title: Transdermal bicarbonate therapy increass intra-tumoral pH and elicits amti-tumor responses in bladder cancer

city: Tampa

first: jiqiang

last: yao

address: 12902 USF Magnolia Drive

institute: moffitt cancer center

summary: Introduction: Tumor acidosis is a hallmark of cancer that leads to abrogation of T cell function and cancer progression. Oral sodium bicarbonate therapy for alkalization of the extracellular tumor pH has shown moderate positive effects in tumor models. However, its applicability in the clinic is very limited due to the unreasonably high dosage required and gastrointestinal disturbances that arise. In this study, we assessed the functional effects of acidity on T cells. Methods: We show that acidity alters T cell proliferation, migration and effector functions as well as transcriptional programming using in vitro culture techniques and RNA sequencing. We then tested the potency of a proprietary transdermal formulation, DYV800, containing sodium bicarbonate to increase the extracellular tumor pH (pHe) and augment anti-tumor immune responses in a murine model of bladder cancer. The tumor pH was assessed using Chemical Exchange Saturation Transfer Magnetic Resonance Imaging (CEST-MRI) and antitumor immune responses via flow cytometry. Results: We found that transdermal DYV800 significantly reduced tumor burden and improved antigen-specific CD8+ T cell responses. Chemical Exchange Saturation Transfer Magnetic Resonance Imaging (CEST-MRI) of treated tumors showed an increase in intra-tumoral pH of bladder tumors, and this therapy also alkalizes the urine. Discussion/Conclusion: The transdermal delivery of DYV800 led to durable anti-tumor immune responses and is more clinically applicable to combat acidity in bladder cancer than oral bicarbonate. Targeting acidosis in the bladder tumor microenvironment has the potential to enhance T cell responses and improve anti-tumor immunity.

overall_design: Isolated CD8⁺ pmel T cells were plated in pH 7.4 or pH 6.6 media. After a 48-hour incubation, RNA was extracted using the RNeasy Mini Kit (Qiagen, cat# 74104). RNA quantity and purity were assessed using NanoDrop spectrophotometer (Thermo Fisher), and five optimal samples per condition were selected. RNA concentration was quantified using Qubit Fluorometer (Thermo Fisher Scientific), and integrity assessed with Agilent TapeStation 4200 (Agilent Technologies; RRID:SCR_019547). RNA-seq libraries were prepared using NuGEN Universal Mouse RNA-Seq Library Preparation Kit (Tecan Genomics) with 100 ng input RNA. Library quality was confirmed via TapeStation and quantified using KAPA Library Quantification Kit (Roche). Libraries were sequenced on Illumina NovaSeq 6000 platform using SP 200-cycle reagent kit, generating ~100 million 100-base paired-end reads per sample. Adapter sequences were removed using BBMerge (v37.02) and Cutadapt (v1.8.1; RRID:  SCR_011841). Reads were aligned to mouse genome (mm10) using STAR aligner (v2.7.7a), and gene expression quantified using RSEM (v1.3.0) with GENCODE annotation. Differential expression analysis was performed using DESeq2 (RRID: SCR_000154) on genes with basemean >100. Results were analyzed using Qiagen's Ingenuity Pathway Analysis (IPA; RRID:SCR_008653) for genes with padj <0.05 and |log2 fold change| >1. Downregulated genes (padj <0.05, log2 fold change <-1) were analyzed using ShinyGO v0.77 (RRID:SCR_019213) against murine TF.Target.RegNetwork database. Gene Set Enrichment Analysis (GSEA v4.2.3, Broad Institute) was performed on complete datasets using MSigDB Hallmark gene sets.

first: Oluwaseyi

last: Oluwatola

first: Sarah

last: Bazargan

first: Pietro

last: Irrera

first: Darwin

last: Chang

first: Ashley

last: Thomas

first: Jamie

last: Blauvelt

first: Matthew

last: Beatty

first: MacLean

last: Hall

first: Chrisropher

last: Whelan

first: Veronica

last: Estrella

first: Katatzyna

last: Rejniak

first: Michael

last: Poch

first: Nathan

last: Fitzsimmons

first: Ryan

last: Beal

first: Arig

last: Ibrahim-Hashim

first: Shari

last: Pilon-Thomas


title: transcriptional analysis of unfrozen and cryopreserved liver organoids

status: Public on Mar 06 2026

city: Shanghai

first: Lijian

last: Hui

address: yueyang road 320

department: SIBCB

country: China

institute: CEMCS, CAS

type: Expression profiling by high throughput sequencing

summary: To analyze the transcriptional changes of unfrozen and cryopreserved liver organoids in cryovials and cryobags using the established CRYSTL cryopreservation protocol, we found that the transcription fidelity was well preserved after cryopreservation.

