Whole-transcriptome Profiling Research Articles

Transcriptomic Profiling and Tumor Microenvironment Classification Reveal Unique and Dynamic Immune Biology in HIV-Associated Kaposi Sarcoma.

Kaposi Sarcoma (KS) is a vascular tumor originating from endothelial cells and is associated with human herpesvirus 8 (KSHV) infection. It disproportionately affects populations facing health disparities. Although antiretroviral therapy (ART) has improved KS control in people with HIV (PWH), treatment options for advanced KS remain limited. This study investigates the tumor microenvironment (TME) of KS through whole-transcriptomic profiling, analyzing changes over time and differences based on HIV status. The TME was categorized into four subtypes: immune-enriched (IE), non-fibrotic, immune-enriched/fibrotic (IE/F), fibrotic (F) and immune-depleted (D). Nine KS patients (four HIV-negative and five HIV-positive) were enrolled in the study. Longitudinally collected KS samples from three patients (one HIV-negative and two HIV-positive) allowed for the investigation of dynamic TME changes within individual patients. The immune cellular composition was determined using deconvolution and compared to a cohort of non-KS patients. Our findings revealed that all KS samples, regardless of HIV status, were enriched in endothelial cells. Compared to non-KS tissues, the KS samples contained a higher percentage of NK and CD8+ T cells. HIV-negative KS samples displayed the IE and IE/F TME subtypes, while HIV-positive samples exhibited IE, IE/F, and F subtypes. Over the course of the disease, a decrease in angiogenic signatures was observed in two HIV-positive KS patients. Notably, HIV-negative KS samples showed alterations in NK cell-mediated immunity and cytotoxic response pathways, whereas HIV-positive samples exhibited changes in growth regulation and protein kinase activity pathways at the time of initial diagnosis. The gene expression of immune checkpoints, including CD274 (PD-L1) and PDCD1LC2 (PD-L2), was comparable between HIV-positive and HIV-negative KS samples at diagnosis. Furthermore, sequencing identified a shared TCRβ chain in all patients analyzed, indicating a T-cell immune response to a common antigen. This study demonstrates unique transcriptomic features and TME subtypes in KS that differ based on HIV status. Additionally, it illustrates longitudinal dynamic changes in the gene signatures and TME subtypes in individual patients. The identification of a shared TCRβ chain suggests that immune T cells in KS patients may target a common antigen. Future studies should further explore the immune microenvironment and unique T cell clonotypes, which could pave the way for the development of novel therapeutic strategies for KS patients.

Gene expression profiles in human dental pulp stem cells treated short-term with lipopolysaccharides before and after osteoinduction.

Dental pulp stem cells (DPSCs) are essential for reparative dentinogenesis following damage or infection. DPSCs surrounding the blood vessels in the central region of the dental pulp actively proliferate after tooth injury and differentiate into new odontoblast-like cells or odontoblasts to form reparative dentin. However, the signaling pathways involved in undifferentiated and osteodifferentiated DPSCs under inflammatory conditions remain unclear. This study aimed to compare the expression profiles of immortalized undifferentiated and osteo-differentiated human DPSCs (hDPSCs) treated with and without lipopolysaccharide (LPS) to elucidate the molecular regulatory mechanisms involved in inflammatory conditions. We investigated the differences between undifferentiated and osteodifferentiated hDPSCs in response to LPS. RNA-seq analyses of undifferentiated and osteodifferentiated hDPSCs were performed with and without LPS. Whole-transcriptome profiling revealed distinct differences in the expression patterns of LPS-treated undifferentiated and osteodifferentiated DPSCs. Death-associated protein kinase 1 levels downregulated in LPS-treated osteodifferentiated cells, inhibiting apoptosis and enhancing cell survival. After LPS treatment, osteodifferentiated DPSCs exhibited higher expression levels of various inflammatory cytokines and chemokines than undifferentiated DPSCs. This study provides valuable transcriptomic data as a critical resource for uncovering potential therapeutic targets to enhance cell survival and regulate inflammation within the dental pulp. By elucidating the key molecular mechanisms and identifying specific gene expression changes linked to inflammatory and immune responses, these findings provide significant insights into osteo-differentiated hDPSCs.

