Cell RNA Analysis Research Articles

Characterising plasmacytoid and myeloid AXL+ SIGLEC-6+ dendritic cell functions and their interactions with HIV.

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered.

Single cell RNA analysis uncovers the cell differentiation and functionalization for air breathing of frog lung

The evolution and development of vertebrate lungs have been widely studied due to their significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring their evolutionary importance in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development remains poorly understood. Using single-cell RNA sequencing, we identify 13 cell types in the developing lungs of a land-dwelling frog (Microhyla fissipes). We elucidate the differentiation trajectories and mechanisms of mesenchymal cells, identifying five cell fates and their respective driver genes. Using temporal dynamics analyses, we reveal the gene expression switches of epithelial cells, which facilitate air breathing during metamorphosis. Furthermore, by integrating the published data from another amphibian and two terrestrial mammals, we illuminate both conserved and divergent cellular repertoires during the evolution of tetrapod lungs. These findings uncover the frog lung cell differentiation trajectories and functionalization for breathing in air and provide valuable insights into the cell-type evolution of vertebrate lungs.

Multi-omics analysis reveals cuproptosis and mitochondria-based signature for assessing prognosis and immune landscape in osteosarcoma.

Osteosarcoma (OSA), the most common primary mesenchymal bone tumor, is a health threat to children and adolescents with a dismal prognosis. While cuproptosis and mitochondria dysfunction have been demonstrated to exert a crucial role in tumor progression and development, the mechanisms by which they are regulated in OSA still await clarification. Two independent OSA cohorts containing transcriptome data and clinical information were collected from public databases. The heterogeneity of OSA were evaluated by single cell RNA (scRNA) analysis. To identify a newly molecular subtype, unsupervised consensus clustering was conducted. Cox relevant regression methods were utilized to establish a prognostic gene signature. Wet lab experiments were performed to confirm the effect of model gene in OSA cells. We determined 30 distinct cell clusters and assessed OSA heterogeneity and stemness scRNA analysis. Then, univariate Cox analysis identified 24 candidate genes which were greatly associated with the prognosis of OSA. Based on these prognostic genes, we obtained two molecular subgroups. After conducting step Cox regression, three model genes were selected to construct a signature showing a favorable performance to forecast clinical outcome. Our proposed signature could also evaluate the response to chemotherapy and immunotherapy of OSA cases. We generated a novel risk model based on cuproptosis and mitochondria-related genes in OSA with powerful predictive ability in prognosis and immune landscape.

A workflow for the enrichment, the identification, and the isolation of non-apoptotic single circulating tumor cells for RNA sequencing analysis.

Circulating tumor cells (CTCs) are constantly shed by tumor tissue and can serve as a valuable analyte for a gene expression analysis from a liquid biopsy. However, a high proportion of CTCs can be apoptotic leading to rapid mRNA decay and challenging the analysis of their transcriptome. We established a workflow to enrich, to identify, and to isolate single CTCs including the discrimination of apoptotic and non-apoptotic CTCs for further single CTC transcriptome analysis. Viable tumor cells-we first used cells from breast cancer cell lines followed by CTCs from metastatic breast cancer patients-were enriched with the CellSearch system from diagnostic leukapheresis products, identified by immunofluorescence analysis for neoplastic markers, and isolated by micromanipulation. Then, their cDNA was generated, amplified, and sequenced. In order to exclude early apoptotic tumor cells, staining with Annexin V coupled to a fluorescent dye was used. Annexin V staining intensity was associated with decreased RNA integrity as well as lower numbers of total reads, exon reads, and detected genes in cell line cells and CTCs. A comparative RNA analysis of single cells from MDA-MB-231 and MCF7 cell lines revealed the expected differential transcriptome profiles. Enrichment and staining procedures of cell line cells that were spiked into blood had only little effect on the obtained RNA sequencing data compared to processing of naïve cells. Further, the detection of transcripts of housekeeping genes such as GAPDH was associated with a significantly higher quality of expression data from CTCs. This workflow enables the enrichment, detection, and isolation of single CTCs for individual transcriptome analyses. The discrimination of apoptotic and non-apoptotic cells allows to focus on CTCs with a high RNA integrity to ensure a successful transcriptome analysis.

