in the RP mouse model. In this study, we find that mouse SCLC cells derived from RP autochthonous mouse model with adherent growth pattern are composed of Mes and Epi subpopulations. The Mes cells have increased ability to form colonies in soft agar and harbored stronger metastatic capability in vivo when compared to the Epi cells. Gene Set Enrichment Analysis (GSEA) reveals that the TGF-β signaling is enriched in the Mes cells. Inhibition of the TGF-β signaling through either ectopic expression of dominant negative Tgfbr2 (Tgfbr2-DN) or treatment with Tgfbr1 inhibitor SD-208 consistently greatly abrogates the tumor metastasis in allograft assays. Moreover, genetic deletion of Tgfbr2 or Smad4, key components of the TGF-β signaling pathway, dramatically attenuates SCLC metastasis in the RP autochthonous mouse model of SCLC. Our results suggest the critical role of TGF-β signaling in SCLC heterogeneity and metastasis. Importantly, inhibition of TGF-β signaling significantly abrogates SCLC metastasis, providing a potential therapeutic avenue for SCLC management in clinic.