EPCO-48. RESOLVING INTRATUMORAL TRANSCRIPTIONAL HETEROGENEITY ACROSS THE CORE-EDGE SPATIAL AXIS IN GLIOBLASTOMA

Abstract Intratumoral heterogeneity promotes tumor progression, treatment resistance, and recurrence in glioblastoma (GBM), and the cellular tumor core and infiltrating edge represent transcriptionally distinct cellular ecosystems. In this study, we characterize and validate the molecular differences across the core-edge spatial axis in GBM by applying whole-transcriptome GeoMx Digital Spatial Profiling (DSP) to 5 IDH-wildtype tumors. Three 3-micron tissue cores were extracted from each tumor and arranged into a tissue microarray (TMA). Core and edge regions of interest (ROIs) were chosen through coregistration of H&E and immunofluorescent TMA scans. Following DSP, data for 44 ROIs (30 core and 14 edge) and 8530 RNA targets were bioinformatically analyzed. Single-sample gene set enrichment analysis aligned the DSP core and edge regional signatures to those of publicly annotated datasets, substantiating our profiling methodology. Unsupervised analyses revealed ROI clusters unique to each tumor specimen for both pre- and post-normalized data. To mitigate a potential patient-level effect in subsequent differential gene expression and pathway analyses, we leveraged a linear mixed-effect model using core versus edge as a fixed effect and tumor specimen as a random effect. Our analyses showed upregulation of epigenomic markers in core ROIs and neuronal markers in edge ROIs. Further, core and edge signatures were incongruent with the canonical GBM subtypes (Mesenchymal, Classical, Proneural) and malignant cell states (OPC-like, NPC-like, AC-like, MES-like). Cellular deconvolution analysis highlighted differential enrichment of neoplastic cells in core ROIs and neuroglia (astrocytes and oligodendrocytes) in edge ROIs. In summary, we leverage spatial profiling to further validate the geographic molecular heterogeneity of GBM, which is characterized by divergent neuronal programs within the infiltrating edge.

Spatial Transcriptomics of IPMN Reveals Divergent Indolent and Malignant Lineages.

Intraductal papillary mucinous neoplasms (IPMN) occur in 5-10% of the population, but only a small minority progress to pancreatic ductal adenocarcinoma (PDAC). The lack of accurate predictors of high-risk disease leads both to unnecessary operations for indolent neoplasms as well as missed diagnoses of PDAC. Digital spatial RNA profiling (DSP-RNA) provides an opportunity to define and associate transcriptomic states with cancer risk. Whole-transcriptome DSP-RNA profiling was performed on 10 IPMN specimens encompassing the spectrum of dysplastic changes from normal duct to cancer. Ductal epithelial regions within each tissue were annotated as normal duct (NL), low-grade dysplasia (LGD), high-grade dysplasia (HGD), or invasive carcinoma (INV). Gene expression count data was generated by Illumina sequencing and analyzed with R/Bioconductor. Dimension reduction analysis exposed three clusters reflecting IPMN transcriptomic states denoted "normal-like" ( cNL ), "low-risk" ( cLR ) and "high-risk" ( cHR ). In addition to specific marker genes, the three states exhibited significant enrichment for the exocrine, classical, and basal-like programs in PDAC. Specifically, exocrine function diminished in cHR , classical activation distinguished neoplasia from cNL , and basal-like genes were specifically upregulated in cHR . Intriguingly, markers of cHR were detected in NL and LGD regions from specimens with PDAC but not low-grade IPMN. DSP-RNA of IPMN revealed low-risk (indolent) and high-risk (malignant) expression programs that correlated with the activity of exocrine and basal-like PDAC signatures, respectively, and distinguished pathologically low-grade from malignant specimens. These findings contextualize IPMN pathogenesis and have the potential to transform existing risk stratification models. Current consensus guidelines for management of intraductal papillary mucinous neoplasms (IPMN) of the pancreas utilize clinical and radiographic criteria for risk stratification. Unfortunately, the estimated positive predictive value of these criteria for IPMN-associated pancreatic ductal adenocarcinoma (PDAC) is under 50%, indicating that over half of pancreatectomies are performed for benign disease. Moreover, nearly 15% of patients who were deemed "low risk" by the same criteria harbored PDAC. Surgical resection of IPMN has maximal benefit when performed prior to the development of PDAC, as evidence of carcinoma has been associated with a high rate of recurrence and poor overall survival. Thus, the development of molecular diagnostics that improve the accuracy of IPMN risk classification would have immediate relevance for patient care, both in terms of better selecting patients for potentially curative operations, as well as sparing patients with low-risk lesions from invasive procedures.

Spatially Resolved Whole-Transcriptomic and Proteomic Profiling of Lung Cancer and Its Immune Microenvironment According to PD-L1 Expression.

The expression of PD-L1 on tumor cells (TC) is used as an immunotherapy biomarker in lung cancer, but heterogeneous intratumoral expression is often observed. To better understand heterogeneity in the lung cancer tumor microenvironment, we performed proteomic and whole-transcriptomic digital spatial profiling analyses of TCs and immune cells (IC) in spatially matched areas based on tumor PD-L1 expression and the status of the immune microenvironment. We validated our findings using IHC, data from The Cancer Genome Atlas, and immunotherapy cohorts. ICs in areas with high PD-L1 expression on TCs showed more features, indicative of immunosuppression and exhaustion, than ICs in areas with low PD-L1 expression on TCs. TCs highly expressing PD-L1 within immune-inflamed areas showed upregulation of proinflammatory processes, whereas TCs highly expressing PD-L1 within immune-deficient areas showed upregulation of various metabolic processes. Using differentially expressed genes of TCs between the immune-inflamed and immune-deficient areas, we identified a prognostic gene signature for lung cancer. In addition, we found that a high ratio of CD8+ cells to M2 macrophages predicted favorable outcomes in patients with PD-L1-expressing lung cancer after immune checkpoint inhibitor therapy. Overall, this study demonstrates that TCs and ICs have distinct spatial features within the lung tumor microenvironment that are related to tumor PD-L1 expression and IC infiltration.