overall_design: unfrozen and cryopreserved liver organoidsin cryovials and cryobags using the established CRYSTL cryopreservation protocol

first: Xiang

last: Yuan

first: Hengxin

last: Han

first: Jianxiang

last: Dong

first: Yi

last: Xu


title: Target discrimination and PAM profiling of the Thermotoga maritima type I-B CRISPR system

city: Baltimore

first: Charles

middle: J

last: Lenihan

state: MD

address: 8 S Durham St

country: USA

institute: Johns Hopkins University

summary: Type I-B CRISPR-Cas systems represent the most abundant CRISPR subtype in nature and have emerged as powerful tools for endogenous genome editing in diverse prokaryotes. Here we reconstitute and characterize the type I-B1 system from the thermophile Thermotoga maritima (Tma) using purified components. We demonstrate that Tma Cascade requires standalone Cas11 expression, as the cryptic internal translation start site within cas8b1 is non-functional in E. coli. The reconstituted system exhibits canonical type I function including RNA-guided DNA binding, PAM-dependent target discrimination, Cas3-mediated degradation, and seed region interrogation spanning seven PAM-proximal nucleotides. Using next-generation sequencing-based PAM library screens, we define a YYD consensus PAM (Y = C/T; D = G/A/T) with strong discrimination against the array repeat-adjacent sequence (AAC). Comprehensive PAM profiling reveals context-dependent tolerance for non-consensus sequences and identifies numerous intermediate-activity PAMs that may function in priming. Comparison with other characterized type I-B systems reveals correlation between Cas8b variant and position -3 specificity, conserved pyrimidine preference at position -2, and variability at position -1. This work establishes a thermostable type I-B platform for biotechnological applications and provides insights into evolutionary mechanisms balancing PAM promiscuity with self-discrimination in the most abundant CRISPR-Cas subtype.

overall_design: In vitro characterization of the PAM preferences of a purified CRISPR-Cas system. Assaying target binding by Cascade, as well as target degradation by Cascade and Cas3.

first: John

last: Mallon

first: Sowmyaa

last: Shridhar

first: Scott

last: Bailey


title: Single Cell and Spatial Transcriptomics Identify Novel Immune-Stromal Interactions in Cardiac Allograft Vasculopathy

city: Saint Louis

first: Haewon

last: Shin

state: MO

address: 660 S Euclid

institute: Washington University School of Medicine in St. Louis

summary: Cardiac allograft vasculopathy (CAV) is the leading cause of mortality in heart transplant recipients. Despite the prevalence of CAV, there are no targeted therapeutic options to prevent or reverse disease progression, and patients ultimately require retransplant. CAV is characterized by progressive neointimal hyperplasia in donor heart coronary arteries, leading to luminal obliteration and ultimately allograft failure or sudden cardiac death. Although immune and stromal cell interactions are believed to play a key role in CAV pathogenesis, the specific cellular players and molecular signals driving disease remain undefined. In this study, we leverage single-cell RNA sequencing and spatial transcriptomics of human coronary arteries to transcriptionally characterize CAV and define the neointimal microenvironment. We compare arteries with CAV to atherosclerotic coronary artery disease and non-disease controls to identify a unique CAV transcriptional signature. Integration of single-cell RNA sequencing and spatial transcriptomic datasets revealed that modulated vascular smooth muscle cells and macrophage subsets dominate the CAV neointima and suggest that these cells interact to propagate type 1 interferon (IFN)-mediated inflammation. In a mouse CAV model, we demonstrate that interferon blockade with Ruxolitinib significantly reduced the incidence of CAV and prolonged allograft survival. Collectively, this study offers a novel and detailed characterization of the unique cellular and transcriptional landscape of CAV and identify a candidate pathway that may underly CAV pathogenesis, which could serve as a new therapeutic target for this devastating disease.

