Characterization Of Transcriptome Research Articles

Emerging advances in defining the molecular and therapeutic landscape of small-cell lung cancer.

Small-cell lung cancer (SCLC) has traditionally been considered a recalcitrant cancer with a dismal prognosis, with only modest advances in therapeutic strategies over the past several decades. Comprehensive genomic assessments of SCLC have revealed that most of these tumours harbour deletions of the tumour-suppressor genes TP53 and RB1 but, in contrast to non-small-cell lung cancer, have failed to identify targetable alterations. The expression status of four transcription factors with key roles in SCLC pathogenesis defines distinct molecular subtypes of the disease, potentially enabling specific therapeutic approaches. Overexpression and amplification of MYC paralogues also affect the biology and therapeutic vulnerabilities of SCLC. Several other attractive targets have emerged in the past few years, including inhibitors of DNA-damage-response pathways, epigenetic modifiers, antibody-drug conjugates and chimeric antigen receptor T cells. However, the rapid development of therapeutic resistance and lack of biomarkers for effective selection of patients with SCLC are ongoing challenges. Emerging single-cell RNA sequencing data are providing insights into the plasticity and intratumoural and intertumoural heterogeneity of SCLC that might be associated with therapeutic resistance. In this Review, we provide a comprehensive overview of the latest advances in genomic and transcriptomic characterization of SCLC with a particular focus on opportunities for translation into new therapeutic approaches to improve patient outcomes.

P-085 Transcriptomic and proteomic characterization of unexplained male infertility: alterations in energy metabolism

Abstract Study question Which are the transcriptomic and proteomic profiles of spermatozoa belonging to patients with Unexplained Male Infertility (UMI)? Summary answer Transcriptomic and proteomic profiles of UMI patients differ from the fertile men, having the energy metabolism altered. What is known already About 30% of infertile couples suffer from “Unexplained Infertility” (UI). Considering that the male factor contributes to 50% of infertility cases, it is estimated that half of all couple UI cases are due to unexplained male infertility (UMI). Men are diagnosed with UMI when despite having “normal” seminal parameters they are unable to conceive. Due to the lack of UMI classification criteria and the seminogram-only-based infertility diagnosis, little is known about the mechanisms underlying UMI. Study design, size, duration 80 normozoospermic human semen samples with proven fertility and 80 samples from patients with UMI were used for this study. Ejaculates were obtained from Clínica Quirón Bilbao between 2012 and 2018, and after capacitation they were pooled to perform protein and RNA extractions. Participants/materials, setting, methods For transcriptomic analysis, libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v3. Sequencing was performed on NextSeq 2000 system (1x 50 bp, 50 cycles, P3 kit) at Centre de Regulació Genómica (CRG). For the proteomic analysis, Tandem MassTag (TMT) 6-plex isotopic labeling strategy was adopted, and generated peptides were run in a Q Exactive mass spectrometer. To find differentially expressed genes and proteins between both groups EdgeR and Perseus softwares were employed. Main results and the role of chance In the present study, a total of 156.124 transcripts and 1722 proteins were identified in human sperm. Principal Component Analysis (PCA) confirmed the existence of distinguishable differences in the transcriptomic and proteomic profile of control and UMI samples. Specifically, 3112 differentially expressed transcripts and 385 differentially expressed proteins were identified. Among the differentially expressed proteins, 49 were correlated with RNA expression. Coherent RNA-protein signatures were mainly related to axonemal dynein complex assembly and flagellated sperm motility. Proteins involved in energy metabolism were enriched in UMI protein signatures but were not well correlated with the RNA. Furthermore, genes involved in protein transport and chromosome condensation were highly enriched in UMI group but were not detected by proteomics profiling. According to our results, there is a lack of RNA-protein correlation at human sperm. However, differential transcriptomic and proteomic profiles were found when comparing fertile normozoospermic samples to UMI samples. Concretely, our results suggest that altered energy metabolism may be one of the key factors in patients leading with UMI. Thus, the study of human sperm metabolism may be essential to find new targets of male infertility. Limitations, reasons for caution The lack of a consensus on the classification criteria of UMI makes this group of patients very heterogeneous. Further studies are needed to identify and validate specific transcripts and/or proteins that may alter specific metabolic processes and cause infertility. Wider implications of the findings The study of human sperm metabolism may be essential to understand the causes of many UMI cases. In this context, the identification of key protein or transcripts could be very helpful to predict the success of the assisted reproductive treatments. Trial registration number Not applicable

Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

Transcriptomic Characterization of Key Factors and Signaling Pathways for the Regeneration of Partially Hepatectomized Liver in Zebrafish.