Spatial resolution of the head and neck cancer tumor microenvironment to identify tumor and stromal features associated with therapy response.

Head and neck cancer (HNC) is the seventh most common cancer globally, resulting in 440 000 deaths per year. While there have been advancements in chemoradiotherapy and surgery, relapse occurs in more than half of HNCs, and these patients have a median survival of 10 months and a 2-year survival of < 20%. Only a subset of patients displays durable benefits from immunotherapies in metastatic and recurrent HNC, making it critical to understand the tumor microenvironment (TME) underpinning therapy responses in HNC. To recognize biological differences within the TME that may be predictive of immunotherapy response, we applied cutting-edge geospatial whole-transcriptome profiling (NanoString GeoMx Digital Spatial Profiler) and spatial proteomics profiling (Akoya PhenoCycler-Fusion) on a tumor microarray consisting of 25 cores from 12 patients that included 4 immunotherapy-unresponsive (8 cores) and 2 immunotherapy-responsive patients (5 cores), as well as 6 immunotherapy naïve patients (12 cores). Through high-plex, regional-based transcriptomic mapping of the tumor and TME, pathways involved with the complement system and hypoxia were identified to be differentially expressed in patients who went on to experience a poor immunotherapy response. Single-cell, targeted proteomic analysis found that immune cell infiltration of the cancer cell mass and interactions of CD8 Tcells with tumor and other immune cells were associated with positive immunotherapy response. The relative abundance of specific tumor phenotypes and their interactions with various immune cells was identified to be different between response groups. This study demonstrates how spatial transcriptomics and proteomics can resolve novel alterations in the TME of HNC that may contribute to therapy sensitivity and resistance.

Profiling cell identity and tissue architecture with single-cell and spatial transcriptomics.

Single-cell transcriptomics has broadened our understanding of cellular diversity and gene expression dynamics in healthy and diseased tissues. Recently, spatial transcriptomics has emerged as a tool to contextualize single cells in multicellular neighbourhoods and to identify spatially recurrent phenotypes, or ecotypes. These technologies have generated vast datasets with targeted-transcriptome and whole-transcriptome profiles of hundreds to millions of cells. Such data have provided new insights into developmental hierarchies, cellular plasticity and diverse tissue microenvironments, and spurred a burst of innovation in computational methods for single-cell analysis. In this Review, we discuss recent advancements, ongoing challenges and prospects in identifying and characterizing cell states and multicellular neighbourhoods. We discuss recent progress in sample processing, data integration, identification of subtle cell states, trajectory modelling, deconvolution and spatial analysis. Furthermore, we discuss the increasing application of deep learning, including foundation models, in analysing single-cell and spatial transcriptomics data. Finally, we discuss recent applications of these tools in the fields of stem cell biology, immunology, and tumour biology, and the future of single-cell and spatial transcriptomics in biological research and its translation to the clinic.

Kidney whole-transcriptome profiling in primary antiphospholipid syndrome reveals complement, interferons and NETs-related gene expression.

Pathogenesis of antiphospholipid syndrome (APS) remains poorly elucidated. We aimed to evaluate for the first time kidney transcriptome profiles in primary APS vs systemic lupus erythematosus (SLE) and control subjects. We performed RNA sequencing on archival formalin-fixed paraffin-embedded kidney biopsies from APS (n = 4), SLE (n = 5) and control (n = 3) individuals, differential gene expression analysis (DGEA) and enrichment analysis using gene ontology (GO) and CORUM, KEGG and Reactome pathway databases. Two-dimensional projection showed a distinct gene profile in primary APS vs control kidneys samples, but similar to SLE. DGEA in APS vs controls returned 276 upregulated and 217 downregulated genes, while the comparison between APS and SLE identified 75 upregulated and 111 downregulated genes. In 276 upregulated genes, enriched GO terms were (innate) immune response, inflammatory response, leucocyte and lymphocyte activation, cytokine production and T cell activation. CORUM and KEGG revealed complement-related genes (C3, C4A, C4B). Expression levels showed logFC values of 2.25 (P = 1.58e-05) for C3, 2.17 (P = 2.69e-06) for C4A and 2.135 (P = 3.7e-06) for C4B in APS vs controls, without differences between APS and SLE. Interferon (IFN) alpha/beta signalling was revealed by Reactome. Expression levels of nine IFN-regulated genes found upregulated in APS vs control kidneys (P-values ≤ 0.001 for all). Examining neutrophil-extracellular traps (NETs)-related gene expression, 13 of 15 upregulated NETs-related genes exhibited higher expression in APS vs controls but not vs SLE. Complement, interferon and NETs-related genes are highly expressed in APS kidney tissues, similarly to SLE, pointing out the role of innate immunity in APS nephropathy pathogenesis and potential treatment targets.