A High-Risk Subgroup Multiple Myeloma Classification Based on the Detection of PR Minor Subclones

In the last two decades several classifications of Multiple Myeloma (MM) have been proposed based on gene expression signatures. These include the supervised translocation and cyclin D (TC) molecular subgroup approach or unsupervised methods to elucidate the molecular heterogeneity of MM and identify high risk subgroups. In particular, two RNA based gene expression studies have identified 8 to 10 unique subtypes, including the MS, MAF, CD1, CD2, and PR subtypes. The PR subgroup represents a highly proliferative gene signature that is associated with very poor survival outcomes. However, assigning this PR subgroup based on bulk transcriptome signatures can be challenging giving the mixture of genetic backgrounds that can be observed within this subtype. Furthermore, the presence of a minor PR subclone can often be missed with bulk genomic studies leading to the misclassification of a patient as standard risk. We here postulated that minor PR subclones expand under selective therapeutic pressures to become the predominant clones at subsequent relapses and the presence of PR subclones can impact any of the molecular subgroups pertaining poor survival outcomes. Bulk RNA or single cell RNA (scRNAseq) analysis were performed in bone marrow sorted primary MM cells obtained from 718 patients from the CoMMpass trialand 32 relapse/refractory MM patients, respectively. For scRNAseq, serial samples with paired baseline and relapse samples were available in 18 cases. Bone marrow plasma cells samples were separated from bone marrow aspirates using positive sorting selection of CD138 + cells. For bulk RNA-seq we used the mRNA HyperPrep kit (Kapa Biosystems) with RNA from 50,000 cells.For scRNA studiesunbiased mRNA profiling was conducted using the GemCode system (10x Genomics). Cell Ranger and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3' gene and data analysis. Sequencing was performed on Illumina. By performing GSEA at the single cell level using the Zhan gene data set we have classified patients in the appropriate TC groups. FISH data was used to insure accurate calls. For the survival analysis OS was analyzed by the Kaplan-Meier method. In the CoMMpass dataset, with bulk RNA sequencing, the PR subtype contained 7.1% of patients with an admixture of classic genetic subtypes and very poor clinical outcome, with a median OS of 21.3 months. High proliferation index scores were concentrated in this subtype. There was also a strong tendency for patients to transition to the PR subtype at progression, with 25.5% of patients in a non-PR subtype at diagnosis transitioning to PR. Regardless of original subtype, patients that transitioned to the PR subtype rapidly succumbed to their disease with a median OS after the detected progression of 88 days. Using subtype prediction weight we could identify non-PR patients with PR subtype weights above 5% and these patients have a significantly shorter OS of 50 months compared to patients with <5% PR. At the single cell level, investigating the relapsed patients cohort, we have analyzed 92,139 cells and identified 59 clusters. A PR signature (defined as present in ≥15% of cells) was identified in 38% of cases despite baseline favorable prognostic group by FISH cytogenetics (7% HP, 28% t(11;14), and 57% normal FISH). A good concordance with FISH data was observed. In serially relapsed paired samples, all the PR patients maintain their signature overtime and 44% of patients transitioned to PR at subsequent relapse (Fig1A). Survival analyses were performed to evaluate how the presence of minor PR clones at the single cell level relate to clinical outcomes. Notably, the overall survival of patients classified as PR was dramatically reduced with a median OS of 6 months compared to 34 months for non-PR patients (Fig1B). In conclusion, we here show that minor PR clones defined by transcriptional signature through bulk or scRNA may be detectable in patients with variable MM molecular subgroups, including those classified as standard risk by the currently adopted classifications. We also show that the presence of these PR minor clones pertains adverse outcomes with very poor survivals. At relapse, most acquired PR cases originated from standard-risk presentations consistent with a Darwinian clonal evolution. Future classifications should account for the presence of these PR subclones for a better disease prognostication and developments of novel therapeutics.