Liver regeneration induced by partial hepatectomy (PHx) has attracted intensive research interests due to the great significance for liver resection and transplantation. The zebrafish (Danio rerio) is an excellent model to study liver regeneration. In the fish subjected to PHx (the tip of the ventral lobe was resected), the lost liver mass could be fully regenerated in seven days. However, the regulatory mechanisms underlying the liver regeneration remain largely unknown. In this study, gene expression profiles during the regeneration of PHx-treated liver were explored by RNA sequencing (RNA-seq). The genes responsive to the injury of PHx treatment were identified and classified into different clusters based on the expression profiles. Representative gene ontology (GO) enrichments for the early responsive genes included hormone activity, ribosome biogenesis and rRNA processing, etc., while the late responsive genes were enriched in biological processes such as glutathione metabolic process, antioxidant activity and cellular detoxification. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments were also identified for the differentially expressed genes (DEGs) between the time-series samples and the sham controls. The proteasome was overrepresented by the up-regulated genes at all of the sampling time points. Inhibiting proteasome activity by the application of MG132 to the fish enhanced the expression of Pcna (proliferating cell nuclear antigen), an indicator of hepatocyte proliferation after PHx. Our data provide novel insights into the molecular mechanisms underlying the regeneration of PHx-treated liver.

Generation and transcriptomic characterization of MIR137 knockout miniature pig model for neurodevelopmental disorders

BackgroundNeurodevelopmental disorders (NDD), such as autism spectrum disorders (ASD) and intellectual disorders (ID), are highly debilitating childhood psychiatric conditions. Genetic factors are recognized as playing a major role in NDD, with a multitude of genes and genomic regions implicated. While the functional validation of NDD-associated genes has predominantly been carried out using mouse models, the significant differences in brain structure and gene function between mice and humans have limited the effectiveness of mouse models in exploring the underlying mechanisms of NDD. Therefore, it is important to establish alternative animal models that are more evolutionarily aligned with humans.ResultsIn this study, we employed CRISPR/Cas9 and somatic cell nuclear transplantation technologies to successfully generate a knockout miniature pig model of the MIR137 gene, which encodes the neuropsychiatric disorder-associated microRNA miR-137. The homozygous knockout of MIR137 (MIR137–/–) effectively suppressed the expression of mature miR-137 and led to the birth of stillborn or short-lived piglets. Transcriptomic analysis revealed significant changes in genes associated with neurodevelopment and synaptic signaling in the brains of MIR137–/– miniature pig, mirroring findings from human ASD transcriptomic data. In comparison to miR-137-deficient mouse and human induced pluripotent stem cell (hiPSC)-derived neuron models, the miniature pig model exhibited more consistent changes in critical neuronal genes relevant to humans following the loss of miR-137. Furthermore, a comparative analysis identified differentially expressed genes associated with ASD and ID risk genes in both miniature pig and hiPSC-derived neurons. Notably, human-specific miR-137 targets, such as CAMK2A, known to be linked to cognitive impairments and NDD, exhibited dysregulation in MIR137–/– miniature pigs. These findings suggest that the loss of miR-137 in miniature pigs affects genes crucial for neurodevelopment, potentially contributing to the development of NDD.ConclusionsOur study highlights the impact of miR-137 loss on critical genes involved in neurodevelopment and related disorders in MIR137–/– miniature pigs. It establishes the miniature pig model as a valuable tool for investigating neurodevelopmental disorders, providing valuable insights for potential applications in human research.

Integrating metabolic profiling of pancreatic juice with transcriptomic analysis of pancreatic cancer tissue identifies distinct clinical subgroups.