Far from Their Origins: A Transcriptomic Investigation on How 2,4-Di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl) Phenol Affects Rainbow Trout Alevins.

Benzotriazole ultraviolet stabilizers (BUVSs) are a group of widely used chemicals added to a variety of consumer (e.g., plastics) and industrial (e.g., metal coating) goods. Although detected globally as an environmentally persistent pollutant, BUVSs have received relatively little toxicological attention and only recently have been acknowledged to affect development and the endocrine system in vivo. In our previous study, altered behavior, indicative of potential neurotoxicity, was observed among rainbow trout alevins (day 14 posthatching) that were microinjected as embryos with a single environmentally relevant dose of 2,4-di-tert-butyl-6-(5-chloro-2H-benzotriazol-2-yl) phenol (UV-327). In the present follow-up study, we performed whole-transcriptome profiling (RNA sequencing) of newly hatched alevins from the same batch. The primary aim was to identify biomarkers related to behavior and neurology. Dose-specifically, 1 to 176 differentially expressed genes (DEGs) were identified. In the group presenting altered behavior (273.4 ng g-1), 176 DEGs were identified, yet only a fraction was related to neurological functions, including water, calcium, and potassium homeostasis; acetylcholine transmission and signaling; as well insulin and energy metabolism. The second objective was to estimate the transcriptomic point of departure (tPOD) and assess if point estimate(s) are protective of altered behavior. A tPOD was established at 35 to 94 ng UV-327 g-1 egg, making this tPOD protective of behavioral alterations. Holistically, these transcriptomic alterations provide a foundation for future research on how BUVSs can influence rainbow trout alevin development, while providing support to the hypothesis that UV-327 can influence neurogenesis and subsequent behavioral endpoints. The exact structural and functional changes caused by embryonic exposure to UV-327 remain enigmatic and will require extensive investigation before being deciphered and understood toxicologically. Environ Toxicol Chem 2024;43:2026-2038. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Whole-transcriptome profiling reveals potential biomarkers for the reversal of thymic epithelial cell senescence by umbilical cord mesenchymal stem cells.

Reduced numbers and dysfunction of thymic epithelial cells (TECs) are important factors of thymic degeneration. Previous studies have found that umbilical cord mesenchymal stem cells (UCMSCs) reverse the structure and function of the senescent thymus in vivo. However, the transcriptomic regulation mechanism is unclear. TECs were cultured with H2O2 for 72 hours to induce senescence. UCMSCs were cocultured with senescent TECs for 48 hours to detect SA-β-gal, P16 and Ki67. The cocultured TECs were collected for lncRNA, mRNA and miRNA sequencing to establish a competitive endogenous regulatory network (ceRNA). And RT-qPCR, immunofluorescence staining, and western blot were used to identified key genes. Our results showed that H2O2 induced TEC aging and that UCMSCs reversed these changes. Compared with those in aged TECs, 2260 DE mRNAs, 1033 DE lncRNAs and 67 DE miRNAs were differentially expressed, and these changes were reversed by coculturing the cells with UCMSCs. Differential mRNA enrichment analysis of ceRNA regulation revealed that the PI3K-AKT pathway was a significant signaling pathway. UCMSC coculture upregulated VEGFA, which is the upstream factor of the PI3K-AKT signaling pathway, and the expression of the key proteins PI3K and AKT. Thus, the expression of the cell cycle suppressor P27, which is downstream of the PI3K-AKT signaling pathway, was downregulated, while the expression of the cell cycle regulators CDK2 and CCNE was upregulated. UCMSC coculture upregulated the expression of VEGFA, activated the PI3K-AKT signaling pathway, increased the expression of CDK2 and CCNE, decreased the expression of P27, and promoted the proliferation of TECs.