Immune and Genome Profiling of Myeloma Patients Treated with Sequential Immunotherapies Reveal Differential Non-Overlapping Mechanisms of Resistance

Chimeric antigen receptor T cells (CAR T) or bispecific T cell engagers (TCE) targeting multiple myeloma (MM) antigens result in deep responses in relapsed patients. Nevertheless, resistance invariably emerges requiring salvage treatments. Sequential therapies with CAR T or TCE, in patients with or without prior anti-BCMA therapy exposure, reported durable responses. However the genomic and molecular determinants of response to sequential immunotherapies are poorly defined. To define the molecular determinants of response to sequential immunotherapies we identified patients treated with more than one anti-BCMA or -GPRC5D (n=8) regimens. Bone marrow (BM) and peripheral blood (PB) mononuclear cells from these patients were collected prior to each therapy and at the time of progression. Sorted CD138+ cells were subjected to whole genome sequencing (WGS) or single cell CNV (scCNV) and single cell RNA (scRNA) analysis. CD3+ T cells were subjected to CITEseq and functional in vitro cytotoxicity assays. Patient 1 received anti-BCMA CAR T (Ide-cel) with VGPR lasting 7 months (mos) (Figure 1). He subsequently received elranatamab with no response. CITEseq profiling of T cells at progression post CAR T did not reveal expansion of exhausted cytotoxic or regulatory T cells. However, WGS and scCNV of MM cells identified biallelic TNFRSF17 deletion consistent with antigenic escape as cause of resistance. Patient 2 received Ide-cel (DOR = 3.5 mos) followed by elranatamab with no response. CD138+ MM cells retained BCMA expression and in vitro studies with in-house manufactured anti-BCMA CAR T efficiently eliminated his MM cells. Profiling of T cells showed terminal T cells exhaustion (CD45RA +, TOX +, TIGIT +, PDCD1 + and LAG3 +). In contrast to recipients of CAR T, antigenic loss was commonly observed post TCE therapy. Patient 3 received 3 sequential therapies with Ide-cel (DOR = 3 mos), teclistamab (DOR = 6 mos), and then talquetamab with daratumumab with an ongoing response of 11 mos. While T cell exhaustion was not observed, functional loss of BCMA on MM cells was detected post teclistamab, with 2 coexisting clones harboring TNFRSF17 extracellular domain mutations (p.Pro34del, or p.Ser30del) that abrogated teclistamab binding. Similarly in patient 4 progressing on elranatamab (DOR = 21 mos), there was no evidence of T cell exhaustion but rather acquired BCMA mutation (p.Pro34del) in MM cells. Consistent with fit T cells profile, he has an ongoing response (13+ mos) to talquetamab and daratumumab in his next line of therapy. Patient 5 with penta-refractory disease and high disease burden (> 90% BM infiltration) had no response to elranatamab, however achieved an ongoing sCR (DOR = 30 mos) with talquetamab, daratumumab and pomalidomide (Tal-DP). PB T cells did not show exhaustion at the time of progression on elranatamab. A similar pattern was observed in patient 6 with high disease burden and no response to teclistamab but achieved lasting VGPR to Tal-DP (DOR=24 mos) in the next line followed by an acquired biallelic loss of GPRC5D at progression. Patient 7 had a 17 mos remission to talquetamab and daratumumab and similarly to patient 6, he acquired biallelic loss of GPRC5D at progression. He subsequently received elranatamab with no response with elevated serum soluble BCMA (sBCMA=638 ng/ml) level and phenotypic and functional evidence of T cell exhaustion. Patients 5, 6 and 7 illustrate the impact of disease burden and the ensuing sBCMA sink effect on the efficacy of anti-BCMA TCEs. Lastly patient 8 had lasting remissions to elranatamab (DOR 13 mos) and subsequently to talquetamab (DOR 13 mos) with no evidence of T cells exhaustion but rather antigenic escape with acquired functional antigenic losses with clonal BCMA (p.Arg27Pro) and subsequently GPRC5D (p.Asp239Asn) mutation. Highlighting the lack of cross resistance between anti-BCMA therapies, our in vitro studies showed that while mutant BCMA (p.Ser30del or p.Pro34del) abrogated teclistamab mediated T cell killing, T cells transduced with teclistamab based scFv CAR retained cytolytic activity. Therefore, TCE resistance derived from BCMA mutations does not preclude retreatment with another anti-BCMA TCE or CAR T. We here describe variable non-overlapping mechanisms mediating resistance to sequential TCE and CAR T therapies. Dynamic surveillance for antigenic escape and functional evaluation of T cell fitness will optimize immunotherapy sequencing.