overall_design: Ethical Approval for Human Specimens Acquisition of coronary artery samples for spatial transcriptomics was approved by the Washington University institutional review board (IRB 201104172). Coronary arteries were collected at time of cardiac explant and were fixed in PFA and embedded in paraffin blocks. Blocks were stored at 4°C, covered from light.   Human coronaries were isolated from human heart explants and were categorized as control (no evidence of coronary artery disease), CAD (coronary artery disease), or CAV (evidence of cardiac allograft vasculopathy).  DV200 (percentage of total RNA fragments >200 nucleotides) values for each tissue were calculated and tissues with DV200 values of <15% were excluded from downstream sample preparation. Visium CytAssist Spatial Gene Expression for FFPE protocol was used to prepare Hematoxylin and Eosin (H&E) stained tissue sections for use with Genomics Visium CytAssist Spatial Gene Expression for FFPE assay. In brief, paraffin embedded coronaries were hydrated in ice bath prior to sectioning. 5um slices were sectioned with a clean blade on a microtome and sections were placed on charged microscope slides. Slides were baked for 3 hours at 42°C and were placed in desiccant beads overnight prior to deparaffinization and staining. Tissues were passed through multiple exchanges of xylenes/ethanol for dehydration/rehydration prior to staining with H&E per Visium Spatial Gene Expression for FFPE tissue staining instructions. Capture regions were demarcated, and high-resolution images were obtained with the Zeiss Axioscan Z1 (Washington University Center for Cellular Imaging) prior to submission for barcoding, sequencing and spatial alignment via Space Ranger at The McDonnell Genome Institute.

first: Macee

middle: C

last: Owen

first: Daniel

middle: Y

last: Li

first: Wenduo

last: Gu

first: Alekhya

last: Parvathaneni

first: Farid

middle: F

last: Kadyrov

first: Xiaoran

last: Wang

first: Maura

last: Sticco-Ivins

first: Hao

last: Dun

first: Sariah

last: Hyacinth

first: Samantha

last: Giangrasso

first: Caroline

last: Chou

first: Chieh-Yu

last: Lin

middle: T

last: Cain

first: Albert

last: Pedroza

first: Alex

last: Dalal

first: Karim

last: Sallam

first: Jack

last: Boyd

first: Joseph

last: Woo

first: Junedh

last: Amrute

first: Paul

last: Cheng

first: Kory

last: Lavine

first: Benjamin

last: Kopecky


title: A Bioorthogonal Nanoconcentrisome System for Epigenetic and Immunometabolic Reprogramming in Aged Bone Repair

city: hangzhou

first: Zhao

last: lan

state: Zhejiang Province

address: Dinglan Street

institute: Hangzhou Traditional Chinese Medicine Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Despite the promising potential of stem cell–based therapies in regenerative medicine, their therapeutic instability remains a major challenge, often resulting in inconsistent clinical outcomes. Achieving robust tissue regeneration requires precise control over the behavior of key cellular mediators—such as macrophages and progenitor cells—particularly at the epigenetic level, which remains an unmet need. Here, by applying single-cell RNA sequencing to systematically profile cellular heterogeneity during tissue regeneration, we identified a pronounced depletion of S-adenosylmethionine (SAM), a central epigenetic metabolite, in impaired bone tissues. We hypothesize that SAM depletion contributes to inadequate modulation of the regenerative microenvironment and associated epigenetic dysregulation. To address this, we developed a compartmentalized, fusogenic nanovesicle system (SMNV), termed the “nanoconcentrisome,” composed of SAM-loaded fusogenic liposomes encapsulating mesenchymal stem cell–derived nanovesicles (MNVs) via bio-orthogonal click chemistry. This dual-compartment architecture enables spatiotemporally controlled cellular modulation through membrane fusion. Upon fusion with macrophages, cytoplasmic delivery of SAM suppresses inflammation, followed by MNV-mediated enhancement of efferocytosis and metabolic reprogramming to establish a pro-regenerative immune niche. Concurrently, fusion with senescent stem cells restores epigenetic integrity via SAM-mediated heterochromatin reinforcement, while MNVs rejuvenate mitochondrial function and promote osteogenic differentiation. This synergistic, cell type–specific modulation significantly enhanced tissue regeneration and effectively repaired bone defects in osteoporotic rats. Collectively, our study presents a precision nanomedicine platform that integrates epigenetic and regenerative reprogramming to counteract aging-associated regenerative decline, with broad translational potential for complex multicellular diseases driven by metabolic dysfunction.