Metabolic reprogramming is a hallmark feature of pancreatic ductal adenocarcinoma (PDAC). A pancreatic juice (PJ) metabolic signature has been reported to be prognostic of oncological outcome for PDAC. Integration of PJ profiling with transcriptomic and spatial characterization of the tumor microenvironment would help in identifying PDACs with peculiar vulnerabilities. We performed a transcriptomic analysis of 26 PDAC samples grouped into 3 metabolic clusters (M_CL) according to their PJ metabolic profile. We analyzed molecular subtypes and transcriptional differences. Validation was performed by multidimensional imaging on tumor slides. Pancreatic juice metabolic profiling was associated with PDAC transcriptomic molecular subtypes (p=0.004). Tumors identified as M_CL1 exhibited a non-squamous molecular phenotype and demonstrated longer survival. Enrichment analysis revealed the upregulation of immune genes and pathways in M_CL1 samples compared to M_CL2, the group with worse prognosis, a difference confirmed by immunofluorescence on tissue slides. Enrichment analysis of 39 immune signatures by xCell confirmed decreased immune signatures in M_CL2 compared to M_CL1 and allowed a stratification of patients associated with longer survival. PJ metabolic fingerprints reflect PDAC molecular subtypes and the immune microenvironment, confirming PJ as a promising source of biomarkers for personalized therapy.

Transcriptomic and epigenetic characterization of mucinous adenocarcinoma of the cervix.

e17518 Background: Mucinous adenocarcinoma of the cervix (MAC) is a rare and aggressive histologic subtype of cervical cancer with poorer prognosis than its more common counterparts, usual type endocervical adenocarcinoma (UEA) and squamous cell carcinoma (SCC). MAC is also known to be less responsive to standard chemotherapy and radiation approaches used to treat cervical cancer. In this study, we sought to identify transcriptomic and epigenetic features of MAC that distinguish MAC from UEA and SCC, and to identify features that may contribute to poorer prognosis in MAC compared to UEA and SCC. Methods: Clinical data, gene expression, miRNA expression, and methylation data for 17 MAC, 27 UEA, and 247 SCC from The Cancer Genome Atlas Cervical Cancer Project were obtained and analyzed. Clinical data were compared for the histologic groups using Fisher’s Exact, ANOVA, and Kruskal-Wallis statistics where appropriate. Kaplan-Meier curves were used for survival analyses. Gene expression data was analyzed using DESeq2 for differential expression of mRNA. Pathway enrichment analysis of gene expression data was completed using Qiagen IPA Core Analysis software. Comparative marker selection was used to determine differential expression of miRNA, and differential methylation across histologic subtypes. Analyses were completed both by histology and by disease-progression status. Data across all three platforms were integrated to evaluate for potential explanatory relationships of interest. Results: RNAseq analysis revealed a number of genes differentially expressed in MAC as compared to UEA and SCC. Several of these genes were also found to be differentially expressed in samples from patients with disease progression compared to those without known disease progression including increased expression of PPARGC1A, SLC2A1, and ZNF703 and decreased expression of NR4A3. Analysis also revealed hypomethylation at CpG probe sites mapping to SLC2A1 and decreased expression of miRNAs known to target SLC2A1 and PPARGC1A in MAC tumors . Pathways involving translation regulation, EIF2 signaling, and oxidative phosphorylation were downregulated in MAC whereas pathways involving mitochondrial dysfunction, glucose metabolism, and estrogen receptor signaling were upregulated in MAC. Downregulation of immune and inflammatory response pathways and upregulation of tumor-supporting macrophage activation was seen in MAC tumors with disease progression. Conclusions: We identified novel transcriptomic and epigenetic features and altered pathways in MAC, as well as those features and pathways associated with poorer prognosis in this population. These findings may contribute to further confirmatory research and lead to novel therapeutic strategies for this rare cancer.