Interaction of Mesonivirus and Negevirus with arboviruses and the RNAi response in Culex tarsalis-derived cells

BackgroundMosquito-specific viruses (MSVs) comprise a variety of different virus families, some of which are known to interfere with infections of medically important arboviruses. Viruses belonging to the family Mesoniviridae or taxon Negevirus harbor several insect-specific viruses, including MSVs, which are known for their wide geographical distribution and extensive host ranges. Although these viruses are regularly identified in mosquitoes all over the world, their presence in mosquitoes in Germany had not yet been reported.MethodsA mix of three MSVs (Yichang virus [Mesoniviridae] and two negeviruses [Daeseongdong virus and Dezidougou virus]) in a sample that contained a pool of Coquillettidia richiardii mosquitoes collected in Germany was used to investigate the interaction of these viruses with different arboviruses in Culex-derived cells. In addition, small RNA sequencing and analysis of different mosquito-derived cells infected with this MSV mix were performed.ResultsA strain of Yichang virus (Mesoniviridae) and two negeviruses (Daeseongdong virus and Dezidougou virus) were identified in the Cq. richiardii mosquitoes sampled in Germany, expanding current knowledge of their circulation in central Europe. Infection of mosquito-derived cells with these three viruses revealed that they are targeted by the small interfering RNA (siRNA) pathway. In Culex-derived cells, co-infection by these three viruses had varying effects on the representative arboviruses from different virus families (Togaviridae: Semliki forest virus [SFV]; Bunyavirales: Bunyamwera orthobunyavirus [BUNV]; or Flaviviridae: Usutu virus [USUV]). Specifically, persistent MSV co-infection inhibited BUNV infection, as well as USUV infection (but the latter only at specific time points). However, the impact on SFV infection was only noticeable at low multiplicity of infection (MOI 0.1) and at specific time points in combination with the infection status.ConclusionsTaken together, these results are important findings that will lead to a better understanding of the complex interactions of MSVs, mosquitoes and arboviruses.Graphical

Regulation of microRNA by circular RNA.

Circular (circ)RNAs have emerged as novel regulators of gene expression through various mechanisms. However, most publications focus on functional circRNAs regulating target gene expression by interacting with micro (mi)RNAs and acting as competing endogenous RNAs (ceRNAs). Although the theory of miRNA sponging by ceRNAs suggests the inhibition of miRNA activity, many studies are biased toward the selection of miRNAs showing a reverse expression pattern compared with circRNA expression. Although several computational tools and molecular assays have been used to predict and validate the interaction of miRNAs with circRNAs, the actual validation of functional in vivo interactions needs careful consideration of molecular experiments with specific controls. As extensive research is being performed on circRNA, many questions arise on the functional significance of circRNA-miRNA interactions. We hope the critical discussion on the criteria for selecting circRNA-miRNA pairs for functional analysis and providing standard methods for validating circRNA-miRNA interactions will advance our understanding of circRNAs as novel gene regulators. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Translation > Regulation RNA Methods > RNA Analyses in Cells.

Unsuspected consequences of synonymous and missense variants in OCA2 can be detected in blood cell RNA samples of patients with albinism.

Oculocutaneous albinism type 2 (OCA2) is the second most frequent form of albinism and represents about 30% of OCA worldwide. As with all types of OCA, patients present with hypopigmentation of hair and skin, as well as severe visual abnormalities. We focused on a subgroup of 29 patients for whom genetic diagnosis was pending because at least one of their identified variants in or around exon 10 of OCA2 is of uncertain significance (VUS). By minigene assay, we investigated the effect of these VUS on exon 10 skipping and showed that not only intronic but also some synonymous variants can result in enhanced exon skipping. We further found that excessive skipping of exon 10 could be detected directly on blood samples of patients and of their one parent with the causal variant, avoiding invasive skin biopsies. Moreover, we show that variants, which result in lack of detectable OCA2 mRNA can be identified from blood samples as well, as shown for the most common OCA2 pathogenic missense variant c.1327G>A/p.(Val443Ile). In conclusion, blood cell RNA analysis allows testing the potential effect of any OCA2 VUS on transcription products. This should help to elucidate yet unsolved OCA2 patients and improve genetic counseling.