overall_design: ATAC-seq profiling of Doxorubicin-induced rMSCs and SMNV intervention in senescence of rMSCs

first: Lan

last: Zhao

first: Kaiyu

last: Liang


title: Integrated metabolome and transcriptome profiling demonstrates dynamic regulatory roles of hormones in direct-seeding rice

first: Yang

last: Hang

address: 1000 Jinqi Road, Fengxian District

institute: Shanghai Academy of Agricultural Sciences

summary: Direct-seeding rice faces the prominent challenge of low seedling emergence vigor, particularly under deep-sowing mechanical resistance and hypoxic conditions. Although some physiological traits are known, the systemic molecular networks determining superior emergence remain elusive. Here, we integrated metabolomic and transcriptomic analyses to compare the elite direct-seeding variety ChongShang2022 (CS2022) with the control Huxiangruan450 (HXR450). Weighted gene co-expression network analysis (WGCNA) identified germination-associated metabolic modules. Hub metabolite analysis revealed that the accelerated germination of CS2022 correlates with a higher accumulation of cytokinins (zeatin and cis-zeatin-9-N-glucoside), known for antagonizing abscisic acid (ABA)-induced dormancy, alongside key amino acids (e.g., L-lysine) and structural sphingolipids. Physiological validation confirmed the functional significance of these hubs, demonstrating that exogenous trans-zeatin and L-lysine significantly promoted seed germination in a dose-dependent manner. Notably, CS2022 exhibited heightened sensitivity, achieving maximal promotion at concentrations approximately 10-fold lower than HXR450. Targeted LC-MS/MS assays further demonstrated that CS2022 maintains a significantly higher GA20/ABA ratio during germination by accumulating the key precursor GA20 and deactivating free ABA into ABA-glucosyl ester. This hormonal homeostasis couples with elevated α-amylase activity, accelerating energy mobilization. At the seedling stage, multi-omics integration suggests an optimized growth-defense trade-off in CS2022. Auxin signaling supports rapid elongation, while the upregulation of jasmonic acid (JA) precursor transcripts contrasts with restricted accumulation of bioactive signals (e.g., JA-Ile). This potential signal buffering mechanism likely mitigates growth arrest. Additionally, lipid remodeling involving sphingolipids and waxes may contribute to hypoxia tolerance. Altogether, this study delineates a correlative regulatory network where dynamic hormone buffering, redirected metabolic flux, and adaptive lipid remodeling synergistically maximize direct-seeding rice emergence vigor, providing mechanistic insights and candidate modules for breeding.

overall_design: We performed transcriptome sequencing on 5 days after sowing seedlings following direct seeding of both varieties and conducted a multi-omics integrated analysis.

first: Hang

last: Yang

first: Zejun

last: Hu

first: Ying

last: Yan


title: Lymphocyte-derived cholinergic circuits modulate germinal center output and B cell activation

city: Toronto

first: Liam

middle: D

last: Hendrikse

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

summary: Germinal centers (GCs) are specialized lymphoid structures in which activated B cells undergo clonal selection and B cell receptor (BCR) somatic hypermutation to generate high-affinity antibodies. Previous work has shown that T cells expressing choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine (ACh), are linked to the production of high-affinity antibodies in the GC response. However, whether B cells in the GC also express ChAT, and the details of the interplay of cholinergic circuits within the GC, remain unclear. Here, we show that ChAT expressed by GC B cells contributes to the early accumulation of high-affinity GC B cells following antigen encounter. We identify key transcriptional regulators of ChAT expression in GC B cells, and demonstrate that ACh receptor (AChR) expression is dynamically coordinated during B cell activation. In vitro, we show that ACh binding to muscarinic AChRs limits plasma cell differentiation and dampens BCR signal transduction to fine-tune the threshold for affinity-based positive selection. Together, these findings reveal a previously unrecognized regulatory axis that operates early during GC selection and employs cholinergic signals to shape B cell fate decisions and humoral immunity.

overall_design: Comparative RNA-seq profiling of ChAT-GFP+ and ChAT-GFP- subsets of splenic GC B cells (CD19+, CD95+, CD38lo) and TFH cells (CD3+, PD1+, Cxcr5+) derived from ChAT-GFP-Tg mice on day 8 after SRBC immunization.

first: Duygu

last: Nechanitzky

first: Logan

middle: K

last: Smith

first: Robert

first: Tak

middle: W

last: Mak


title: Keratin 5 expression defines cancer-propagating cells that are sustained by a hierarchically related osteopontin-producing niche in high-grade serous ovarian carcinoma