Abstract 3766: Utilizing whole-transcriptome digital spatial profiling for glioblastoma clinical biomarker discovery

Abstract Objective: IDH-wildtype glioblastoma (GBM) is the most aggressive brain tumor in adults. Notable for substantial inter- and intra-tumoral heterogeneity, GBM is highly resistant to standard-of-care treatment. Emerging spatial omics technologies, such as GeoMx Digital Spatial Profiling (DSP), offer novel approaches to interrogate the molecular features of regionally defined cellular ecosystems in cancer. Here, we leverage DSP to study whole-transcriptome gene expression signatures in a spatially defined manner across five IDH-wildtype GBMs. Methods: Formalin-fixed paraffin-embedded tumors were assessed histologically and three spatially distinct areas from each tumor were used to construct a custom “spatial” tissue microarray. From each set of three areas, we identified nine spatially distinct regions of interest (ROIs) for bulk transcriptomic analysis according to two histopathological features: distribution (cellular tumor core or tumor periphery) and vascular proximity (near or far). We evaluated normalized target expression data statistically using linear mixed modeling (LMM) with Benjamini-Hochberg correction. Results: 44 profiled ROIs and 8531 transcriptomic targets were analyzed following quality control trimming. 3D principal component analysis and unsupervised hierarchical clustering analysis showed clustering according to tumor specimen as well as distribution (core versus periphery), thereby substantiating the presence of inter- and intra-tumoral transcriptional heterogeneity. LMM-based differential gene expression analysis identified distinct molecular signatures when comparing core and periphery. Specifically, we observed elevated log2-transformed expression of migration and proliferation markers including LAMC1 (p=0.00297) and FABP7 (p=0.00434) in core ROIs and neurodevelopmental markers including NTRK2 (p=0.00734) and DAAM2 (p=0.00263) in periphery ROIs. Additionally, reactome analysis demonstrated considerable enrichment for the aberrant programmed cell death pathway (R-HSA-9645723, coverage=71.84%) in core ROIs and the MHC class II antigen presentation pathway (R-HSA-2132295, coverage=69.11%) in periphery ROIs. Interestingly, differential gene expression analysis did not reveal any biologically significant targets when subsetting for vascular proximity within cellular tumor core ROIs. However, reactome analysis of this regional subset demonstrated upregulation of various cellular proliferation pathways (G2/M transition, APC/C activity, DNA synthesis, etc.) within vessel-near ROIs when compared with vessel-far ROIs. Conclusions: We preliminarily characterize the molecular heterogeneity of GBM using a spatially guided approach. Ongoing analyses are focused on dataset validation, mixed cell deconvolution, further regional genotyping, and potential biomarkers. Citation Format: Adam Luo, Kelly Casella, Shivang Sharma, Ezra Baraban, Calixto-Hope Lucas, Eugene Shenderov. Utilizing whole-transcriptome digital spatial profiling for glioblastoma clinical biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3766.

Whole-transcriptome profiling and identification of cold tolerance-related ceRNA networks in japonica rice varieties.

Low-temperature stress negatively impacts rice yield, posing a significant risk to food security. While previous studies have explored the physiological and linear gene expression alterations in rice under low-temperature conditions, the changes in competing endogenous RNA (ceRNA) networks remain largely unexamined. We conducted RNA sequencing on two japonica rice varieties with differing cold-tolerance capabilities to establish ceRNA networks. This enabled us to investigate the transcriptional regulatory network and molecular mechanisms that rice employs in response to low-temperature stress. We identified 364 differentially expressed circular RNAs (circRNAs), 224 differentially expressed microRNAs (miRNAs), and 12,183 differentially expressed messenger RNAs (mRNAs). WRKY family was the most prominent transcription factor family involved in cold tolerance. Based on the expression patterns and targeted relationships of these differentially expressed RNAs, we discerned five potential ceRNA networks related to low-temperature stress in rice: osa-miR166j-5p from the miR166 family was associated with cold tolerance; osa-miR528-3p and osa-miR156j-3p were linked to stress response; and osa-miR156j-3p was involved in the antioxidant system. In addition, Os03g0152000 in the antioxidant system, as well as Os12g0491800 and Os05g0381400, correlated with the corresponding stress response and circRNAs in the network. A gene sequence difference analysis and phenotypic validation of Os11g0685700 (OsWRKY61) within the WRKY family suggested its potential role in regulating cold tolerance in rice. We identified Os11g0685700 (OsWRKY61) as a promising candidate gene for enhancing cold tolerance in japonica rice. The candidate miRNAs, mRNAs, and circRNAs uncovered in this study are valuable targets for researchers and breeders. Our findings will facilitate the development of cold-tolerant rice varieties from multiple angles and provide critical directions for future research into the functions of cold-tolerance-related miRNAs, mRNAs, and circRNAs in rice.

Whole-Transcriptome Profiling and Functional Prediction of Long Non-Coding RNAs Associated with Cold Tolerance in Japonica Rice Varieties.