Transcriptomic and epigenetic characterization of mucinous adenocarcinoma of the cervix.

e17518 Background: Mucinous adenocarcinoma of the cervix (MAC) is a rare and aggressive histologic subtype of cervical cancer with poorer prognosis than its more common counterparts, usual type endocervical adenocarcinoma (UEA) and squamous cell carcinoma (SCC). MAC is also known to be less responsive to standard chemotherapy and radiation approaches used to treat cervical cancer. In this study, we sought to identify transcriptomic and epigenetic features of MAC that distinguish MAC from UEA and SCC, and to identify features that may contribute to poorer prognosis in MAC compared to UEA and SCC. Methods: Clinical data, gene expression, miRNA expression, and methylation data for 17 MAC, 27 UEA, and 247 SCC from The Cancer Genome Atlas Cervical Cancer Project were obtained and analyzed. Clinical data were compared for the histologic groups using Fisher’s Exact, ANOVA, and Kruskal-Wallis statistics where appropriate. Kaplan-Meier curves were used for survival analyses. Gene expression data was analyzed using DESeq2 for differential expression of mRNA. Pathway enrichment analysis of gene expression data was completed using Qiagen IPA Core Analysis software. Comparative marker selection was used to determine differential expression of miRNA, and differential methylation across histologic subtypes. Analyses were completed both by histology and by disease-progression status. Data across all three platforms were integrated to evaluate for potential explanatory relationships of interest. Results: RNAseq analysis revealed a number of genes differentially expressed in MAC as compared to UEA and SCC. Several of these genes were also found to be differentially expressed in samples from patients with disease progression compared to those without known disease progression including increased expression of PPARGC1A, SLC2A1, and ZNF703 and decreased expression of NR4A3. Analysis also revealed hypomethylation at CpG probe sites mapping to SLC2A1 and decreased expression of miRNAs known to target SLC2A1 and PPARGC1A in MAC tumors . Pathways involving translation regulation, EIF2 signaling, and oxidative phosphorylation were downregulated in MAC whereas pathways involving mitochondrial dysfunction, glucose metabolism, and estrogen receptor signaling were upregulated in MAC. Downregulation of immune and inflammatory response pathways and upregulation of tumor-supporting macrophage activation was seen in MAC tumors with disease progression. Conclusions: We identified novel transcriptomic and epigenetic features and altered pathways in MAC, as well as those features and pathways associated with poorer prognosis in this population. These findings may contribute to further confirmatory research and lead to novel therapeutic strategies for this rare cancer.

Integrated genomic and transcriptomic characterization of neuroendocrine carcinomas for improved treatment decision-making.

e15138 Background: Neuroendocrine carcinomas (NECs) are poorly characterized due to their innate heterogeneity, thus limiting treatment and clinical trial options for patients. Here, we used whole exome sequencing (WES) and RNA sequencing (RNA-seq) to explore the genetic and microenvironmental landscape of NECs in order to understand the unique features of different NEC types, with the aim of improving treatment modalities. Methods: We analyzed 51 NEC clinical samples morphologically verified by a pathologist: lung NEC (20 small cell NEC and 1 large cell NEC cases), gastroenteropancreatic NEC (GEP, 12 cases), cancer of unknown primary (7 cases), Merkel cell carcinoma (MCC, 5 cases), genitourinary tract NEC (3 cases), and breast NEC (3 cases). Findings on tumor mutational burden (TMB, mut/Mb), microsatellite instability (MSI), gene expression signatures, and tumor microenvironment (TME) profiling were reported. Small cell lung cancer (SCLC) subtyping was performed as reported by Gay et al. on the entire NEC cohort. Results: Half of the patients with lung NEC and 84% of those with extrapulmonary NEC satisfied the molecular inclusion criteria for clinical trials, with the most frequent criterion being KRAS G12X or G13X mutations. Among the extrapulmonary NEC samples, 13.7% were deemed eligible for targeted therapy, specifically for cases with BRAF V600E mutation and/or high TMB (> 10 mut/Mb). We detected varying TMB and distinct gene expression features across the entire cohort. Specifically, we found 2 MCC cases to be Merkel cell polyomavirus-positive with low TMB and devoid of UV-associated mutations. One GEP-NEC case exhibited a high MSI (MSI-high) status. We detected 6 SCLC cases (3 non-smokers, 3 former smokers) with surprisingly low levels of tobacco-associated mutations (<10% of all mutations). Two GEP-NEC cases had high levels of mutations associated with tobacco smoking. While TME profiling showed no significant difference in cell population makeup across all samples, transcriptional SCLC subtyping revealed significant differences among the cases. Inflammatory SCLC (SCLC-I) samples showed statistically significant enrichment of all subpopulations of the immune microenvironment (i.e., B cells, T cells, NK cells), supporting the use of immune checkpoint blockade for treatment. The POU2F3+ SCLC (SCLC-P) samples showed low expression of all neuroendocrine diagnostic markers (i.e., INSM1, NCAM1, SYP, CHGA). Conclusions: Given the heterogeneous molecular features across diverse NEC tumors, a uniform treatment strategy is unlikely to benefit all NEC patients. Hence, our ability to detect these unique molecular features is crucial for matching individual NEC patients with specific clinical trials and targeted therapies that may improve their outcomes. Thus, a concerted effort using in-depth genomic, transcriptomic, and TME profiling for NEC tumors is warranted.