city: Ithaca

first: Coulter

last: Ralston

state: NY

address: 237 Tower Rd

department: Biomedical Engineering

institute: Cornell University

summary: High-grade serous carcinoma (HGSC) is the most common and aggressive form of ovarian cancer. Advanced HGSCs display marked cellular heterogeneity. A subset of tumor cells functions as cancer-propagating cells (CPCs, also referred to as cancer stem cells), which are highly tumorigenic and exhibit stem cell-associated properties, including self-renewal and chemoresistance. In contrast, a substantial fraction of HGSC cells is non-tumorigenic. The role of these non-cancer-propagating cells (non-CPCs) and their relationship to CPCs remain poorly understood. Here, we demonstrate that neoplastic cells expressing the intermediate filament protein keratin 5 (KRT5) represent bona fide CPCs. KRT5⁺ cells form cancer organoids over successive passages, are tumorigenic in serial dilution xenograft assays, and are resistant to the antineoplastic agents doxorubicin and cisplatin. Single-cell lineage-tracing experiments show that KRT5⁺ CPCs give rise to KRT5⁻ cells. KRT5⁺ and KRT5⁻ populations exhibit distinct gene expression profiles, with KRT5⁻ cells characterized by expression of SPP1, which encodes the secreted factor osteopontin (OPN). Treatment with OPN enhances HGSC organoid growth and chemoresistance, whereas SPP1 knockdown reverses these effects. Together, these findings support a model in which HGSC contains two hierarchically related cell populations: KRT5⁺, OPN-responsive CPCs and KRT5⁻, non-tumorigenic cells that form a niche producing OPN. Targeting pathways that sustain both stem-like tumor cells and their supportive niche may enable reduced dosing of highly toxic chemotherapeutic agents while enhancing therapeutic efficacy in HGSC.

overall_design: RNA-seq analysis to explore how the transcriptome of high-grade serous ovarian carcinoma cell line, SKOV3, after sorting the cells based on KRT5 positivity.

first: Mallikarjun

last: Bidarimath

middle: R

first: Ian

middle: M

last: Rose

first: Alexander

last: Nikitin


title: Developmental control of DNA damage responses in α- and β-cells shapes the selective beta-cell susceptibility in diabetes [scRNA-Seq]

city: Duarte

first: Sangeeta

last: Dhawan

state: CA

address: 1500 E Duarte Rd

department: Translational Research and Cellular Therapeutics

institute: City of Hope

summary: Accumulation of DNA damage drives β-cell dysfunction, senescence, and death in Type 1 and Type 2 diabetes. While α-cell dysfunction also contributes to disease pathology, they remain remarkably resistant to senescence and cell-death. The mechanisms underlying these differential responses to diabetogenic stress, particularly differences in their DNA damage vulnerability, remain unclear. We demonstrate that replication introduces a window of genomic vulnerability in both α- and β-cells during neonatal growth, with α-cells exhibiting higher replication rates and DNA damage. We show that neonatal β-cells resolve DNA damage more efficiently during mitosis and favor error-free repair, while α-cells compensate for their higher DNA damage vulnerability through increased cellular turnover. Using mouse models of overnutrition and diabetes, we show that β-cells exhibit greater vulnerability to terminal DNA damage and impaired repair capacity under diabetogenic stress, with compensatory replication amplifying this vulnerability. We demonstrate that developmental epigenetic programs shape the differential DNA damage vulnerability of postnatal β- and α-cells. Loss of de novo DNA methyltransferase Dnmt3a in pancreatic progenitors selectively increases the DNA damage vulnerability of β-cells from neonatal growth through adulthood. Our findings uncover novel developmental mechanisms that shape the distinct DNA damage responses of postnatal β- and α-cells during growth and diabetes.

overall_design: Cells were captured using the Chromium X system and Next GEM Single Cell 3’ Reagent Kits v3. (10x Genomics), per manufacturer’s protocol, yielding ~5,800 and ~18,000 cells from the small and large neonatal islet samples respectively. Final sequencing libraries were assessed using the Agilent High Sensitivity DNA Chip and quantified with the Qubit High Sensitivity DNA Assay Kit (Thermo Fisher). Libraries were sequenced on an Illumina NovaSeq 6000 platform with the S4 Reagent Kit v1.5 (Illumina) at TGen, using a paired end read sequencing of 28 cycles for Read 1, 101 cycles for Read 2, and 10 cycles each for i7 and i5 index reads. Image analysis was performed with Real-time analysis (RTA) v3.4.4 software.

first: Sneha

middle: S

last: Varghese

first: Alessandro

middle: G

last: Hernandez-De La Peña

first: Aparamita

last: Pandey

first: Laura

last: Anchondo

first: Xiwei

last: Wu

first: Supriyo

last: Bhattacharya


title: Dysregulated TGFb-ERK Signaling Drives Aberrant Extracellular Matrix Production in Noonan Syndrome-Associated Pulmonary Valve Stenosis

city: New York

first: Clifford

middle: Z.