A novel classifier combining G protein-coupled receptors and the tumor microenvironment is associated with survival status in glioblastoma.

Background: Numerous studies have highlighted the crucial role of G protein-coupled receptors (GPCRs) in tumor microenvironment (TME) remodeling and their correlation with tumor progression. However, the association between GPCRs and the TME in glioblastoma (GBM) remains largely unexplored. Methods: In this study, we investigated the expression profile of GPCRs in GBM using integrated data from single-cell RNA sequencing and bulk sequencing. Surgical samples obtained from meningioma and GBM patients underwent single-cell RNA sequencing to examine GPCR levels and cell-cell interactions. Tumor microenvironment (TME) score is calculated by the infiltrated immune cells with CIBERSORT. Results: Our findings revealed a predominantly increased expression of GPCRs in GBM, and demonstrated that the classification of GPCRs and TME is an independent risk factor in GBM. Patients with high GPCR expression in the tumor tissue and low TME score exhibited the worst outcomes, suggesting a potentially aggressive tumor phenotype. On the other hand, patients with low GPCR expression in the tumor tissue and high TME score showed significantly better outcomes, indicating a potentially more favorable tumor microenvironment. Furthermore, the study found that T cells with high GPCR levels displayed extensive cell-cell connections with other tumor and immune cells in the single cell RNA analysis, indicating their potential involvement in immune escape. Conclusion: In conclusion, GPCRs in combination with TME classification can serve as prognostic markers for GBM. GPCRs play an essential role in tumor progression and the TME in GBM.

Insights gained from single-cell RNA analysis of murine endothelial cells in aging hearts

Aging is the strongest risk factor for cardiovascular disease, with progressive decline in the function of vascular endothelial cells (ECs) with age. Systematic analyses of the effects of aging on different cardiac EC types remain limited. Here, we constructed a scRNA atlas of EC transcriptomes in young and old mouse hearts. We identified 10 EC subclusters. The multidimensionally differential genes (DEGs) analysis across different EC clusters shows molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the decrease in angiogenesis and cytoskeletal support capacity of aged ECs. And we performed an in-depth analysis of 3 special ECs, Immunology, Proliferating and Angiogenic. The Immunology EC seems highly associated with some immune regulatory functions, which decline with aging at different degrees. Analysis of two types of neovascular ECs, Proliferating, Angiogenic, implied that Angiogenic ECs can differentiate into multiple EC directions after initially originating from proliferating ECs. And aging leads to a decrease in the ability of vascular angiogenesis and differentiation. Finally, we summarized the effects of aging on cell signaling communication between different EC clusters. This cardiac EC atlas offers comprehensive insights into the molecular regulations of cardiovascular aging, and provides new directions for the prevention and treatment of age-related cardiovascular disease.

Single cell RNA analysis of the left–right organizer transcriptome reveals potential novel heterotaxy genes

The establishment of left–right patterning in mice occurs at a transient structure called the embryonic node or left–right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0–1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.

FAM46C-mediated tumor heterogeneity predicts extramedullary metastasis and poorer survival in multiple myeloma.

Cancers originate from a single cell according to Nowell's theory of clonal evolution. The enrichment of the most aggressive clones has been developed and the heterogeneity arises for genomic instability and environmental selection. Multiple myeloma (MM) is a multiple relapse plasma cell cancer generated from bone marrow. Although there were accumulating researches in multiple myeloma pathogenesis, the heterogeneity remains poorly understood. The participants enrolled in this study were 4 EMP+ (EMP, Extramedullary plasmacytoma) and 2 EMP- primarily untreated MM patients. Single cell RNA sequencing and analysis were conducted for the single cell suspension, which was sorted by flow cytometry from peripheral blood mononuclear cells or bone marrow cells. In our research, the results of single cell RNA sequencing show that FAM46C determines MM tumor heterogeneity predicting extramedullary metastasis by influencing RNA stability. Further, we integrated and analyzed 2280 multiple myeloma samples from 7 independent datasets, which uncover that FAM46C mediated tumor heterogeneity predicts poorer survival in multiple myeloma.