Insights into the heterogeneity of the tumor microenvironment in lung adenocarcinoma and squamous carcinoma through single‐cell transcriptomic analysis: Implications for distinct immunotherapy outcomes

AbstractBackgroundImmune checkpoint blockade has emerged as a key strategy to the therapy landscape of non‐small cell lung cancer (NSCLC). However, notable differences in immunotherapeutic outcomes exist between the two primary NSCLC subtypes: lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). This disparity may stem from the tumor immune microenvironment's heterogeneity at the transcriptome level.MethodsBy integrative analysis of transcriptomic characterization of 38 NSCLC patients by single‐cell RNA sequencing, the present study revealed a distinct tumor microenvironment (TME) between LUAD and LUSC, with relevant results further confirmed in bulk transcriptomic and multiplex immunofluorescence (mIF) validation cohort of neoadjuvant immunotherapy patients.ResultsLUAD exhibited a more active immune microenvironment compared to LUSC. This included highly expression of HLA I/II in cancer cells, reinforced antigen presentation potential of dendritic cells and enhanced cytotoxic activity observed in T/NK cells. In LUSC, cancer cells highly expressed genes belonging to the aldo‐keto reductases, glutathione S‐transferases and aldehyde dehydrogenase family, negatively correlating with immunotherapy outcomes in the validation cohort of our center. Further analysis revealed elevated infiltrated cancer‐associated fibroblasts (CAFs) in LUSC, which was corroborated in The Cancer Genome Atlas cohort. Corresponding increased infiltration of ADH1B+ CAFs in major pathologic response (MPR) patients and the higher presence of FAP+ CAFs in non‐MPR patients were demonstrated by multiplex mIF. Moreover, upregulating immunosuppressive extracellular matrix remodeling was identified in LUSC.ConclusionsThese comprehensive analyses advance the understanding of the differences in TME between LUAD and LUSC, offering insights for patient selection and developing subtype‐specific treatment strategies.

LRRK2 G2019S variant is associated with transcriptional changes in Parkinson's disease human myeloid cells under proinflammatory environment.

The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously expressed, is highly abundant in cells of the innate immune system. Given the importance of central and peripheral immune cells in the development of PD, we sought to investigate the consequences of the G2019S mutation on microglial and monocyte transcriptome and function. We have generated large-scale transcriptomic profiles of isogenic human induced microglial cells (iMGLs) and patient derived monocytes carrying the G2019S mutation under baseline culture conditions and following exposure to the proinflammatory factors IFNγ and LPS. We demonstrate that the G2019S mutation exerts a profound impact on the transcriptomic profile of these myeloid cells, and describe corresponding functional differences in iMGLs. The G2019S mutation led to an upregulation in lipid metabolism and phagolysosomal pathway genes in untreated and LPS/IFNγ stimulated iMGLs, which was accompanied by an increased phagocytic capacity of myelin debris. We also identified dysregulation of cell cycle genes, with a downregulation of the E2F4 regulon. Transcriptomic characterization of human-derived monocytes carrying the G2019S mutation confirmed alteration in lipid metabolism associated genes. Altogether, these findings reveal the influence of G2019S on the dysregulation of the myeloid cell transcriptome under proinflammatory conditions.

Physiological and transcriptome characterization provide new sights into cadmium tolerance and accumulation mechanisms in Tagetes patula L.