last: Liu

address: 1470 Madison Ave, Hess 8-301

institute: Icahn School of Medicine at Mount Sinai

summary: Pulmonary valve stenosis (PVS) is the most common congenital heart defect in Noonan syndrome (NS) and related RASopathies, yet the molecular mechanisms that link the underlying pathogenic variants to the valve pathology remains poorly defined. Here, we utilized a human iPSC-based valve differentiation platform to generate the cardiac valve cell lineage—including fibrosa and spongiosa valve interstitial cell (VIC) subtypes. Applying this model to CRISPR-edited iPSCs carrying NS- and Noonan syndrome with multiple lentigines (NSML)-associated variants, we identified early mesodermal and endocardial specification defects in all lines. Additionally, we found that NS-iPSCs exhibited fibrosa VIC-specific defects in endothelial-to-mesenchymal transition (EndMT), which was most pronounced in PTPN11N308D (N308D) cells. Through single-cell transcriptomics, we revealed widespread dysregulation of extracellular matrix (ECM) programs in N308D fibrosa VICs, including increased expression of collagens and proteoglycans, as well as dysregulation of multiple genes involved in ECM remodeling. We also detected activation of RAS-MAPK, TGFb, and fibrosis-associated pathways in our transcriptional dataset. We then characterized the global signaling changes in N308D fibrosa VICs through mass spectrometry-based phosphoproteomic profiling, which identified coordinated increases in ERK, PKC, and stress-related kinases, as well as enhanced activity at the TGFb receptor. Functionally, we observed that N308D fibrosa VICs had an exaggerated upregulation of ECM genes in the presence of TGFb2 ligand, suggesting that these cells are hypersensitive to TGFb stimulation. Furthermore, we demonstrated that this pathological ECM-program occurs independently of BAMBI, a negative regulator of TGFb signaling that was found to be decreased in N308D fibrosa VICs. Lastly, we performed histopathological analyses of stenotic pulmonary valves from two NS infants, which demonstrated marked overproduction and disorganization of ECM, mirroring the findings from our iPSC-based disease model. Together, our data reveals a central mechanism where NS-associated alleles sensitize fibrosa VICs to TGFb, which leads to aberrant downstream TGFb signaling and drives the pathological ECM program in NS-associated PVS.

overall_design: WTC11 human iPSC lines were CRISPR-edited to contain either RAF1 S257L/+, PTPN11 N308D/N308D, or PTPN11 Y279C/+ variants. These iPSCs were then differentiated to endocardial cells, fibrosa valvular interstitial cells, or spongiosa valvular interstitial cells. Live cell samples for each genotype, including unedited control iPSCs (WT), and differentiated cell populations were processed as individual samples, for a total of 12 samples. Single-cell RNAseq was performed using the Chromium platform (10x Genomics) with the 3' gene expression V3 kit, using an input of ~10,000 cells. Gel-bead in emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from GEMs by post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter ligation, and 10X-specific sample indexing following the manufacturer’s protocol. Libraries were quantified using Bioanalyzer (Agilent) and QuBit (Thermofisher) analysis and were sequenced in paired end mode on a NovaSeq instrument (Illumina) targeting a depth of 50,000-100,000 reads per cell. Sequencing data was aligned and quantified using the Cell Ranger Single-Cell Software Suite (version 7.1.0, 10x Genomics) against the provided GRCh38 reference genome.

middle: Z

first: Shrey

last: Patel

first: Daniel

last: Charytonowicz

last: Sebra

first: Bruce

last: Gelb


city: Palo Alto

first: Paul

last: CHENG

address: 870 QUARRY RD EXT, PALO ALTO

institute: Stanford University

summary: Cardiac allograft vasculopathy (CAV) is the leading cause of mortality in heart transplant recipients. Despite the prevalence of CAV, there are no targeted therapeutic options to prevent or reverse disease progression, and patients ultimately require retransplant. CAV is defined by progressive neointimal hyperplasia in donor heart coronary arteries, leading to luminal obliteration and ultimately allograft failure or sudden cardiac death. Although immune and stromal cell interactions are believed to play a key role in CAV pathogenesis, the specific cellular players and molecular signals driving disease remain undefined. In this study, we leverage single-cell RNA sequencing and spatial transcriptomics of human coronary arteries to transcriptionally characterize CAV and define the neointimal microenvironment. We compare arteries with CAV to atherosclerotic coronary artery disease and non-disease controls to identify a unique CAV transcriptional signature.