Low-temperature chilling is a major abiotic stress leading to reduced rice yield and is a significant environmental threat to food security. Low-temperature chilling studies have focused on physiological changes or coding genes. However, the competitive endogenous RNA mechanism in rice at low temperatures has not been reported. Therefore, in this study, antioxidant physiological indices were combined with whole-transcriptome data through weighted correlation network analysis, which found that the gene modules had the highest correlation with the key antioxidant enzymes superoxide dismutase and peroxidase. The hub genes of the superoxide dismutase-related module included the UDP-glucosyltransferase family protein, sesquiterpene synthase and indole-3-glycerophosphatase gene. The hub genes of the peroxidase-related module included the WRKY transcription factor, abscisic acid signal transduction pathway-related gene plasma membrane hydrogen-ATPase and receptor-like kinase. Therefore, we selected the modular hub genes and significantly enriched the metabolic pathway genes to construct the key competitive endogenous RNA networks, resulting in three competitive endogenous RNA networks of seven long non-coding RNAs regulating three co-expressed messenger RNAs via four microRNAs. Finally, the negative regulatory function of the WRKY transcription factor OsWRKY61 was determined via subcellular localization and validation of the physiological indices in the mutant.

Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications.

RNA molecules are not merely a combination of four bases of A, C, G, and U. Chemical modifications occur in almost all RNA species and play diverse roles in gene expression regulation. The abundant cellular RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), are known to have the highest density of RNA modifications, which exert critical functions in rRNA and tRNA biogenesis, stability, and subsequent translation. In recent years, modifications on low-abundance RNA species in mammalian cells, such as messenger RNA (mRNA), regulatory noncoding RNA (ncRNA), and chromatin-associated RNA (caRNA), have been shown to contain multiple different chemical modifications with functional significance. As the most abundant mRNA modification in mammals, N6-methyladenosine (m6A) affects nearly every stage of mRNA processing and metabolism, with the antibody-based m6A-MeRIP-seq (methylated RNA immunoprecipitation sequencing) followed by high-throughput sequencing widely employed in mapping m6A distribution transcriptome-wide in diverse biological systems. In addition to m6A, other chemical modifications such as pseudouridine (Ψ), 2'-O-methylation (Nm), 5-methylcytidine (m5C), internal N7-methylguanosine (m7G), N1-methyladenosine (m1A), N4-acetylcytidine (ac4C), etc. also exist in polyA-tailed RNA in mammalian cells, requiring effective mapping approaches for whole-transcriptome profiling of these non-m6A mRNA modifications. Like m6A, the antibody-based enrichment followed by sequencing has been the primary method to study distributions of these modifications. Methods to more quantitatively map these modifications would dramatically improve our understanding of distributions and modification density of these chemical marks on RNA, thereby bettering informing functional implications. In this Account, aimed at both single-base resolution and modification fraction quantification, we summarize our recent advances in developing a series of chemistry- or biochemistry-based methods to quantitatively map RNA modifications, including m6A, Ψ, m5C, m1A, 2'-O-methylation (Nm), and internal m7G, in mammalian mRNA at base resolution. These new methods, including m6A-SAC-seq, eTAM-seq, BID-seq, UBS-seq, DAMM-seq, m1A-quant-seq, Nm-Mut-seq, and m7G-quant-seq, promise to conduct base-resolution mapping of most major mRNA modifications with low RNA input and uncover dynamic changes in modification stoichiometry during biological and physiological processes, facilitating future investigations on these RNA modifications in regulating cellular gene expression and as potential biomarkers for clinical diagnosis and prognosis. These quantitative sequencing methods allow the mapping of most mRNA modifications with limited input sample requirements. The same modifications on diverse RNA species, such as caRNA, ncRNA, nuclear nascent RNA, mitochondrial RNA, cell-free RNA (cfRNA), etc., could be sequenced using the same methods.

RpoS-Regulated Genes and Phenotypes in the Phytopathogenic Bacterium Pectobacterium atrosepticum.

The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed.

Cytokine-responsive T- and NK-cells portray SARS-CoV-2 vaccine-responders and infection in multiple myeloma patients

Patients with multiple myeloma (MM) routinely receive mRNA-based vaccines to reduce COVID-19-related mortality. However, whether disease- and therapy-related alterations in immune cells and cytokine-responsiveness contribute to the observed heterogeneous vaccination responses is unclear. Thus, we analyzed peripheral blood mononuclear cells from patients with MM during and after SARS-CoV-2 vaccination and breakthrough infection (BTI) using combined whole-transcriptome and surface proteome single-cell profiling with functional serological and T-cell validation in 58 MM patients. Our results demonstrate that vaccine-responders showed a significant overrepresentation of cytotoxic CD4+ T- and mature CD38+ NK-cells expressing FAS+/TIM3+ with a robust cytokine-responsiveness, such as type-I-interferon-, IL-12- and TNF-α-mediated signaling. Patients with MM experiencing BTI developed strong serological and cellular responses and exhibited similar cytokine-responsive immune cell patterns as vaccine-responders. This study can expand our understanding of molecular and cellular patterns associated with immunization responses and may benefit the design of improved vaccination strategies in immunocompromised patients.