Abstract B003: Inactivation mutations of Kmt2c/d license a molecular “field effect” and prime the urothelium for tumorigenesis

Abstract Urothelial carcinoma (UC) is widely recognized to arise from a “field” of precancerous but histologically normal urothelium (HNU). The molecular mechanism that licenses the “field effect” remains elusive. Recent studies revealed prevalent KMT2C and KMT2D loss-of-function (LOF) mutations in HNU and cancer adjacent urothelium, suggesting their potential involvement in UC initiation. Here, we demonstrated that knockout (KO) of Kmt2c and/or Kmt2d in murine urothelial cells induced drastic alterations of cellular states by single cell RNA analysis, but was insufficient to induce robust histological changes. Kmt2c/d loss enhanced organoid formation efficiency, induced epithelial-mesenchymal transition (EMT), and impaired urothelial differentiation, indicating the augmented lineage plasticity after Kmt2c/d KO. Additionally, we identified that Kmt2c/d KO induced a pre-tumorigenic transcriptome with increased enrichments of gene sets associated with inflammation and decreased enrichments of gene sets associated with differentiation. Consequently, loss of Kmt2c/d sensitized urothelial cells to common oncogenic mutations to initiate UC in mouse models. We further observed that KO of Kmt2c/d increased tumorigenic susceptibility in carcinogen-induced UC model. Mechanistically, we observed decreased H3K4me1, H3K27Ac histone marks and decreased enhancer RNA production at the majority of enhancers that correlate with downregulation of urothelial specific lineage gene expression after Kmt2c/d KO. Furthermore, we observed increased Menin deposition on promoters of up-regulated genes. Blockade of Menin-KMT2A complex by small molecule inhibitor partially rescued the EMT and basal differentiation induced by Kmt2c/d KO. Together, our data posit that Kmt2c/d represents a key molecular determinant and their functional loss licenses a molecular “field effect” which primes the urothelium for oncogenic transformation. Citation Format: Naitao Wang, Mohini R. Pachai, Dan Li, Cindy Lee, Sarah Warda, Guojia Xie, Cheng Qian, Wai Pung E. Wong, Juan Yan, Wenhuo Hu, Alison Smith, Kai Ge, Sarat Chandarlapaty, Gopakumar V. Iyer, Jonathan E. Rosenberg, David B. Solit, Hikmat A. AI-Ahmadie, Ping Chi, Yu Chen. Inactivation mutations of Kmt2c/d license a molecular “field effect” and prime the urothelium for tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B003.

Peroxidase is a novel potential marker in glioblastoma through bioinformatics method and experimental validation.

Peroxidase (PXDN), a specific extracellular matrix (ECM)-associated protein, has been determined as a tumor indicator and therapeutic target in various tumors. However, the effects of PXDN in prognostic performance and clinical implications in glioblastoma multiforme (GBM) remains unknown. Here, we assessed PXDN expression pattern and its performance on prognosis among GBM cases from TCGA and CGGA databases. PXDN was up-regulated within GBM samples in comparison with normal control. High PXDN expression was a dismal prognostic indicator in GBM. Single cell RNA analysis was conducted to detect the cell localization of PXDN. We also set up a PPI network to explore the interacting protein associated with PXDN, including TSKU, COL4A1 and COL5A1. Consistently, functional enrichment analysis revealed that several cancer hallmarks were enriched in the GBM cases with high PXDN expression, such as epithelial-mesenchymal transition (EMT), fatty acid metabolism, glycolysis, hypoxia, inflammatory response, and Wnt/beta-catenin signaling pathway. Next, this study analyzed the association of PXDN expression and immunocyte infiltration. PXDN expression was in direct proportion to the infiltrating degrees of NK cells resting, T cells regulatory, M0 macrophage, monocytes and eosinophils. The roles of PXDN on immunity were further estimated by PXDN-associated immunomodulators. In addition, four prognosis-related lncRNAs co-expressed with PXDN were identified. Finally, we observed that PXDN depletion inhibits GBM cell proliferation and migration by in vitro experiments. Our data suggested that PXDN has the potential to be a powerful prognostic biomarker, which might offer a basis for developing therapeutic targets for GBM.