overall_design: We performed single-cell RNAseq of cells from atherosclerotic human coronary arteries, obtained from explanted hearts of patients undergoing heart transplantation. Debris was removed and cells were positively selected for viability using FACS. Cells were then processed by a 10X Chromium instrument using 3’ reagents (version 3 chemistry).

first: Li

last: Daniel Y

first: Gu

last: Wenduo

first: Cheng

last: Paul C


title: Developmental control of DNA damage responses in α- and β-cells shapes the selective beta-cell susceptibility in diabetes [RNA-Seq]

overall_design: Total RNA was extracted from 30-50 hand-picked islets isolated from C57BL/6J mice at P5 (neonatal, separated into small and large islets) and 2.5 months (adult) using the RNeasy Micro Kit (Qiagen). cDNA libraries were prepared from 250 ng total RNA using the KAPA HyperPrep Kit with RiboErase (HMR) (Kapa Biosystems) using manufacturer’s instructions. Libraries were validated using the Agilent Bioanalyzer and quantified with the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). Libraries were sequenced on Illumina HiSeq2500 (2X 101 bp, paired-end mode, SBS v4 kits)


title: DNA methylation stochasticity is linked to transcriptional variability and identifies convergent epigenetic disruption across genetically-defined subtypes of AML

first: Andrew

middle: P

last: Feinberg

address: 855 North Wolfe Street

department: Center for Epigenetics

type: Methylation profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Cancerformer: A CRISPR Screen-benchmarked Multimodal AI Platform for Predication of Cancer Dependencies in Patient-derived Organoids

first: Yu

last: Meng

address: Zhongke Road No.1

institute: ShanghaiTech University

summary: Dissection of cancer dependencies is the central topic of cancer research. Recent advance in artificial intelligence (AI) has provided the opportunies of rapid predication of cancer essential genes. However, these AI models are often limited by the incapability of leveraging multimodal information or insufficient benckmarks, leading to low success rate in physiologically relevant practice. Here, we developed Cancerformer, a multimodal deep learning framework that integrates single-cell RNA sequencing (scRNA-seq), TCGA transcriptomic profiles and protein-protein interaction (PPI) networks to predict cancer gene essentiality. By employing a Transformer architecture to capture gene functional context and Graph Neural Networks to embed topological structures of PPI networks, Cancerformer overcomes the generalization limitations of existing methods. Using the experimental results of CRISPR screen from multiple cancer cell lines HeLa, A549 and U-87MG, we demonstrated that Cancerformer consistently outperformed state-of-the-art baseline models under both gene-wise and sample-wise cross-validation splits as measured by multiple evaluation metrics. In subsequent applications, Cancerformer demonstrated strong generalization ability achieving a 90% experimental verification rate for top candidates in colorectal cancer HCT116 cells. Most importantly, without pre-training on patient-derived organoids (PDOs) data, Cancerformer successfully captured inter-patient heterogeneity in PDOs and revealed a context-specific metabolic dependency on oxidative phosphorylation pathways in 3D culture compared to 2D cell lines. Functional assays on top predicted targets confirmed their essentiality in PDO growth. This study established Cancerformer as a rigorously benchmarked multimodal AI model for predicting cancer dependencies with physiological relevance.

overall_design: Single-cell RNA sequencing (scRNA-seq) was performed on patient-derived tumor organoids (PDOs) from individuals with colorectal cancer. The primary objective of this study is to leverage artificial intelligence (AI) models to analyze single-cell transcriptomic profiles, facilitating the identification and discovery of disease-specific therapeutic targets.


title: Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males [MB49 tumor cells]

city: St. Louis

first: Elise

last: Alspach

address: 1100 South Grand Boulevard

department: MMI

institute: St. Louis University

summary: Sex-based differences in cancer incidence are incompletely understood, but potential roles for the immune system are beginning to emerge. CD4+ T cells play a central role in organizing antitumor immunity. In addition to cytokine production, CD40L expression on CD4+ T cell provides necessary helper signaling to dendritic cells that is required for the priming of cytotoxic tumor-specific CD8+ T cells. Despite these critical functions, the impact of biological sex on the CD4+ T cell response to cancer remains unknown. Here, we demonstrate that impaired immune-mediated tumor control in male compared to female mice is driven by disparate helper CD4+ T cell responses between the sexes. We find that CD40L expression is reduced on CD4+ T cells isolated from males via a mechanism predominantly driven by sex hormones, resulting in decreased CD40 signaling in dendritic cells within tumor-draining lymph nodes. These deficits resulted in decreased helper CD4+ T cell frequencies and impaired CD8+ T cell function within the male tumor microenvironment which could be rescued by targeting the CD40L-CD40 axis. Our findings identify a novel mechanism of CD4+ T cell-based sex differences in the immune response to cancer that impairs tumor control.