Molecular portraits of colorectal cancer morphological regions.

Heterogeneity of colorectal carcinoma (CRC) represents a major hurdle towards personalized medicine. Efforts based on whole tumor profiling demonstrated that the CRC molecular subtypes were associated with specific tumor morphological patterns representing tumor subregions. We hypothesize that whole-tumor molecular descriptors depend on the morphological heterogeneity with significant impact on current molecular predictors. We investigated intra-tumor heterogeneity by morphology-guided transcriptomics to better understand the links between gene expression and tumor morphology represented by six morphological patterns (morphotypes): complex tubular, desmoplastic, mucinous, papillary, serrated, and solid/trabecular. Whole-transcriptome profiling by microarrays of 202 tumor regions (morphotypes, tumor-adjacent normal tissue, supportive stroma, and matched whole tumors) from 111 stage II-IV CRCs identified morphotype-specific gene expression profiles and molecular programs and differences in their cellular buildup. The proportion of cell types (fibroblasts, epithelial and immune cells) and differentiation of epithelial cells were the main drivers of the observed disparities with activation of EMT and TNF-α signaling in contrast to MYC and E2F targets signaling, defining major gradients of changes at molecular level. Several gene expression-based (including single-cell) classifiers, prognostic and predictive signatures were examined to study their behavior across morphotypes. Most exhibited important morphotype-dependent variability within same tumor sections, with regional predictions often contradicting the whole-tumor classification. The results show that morphotype-based tumor sampling allows the detection of molecular features that would otherwise be distilled in whole tumor profile, while maintaining histopathology context for their interpretation. This represents a practical approach at improving the reproducibility of expression profiling and, by consequence, of gene-based classifiers.

Molecular portraits of colorectal cancer morphological regions

Heterogeneity of colorectal carcinoma (CRC) represents a major hurdle towards personalized medicine. Efforts based on whole tumor profiling demonstrated that the CRC molecular subtypes were associated with specific tumor morphological patterns representing tumor subregions. We hypothesize that whole-tumor molecular descriptors depend on the morphological heterogeneity with significant impact on current molecular predictors. We investigated intra-tumor heterogeneity by morphology-guided transcriptomics to better understand the links between gene expression and tumor morphology represented by six morphological patterns (morphotypes): complex tubular, desmoplastic, mucinous, papillary, serrated, and solid/trabecular. Whole-transcriptome profiling by microarrays of 202 tumor regions (morphotypes, tumor-adjacent normal tissue, supportive stroma, and matched whole tumors) from 111 stage II-IV CRCs identified morphotype-specific gene expression profiles and molecular programs and differences in their cellular buildup. The proportion of cell types (fibroblasts, epithelial and immune cells) and differentiation of epithelial cells were the main drivers of the observed disparities with activation of EMT and TNF-α signaling in contrast to MYC and E2F targets signaling, defining major gradients of changes at molecular level. Several gene expression-based (including single-cell) classifiers, prognostic and predictive signatures were examined to study their behavior across morphotypes. Most exhibited important morphotype-dependent variability within same tumor sections, with regional predictions often contradicting the whole-tumor classification. The results show that morphotype-based tumor sampling allows the detection of molecular features that would otherwise be distilled in whole tumor profile, while maintaining histopathology context for their interpretation. This represents a practical approach at improving the reproducibility of expression profiling and, by consequence, of gene-based classifiers.

Unveiling Putative Pathway Enrichment Following Crisantaspase Treatment in AML through RNA Sequencing Analysis