Tagetes patula L. (T. patula) is a Cd hyperaccumulator plant with high ornamental value and strong resistance. Understanding the molecular mechanisms of Cd tolerance in hyper-accumulating plants is important for improving phytoremediation efficiency. This investigation studied to how Cd affected T. patula's physiological characteristics and molecular alterations. With the increase of Cd concentration, chlorophyll content, and root biomass decreased; the activities of antioxidant enzymes such as GST, SOD and POD increased. Initially, Cd accumulated mainly in the root and then transferred upward to the leaves. The molecular mechanisms of T. patula after 150 mg/kg and 250 mg/kg treatments were analyzed by transcriptome. In total, 5429 and 6546 differentially expressed unigenes were identified. Phenylpropanoid biosynthesis and phenylalanine metabolism were identified as key pathways in reaction to Cd stress. In addition, many differential genes involved in the response to Cd stress were identified, including heavy metal transporter protein genes, genes and transcription factors related to Cd detoxification. These results indicate that the response of T. patula to Cd was multifaceted, laying the foundation for studies to improve its tolerance to toxic metals.

Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs which have demonstrated limited effectiveness in OC patients. We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). In the SKOV-3 Luc Hu-mouse model, we assessed the efficacy of PD-1 blockade with pembrolizumab. We observed the presence of human lymphocyte and myeloid cell subsets within the tumors, lymph nodes, blood, and spleens in these models. Notably, these tumors exhibited a high prevalence of tumor-infiltrating macrophages. Furthermore, we identified HDAC class I target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts in the tumors of Hu-mice treated with pembrolizumab. Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer useful information to explore potential mechanisms associated with unresponsive anti-PD-1 treatment in OC.

Integrated sequence-based genomic, transcriptomic, and methylation characterization of the susceptibility to tuberculosis in monozygous twins

BackgroundTuberculosis (TB) is a complex disease with a spectrum of outcomes for more than six decades; however, the genomic and epigenetic mechanisms underlying the highly heritable susceptibility to TB remain unclear. MethodsIntegrated sequence-based genomic, transcriptomic, and methylation analyses were conducted to identity the genetic factors associated with susceptibility to TB in two pairs of Mongolian monozygous twins. In this study, whole-genome sequencing was employed to analyze single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), and copy number variations (CNVs). Gene expression was assessed through RNA sequencing, and methylation patterns were examined using the Illumina Infinium Methylation EPIC BeadChip. The gene–gene interaction network was analyzed using differentially expressed genes. ResultsOur study revealed no significant difference in SNP and InDel profiles between participants with and without TB. Genes with CNVs were involved in human immunity (human leukocyte antigen [HLA] family and interferon [IFN] pathway) and the inflammatory response. Different DNA methylation patterns and mRNA expression profiles were observed in genes participating in immunity (HLA family) and inflammatory responses (IFNA, interleukin 10 receptor [IL-10R], IL-12B, Toll-like receptor, and IL-1B). ConclusionsThe results of this study suggested that susceptibility to TB is associated with transcriptional and epigenetic alternations of genes involved in immune and inflammatory responses. The genes in the HLA family (HLA-A, HLA-B, and HLA-DRB1) and IFN pathway (IFN-α and IFN-γ) may play major roles in susceptibility to TB.

Disruption of memory B-cell trafficking by belimumab in patients with systemic lupus erythematosus.