Abstract P1020: Differential Contribution Of Bone Marrow Cells To The Heart In Steady-state And After Repetitive Ischemia

Multiple studies have implicated the important role of migration of bone marrow (BM)-derived cells in heart after ischemia. Various BM-derived cells have been demonstrated to exert beneficial or detrimental effects in ischemia. But the response of BM to repetitive ischemia (RI) and the difference in contribution of this heterogenous population to the heart after RI remain elusive. Here, we applied an occluder on the left anterior descending coronary arteries of rats and they went under RI over a period of 10-17 days. This RI model induced coronary collateral growth and increased myocardial blood flow in the ischemic region after 10-17 days. We performed single- cell RNA sequencing and analysis of 24103 bone marrow transcriptomes isolated from the heart and bone marrow of rats in steady-state and after RI. Unsupervised clustering of cardiac neutrophils revealed 28 major clusters and 16 different cell types. Compared to steady state heart, proportion of BM derived macrophage mainly CD163 positive population was increased in the heart after RI. The proportion of BM derived natural killer cells was decreased markedly in the RI group. There was a slightly higher percentage of BM derived endothelial cells and lower smooth muscle cell and fibroblast in the RI group. While neutrophil degranulation, leukocyte activation, leukocyte migration are the major biological pathways in both groups, RI group showed activation of regeneration. We report heterogeneity of BM cells migrated to the heart during RI and redefine the BM that respond to heart ischemia and participate in induction of angiogenesis after RI.

Abstract 4076: Single cell RNA analysis reveals heterogeneous sub populations in aggressive variants of prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) represent an aggressive malignancy that results in poor patients outcomes. At a histological level, NEPC exhibits heterogeneous pathologies; nevertheless, the molecular features, drivers and therapeutic implications of this heterogeneity are poorly understood. Single cell technology enables the resolution needed to unveil the complex heterogeneity of NEPC that bulk RNA analysis could not previously detect. Therefore, we aim to characterize the molecular heterogeneity of NEPC at a single cell level to detect common phenotypes across patients and identify new therapeutic targets. We performed single cell RNA sequencing on a novel cohort of nine patient derived xenograft (PDX) models that recapitulate the pathological and clinical heterogeneity of NEPC. Downstream analysis of single cell data was first completed on individual samples to identify distinct subpopulations of cells within each tumor. Then, integration analysis was performed to identify common and unique neuroendocrine populations across the different tumors. We profiled the transcriptome of 19,361 cells captured from the eight NEPCs. On an individual sample analysis, we detected 3 to 8 different subpopulations in each tumor, where every subpopulation displayed distinct biological properties such as epithelial mesenchymal transition, quiescence and stemness. Data integration of all samples revealed 16 subpopulations of tumor cells across all patients, of which 10 populations were primarily unique to one patient. Enriched pathway analyses revealed heterogeneous expression of most cancer hallmarks and oncogenic pathways across subpopulations, although some pathways were common to several neuroendocrine subpopulations such as EMT, P53 and KRAS. The detection of heterogeneous subpopulations in NEPC provides novel insight into why finding effective treatments for these aggressive tumors is challenging. Future work should focus on further evaluation of common druggable pathways that are shared across NEPC subpopulations. Alternatively, efforts could look at combination therapies that could effectively target these heterogeneous tumors. Citation Format: Rosalia Quezada Urban, Shivakumar Keerthikumar, Ashlee K. Clark, Laura H. Porter, Mitchell G. Lawrence, Renea A. Taylor, Gail P. Risbridger, Roxanne Toivanen, David L. Goode. Single cell RNA analysis reveals heterogeneous sub populations in aggressive variants of prostate cancer [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 4076.