Background: Tumor development is intricately influenced by metabolic reprogramming, resulting in the emergence of exploitable vulnerabilities that can be targeted. Glutamine (Gln) depletion has emerged as a promising therapeutic strategy for malignancies, including AML, that heavily rely on Gln for their survival. Erwinia-derived asparaginase, also called crisantaspase, has exhibited anti-AML activity in both preclinical and clinical models by disrupting Gln supply, albeit with the persistence of residual leukemia burden. To identify cellular pathways modulated after crisantaspase-induced Gln depletion, here we report our comprehensive results using a whole transcriptome profiling approach (RNA-Seq) after crisantaspase treatment. Methods: Whole-transcriptomic profiling using RNA-Seq was performed to profile gene expression of MOLM-14 AML cells following crisantaspase treatment. RNA was isolated from each sample and total RNA was utilized for this study. The samples were sequenced at IGS using the Illumina HiSeq sequencing platform. Raw sequencing reads generated for each sample were analyzed using the CAVERN analysis pipeline. Read quality was assessed using the FastQC toolkit to ensure good-quality reads for downstream analyses. P values were generated using a Wald test implemented in DESeq2 and then corrected for multiple hypothesis testing using the Benjamini-Hochberg correction method. Results: We utilized Rylaze, an FDA-approved Erwinia crisantaspase, to deplete exogenous Gln. MOLM-14 cells were treated with Rylaze for 16h, followed by whole-transcriptome/gene expression profiling (GEP) using RNA-seq. The transcriptomic analysis provided information on the regulation of gene expression between vehicle control and Rylaze treatment in AML, as well as on the potential association with several putative pathways including serine family amino acids biosynthetic process (GO: 0009070, p-value = 2.18E-08), regulation of programmed cell death (GO:0043067, p-value = 7.96E-06), and amino acid transmembrane transport (GO:0003333, p-value = 5.10E-05). Furthermore, Figure 1 illustrates the gene ontology terms enriched by the DEGs, offering insights into the underlying molecular mechanisms involved in the response to Rylaze-induced Gln depletion in MOLM-14 cells. For example, the upregulation of genes associated with the serine family amino acid biosynthetic process may suggest a compensatory mechanism to counteract the depletion of Gln induced by Rylaze. By employing generalized linear regression models, we observed significant changes in gene expression following Rylaze (141 genes) compared to the vehicle control. This finding is further illustrated by a heatmap generated from transcriptome data analysis (Figure 2). We identified 141 significantly differentially expressed genes (DEGs); among the Rylaze-treated samples, consisting of 114 up-regulated and 27 down-regulated genes. Specifically, we focused on five genes that exhibited significant modulation in response to the treatment with Rylaze. These genes included CHAC1, ADM2, S100P, CTH and SPOCK2. These genes might play crucial roles in the cellular response to Gln depletion and could potentially serve as biomarkers or therapeutic targets in AML treatment. Validation of these observations was performed using immunoblotting techniques and quantitative real-time polymerase chain reaction (qRT-PCR), which confirmed the significant increase in CHAC1, ADM2, S100P, and CTH, as well as the significant decrease in SPOCK2 (p&amp;lt;0.0001) following Rylaze-induced Gln depletion. These findings contribute to a better understanding of the drug's mode of action and the impact of Gln depletion on cellular processes in AML. Conclusion: This study represents a significant contribution towards advancing our understanding of amino acid-regulated metabolic pathways in AML, with potential implications for other Gln metabolism-dependent cancers as well.

Molecular Mechanisms of Intratumoral Nerve Recruitment and Perineural Invasion Elucidated with Spatial Transcriptomics and CRISPR Activation

Perineural invasion (PNI) is an aggressive manifestation of tumor-nerve interactions associated with postoperative recurrence, metastasis, pain, and decreased survival. Hence, PNI is included in the staging criteria of several malignancies and often an indication for treatment intensification using adjuvant radiotherapy. However, the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown-hindering the development of new therapies targeting this key pathological process. Moreover, prior studies were limited by a lack of cell-type information, spatial context, and/or a fragmented focus on a small number of pathways. Using pancreatic ductal adenocarcinoma (PDAC) as an exemplar given the exceptionally high frequency of PNI in this malignancy, we performed the first comprehensive, cell-type specific, and spatially resolved whole-transcriptome analysis of human PDAC to identify molecular mediators of tumor-nerve crosstalk and PNI. We constructed 12 custom tissue microarrays (TMAs) derived from matched malignant regions with and without tumor-nerve proximity (n = 288 cores). We performed whole-transcriptome digital spatial profiling (DSP) to independently determine mRNA abundance from the malignant, fibroblast, and nerve compartments through optical sectioning. We mapped malignant subtypes we previously identified onto the spatial data and found strong (p<0.0001) positive nerve associations with the mesenchymal, basaloid, and neural-like progenitor subtypes and a negative nerve association with the classical subtype. Numerous genes expressed by malignant cells were enriched (e.g., MMP2, PLXND1, NRP1) or depleted (e.g., SEMA3B) in association with radial distance from nerves, including recapitulation of prior literature. To functionally explore these candidate mediators of tumor-nerve crosstalk, we derived genetically-engineered murine organoids (KrasLSL-G12D/+; Trp53FL/FL; Rosa26-dCas9-VPR) and transduced them with guide RNAs to overexpress subtype-specific transcription factors or candidate genes from the spatial analysis. We quantified (1) cancer cell invasion through extracellular matrix using cultured dorsal root ganglia (DRG) sensory neurons as the chemoattractant, and (2) the role of cancer-intrinsic signaling on nerve recruitment/outgrowth by applying conditioned media or exogenous proteins to cultured DRG sensory neurons and tracking their growth with live imaging. Our results suggest that the mechanisms enabling cancer cells to recruit nerves into the tumor microenvironment are distinct from those facilitating perineural invasion. This study has transformed our understanding of how cancer cells and the peripheral nervous system collaborate to promote tumor growth, survival, and dissemination, and is now guiding prioritization of therapeutic strategies that synergize with adjuvant radiotherapy in the burgeoning field of cancer neuroscience.