Autoreactive memory B cells contribute to chronic and progressive courses in autoimmune diseases like systemic lupus erythematosus (SLE). The efficacy of belimumab (BEL), the first approved biologic treatment for SLE and lupus nephritis (LN), is generally attributed to depletion of activated naïve B cells and inhibition of B cell activation. BEL's effect on memory B cells (MBCs) is currently unexplained. We performed an in-depth cellular and transcriptomic analysis of BEL's impact on the blood MBC compartment in patients with SLE. A retrospective meta-analysis was conducted, pooling flow cytometry data from four randomized trials involving 1245 patients with SLE treated with intravenous BEL or placebo. Then, extensive MBC phenotyping was performed using high-sensitivity flow cytometry in patients with mild/moderate SLE and severe SLE/LN treated with subcutaneous BEL. Finally, transcriptomic characterization of surging MBCs was performed by single-cell RNA sequencing. In BEL-treated patients, a significant increase in circulating MBCs, in a broad range of MBC subsets, was established at week 2, gradually returning to baseline by week 52. The increase was most prominent in patients with higher SLE disease activity, serologically active patients, and patients aged ≤18 years. MBCs had a non-proliferating phenotype with a prominent decrease in activation status and downregulation of numerous migration genes. Upon BEL initiation, an increase of MBCs was firmly established. In the small cohort investigated, circulating MBCs were de-activated, non-proliferative, and demonstrated characteristics of disrupted lymphocyte trafficking, expanding on our understanding of the therapeutic mechanism of B cell-activating factor inhibition by BEL. ClinicalTrials.gov NCT00071487, NCT00410384, NCT01632241, NCT01649765, NCT03312907, NCT03747159.

Transcriptomic characterization of lung pericytes in systemic sclerosis-associated pulmonary fibrosis

Systemic sclerosis (SSc) is a chronic disease characterized by fibrosis and vascular abnormalities in the skin and internal organs, including the lung. SSc-associated pulmonary fibrosis (SSc-PF) is the leading cause of death in SSc patients. Pericytes are key regulators of vascular integrity and endothelial function. The role that pericytes play in SSc-PF remains unclear. We compared the transcriptome of pericytes from SSc-PF lungs (SScL) to pericytes from normal lungs (NORML). We identified 1,179 differentially expressed genes in SScL pericytes. Pathways enriched in SScL pericytes included prostaglandin, PI3K-AKT, calcium, and vascular remodeling signaling. Decreased cyclic AMP production and altered phosphorylation of AKT in response to prostaglandin E2 in SScL pericytes demonstrate the functional consequence of changes in the prostaglandin pathway that may contribute to fibrosis. The transcriptomic signature of SSc lung pericytes suggests that they promote vascular dysfunction and contribute to the loss of protection against lung inflammation and fibrosis.

Immunohistochemical and transcriptomic characterization of T and myeloid cell infiltrates in canine malignant melanoma.

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm2 and 166.7 cells/mm2, respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation.

Comprehensive genomic and transcriptomic characterization of high-grade gastro-entero-pancreatic neoplasms

BackgroundHigh-grade gastro-entero-pancreatic neoplasms (HG GEP-NENs) can be stratified according to their morphology and Ki-67 values into three prognostic classes: neuroendocrine tumors grade 3 (NETs G3), neuroendocrine carcinomas with Ki-67 < 55% (NECs <55) and NECs with Ki-67 ≥ 55% (NECs ≥55).MethodsWe analyzed a cohort of 49 HG GEP-NENs by targeted Next-Generation Sequencing (TrueSight Oncology 500), RNA-seq, and immunohistochemistry for p53, Rb1, SSTR-2A, and PD-L1.ResultsFrequent genomic alterations affected TP53 (26%), APC (20%), KRAS and MEN1 (both 11%) genes. NET G3 were enriched in MEN1 (p = 0.02) mutations, while both NECs groups were enriched in TP53 (p = 0.001), APC (p = 0.002) and KRAS (p = 0.02) mutations and tumors with TMB ≥ 10 muts/Mb (p = 0.01). No differentially expressed (DE) gene was found between NECs <55% and NECs ≥55%, while 1129 DE genes were identified between NET G3 and NECs. A slight enrichment of CD4+ and CD8+ T cells in NECs and of cancer-associated fibroblasts and macrophages (M2-like) in NET G3. Multivariate analysis identified histologic type and Rb1 loss as independent prognostic factors for overall survival.ConclusionsThis study showed that GEP-NET G3 and GEP-NECs exhibit clear genomic and transcriptomic differences, differently from GEP-NECs <55% and GEP-NECs ≥55%, and provided molecular findings with prognostic and potentially predictive value.

Transcriptomic characterization of single cells derived from immortalized human thyroid follicular epithelial cell lines

Transcriptomic characterization of single cells derived from immortalized human thyroid follicular epithelial cell lines

Acinar to β-like cell conversion through inhibition of focal adhesion kinase

Insufficient functional β-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing β-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore β-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.