Subcellular distribution, localization, and function of noncoding RNAs.

Eukaryotic cells contain subcellular organelles with spatiotemporal regulation to coordinate various biochemical reactions. The various organelles perform their essential biological functions by employing specific biomolecules, including nucleic acids. Recent studies have revealed that noncoding RNAs (ncRNAs) are highly compartmentalized in cells and that their spatial distribution is intimately related to their functions. Dysregulation of subcellular ncRNAs can disrupt cellular homeostasis and cause human diseases. Mitochondria are responsible for energy generation to fuel cell growth and proliferation. Therefore, identifying mitochondria-associated ncRNAs helps to reveal new regulatory mechanisms and physiological functions of mitochondria. In this review, we summarize the latest advances in subcellular ncRNAs derived from either the nuclear or mitochondrial genome. We also discuss available biological approaches for investigating organelle-specific ncRNAs. Exploring the distribution and function of subcellular ncRNAs may facilitate the understanding of endomembrane dynamics and provide potential strategies for clinical transformation. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Methods > RNA Analyses in Cells.

Progenitor cells from brown adipose tissue undergo neurogenic differentiation

Multipotent cells derived from white adipose tissue have been shown to differentiate into multiple lineages including neurogenic lineages. However, the high innervation of brown adipose tissue by the sympathetic nervous system suggest it might be a better source of neural precursor cells. To investigate potential differences between white and brown progenitors, we cultured white and brown dedifferentiated fat (wDFAT and brDFAT) cells from mouse and human adipose tissue and compared marker expression of neural precursors, and neuronal and glial cells, using fluorescence-activated cell sorting, bright-field imaging, immunofluorescence, and RNA analysis by qPCR. The results showed that both wDFAT and brDFAT cells had the capacity to generate neuronal and glial-like cells under neurogenic conditions. However, the brDFAT cells exhibited enhanced propensity for neurogenic differentiation. The neurogenic cells were at least in part derived from Adiponectin-expressing cells. TdTomato-expressing cells derived from Adiponectin (Adipoq) CreERT2 -tdTomatoflox/flox mice gave rise to individual cells and cell clusters with neurogenic characteristics. Moreover, human brDFAT cells demonstrated a similar ability to undergo neurogenic differentiation after treatment with neurogenic medium, as assessed by immunofluorescence and qPCR. Together, our results support that brDFAT cells have ability to undergo neurogenic differentiation.

RNA-protein interactomes as invaluable resources to study RNA viruses: Insights from SARS CoV-2 studies.

Understanding the molecular mechanisms of severe respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection is essential for the successful development of therapeutic strategies against the COVID‐19 pandemic. Numerous studies have focused on the identification of host factors and cellular pathways involved in the viral replication cycle. The speed and magnitude of hijacking the translation machinery of host mRNA, and shutting down host transcription are still not well understood. Since SARS‐CoV‐2 relies on host RNA‐binding proteins for the infection progression, several efforts have been made to define the SARS‐CoV‐2 RNA‐bound proteomes (RNA–protein interactomes). Methodologies that enable the systemic capture of protein interactors of given RNA in vivo have been adapted for the identification of the SARS‐CoV‐2 RNA interactome. The obtained proteomic data aided by genome‐wide and targeted CRISPR perturbation screens, revealed host factors with either pro‐ or anti‐viral activity and highlighted cellular processes and factors involved in host response. We focus here on the recent studies on SARS‐CoV‐2 RNA–protein interactomes, with regard to both the technological aspects of RNA interactome capture methods and the obtained results. We also summarize several related studies, which were used in the interpretation of the SARS‐CoV‐2 RNA–protein interactomes. These studies provided the selection of host factors that are potentially suitable candidates for antiviral therapy. Finally, we underscore the importance of RNA–protein interactome studies in regard to the effective development of antiviral strategies against current and future threats.This article is categorized under:RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional ImplicationsRNA in Disease and Development > RNA in DiseaseRNA Methods > RNA Analyses in Cells