overall_design: MB49 tumor cells were cultured prior to being submitted for scRNA seq.

first: Katey

last: Hunt

first: Samantha

last: Cooke

first: Lindsey

last: Kuehm

first: Tyson

middle: A

last: Lobb

first: Emily

last: Ebert

first: Smirthi

last: Beeram

first: Ryan

last: Teague

first: Michelle

last: Brennan

first: Melissa

last: Berrien-Elliott

first: Stephen

last: Ferris


title: PMEPA1 mediates resistance to mTOR inhibitors in triple-negative breast cancer via dual modulation of TGF-β signaling and PI3K/AKT activation

city: hyderabad

first: Ramesh

last: Ummanni

state: Telangana

address: Tarnaka

department: Applied Biology

country: India

institute: CSIR-Indian Institute of Chemical Technology

summary: Acquired resistance to mammalian target of rapamycin inhibitors (mTORi) severely limits their clinical efficacy in triple-negative breast cancer (TNBC), a subtype devoid of targeted therapeutic options. To model acquired resistance, we established two mTORi-resistant TNBC cell lines, MDA-MB-231/DREVE and MDA-MB-231/DRRIDA, through chronic exposure to everolimus and ridaforolimus, respectively. These resistant cells exhibited sustained mTOR signaling, evasion of G1 arrest, enhanced migratory potential, and anchorage-independent growth. Transcriptome profiling by whole RNA Sequencing coupled with GSEA analyses revealed convergent activation of the PI3K/AKT axis and upregulation of TGF-β and KRAS signaling, alongside suppression of IL-2-mediated responses. Integrative analysis identified PMEPA1, a known modulator of TGF-β signaling, as a key driver of resistance. PMEPA1 expression was elevated in resistant cells and correlated with poor survival in TNBC patients. We demonstrated that PMEPA1 confers resistance to mTORi by suppressing canonical SMAD signaling while promoting activation of the PI3K/AKT/mTOR pathway through PTEN downregulation. Targeted silencing of PMEPA1 restored drug sensitivity, reversed epithelial–mesenchymal transition (EMT), and impaired stem-like properties. Pharmacological inhibition of upstream TGF-β signaling with galunisertib downregulated PMEPA1 and synergistically restored mTORi sensitivity in in-vitro and xenograft models, leading to significant tumor regression. Histological analysis confirmed reduced proliferation, angiogenesis, and PMEPA1 expression while necrotic foci increased in combination treated tumors. These findings establish PMEPA1 as a dual modulator of canonical and non-canonical TGF-β signaling, and a critical mediator of mTORi resistance in TNBC. Targeting the TGF-β/PMEPA1 axis represents a promising strategy to overcome resistance and improve outcomes in patients with mTORi-refractory TNBC

overall_design: RNA-sequencing was performed on MDA-MB-231/WT and two resistant derivatives, MDA-MB-231/DREVE (cultured in 20 μM Everolimus) and MDA-MB-231/DRRIDA (cultured in 30 μM Ridaforolimus), to profile transcriptional changes associated with acquired mTOR inhibitor resistance

first: Chithrapournami

last: M S

first: Biswajit

last: Biswas

first: Andugulapati

last: Balagi


title: Blimp-1 shapes germinal center B-cell clonal diversity by gating chromatin accessibility during light-to-dark zone transition

city: Pittsburgh

first: Harinder

last: Singh

state: PA

address: 5051 Centre Avenue

department: Center for Systems Immunology and Department of Immunology

institute: University of Pittsburgh

summary:
Extensive studies have delineated signaling systems and transcription factors (TFs) that positively regulate GC B cell responses. In the LZ, T follicular helper (Tfh) derived CD40L and ICOS along with B cell receptor (BCR) signals license B cell clones to pass through the G1-S checkpoint; the cells then re-enter the DZ for clonal expansion and AID mediated somatic hypermutation (SHM). Among TFs that promote GC initiation, selection, and output, BCL6 programs GC identity. MYC licenses positively selected LZ cells for re-entry and proliferation in the DZ, whereas FOXO1 controls the program underlying rapid cell divisions. PU.1 and SPIB via interactions with IRF8 help maintain GC identity and selection, acting on ETS–IRF composite elements (EICE). BATF supports robust GC responses and class switching. The POU TF module comprised of OCT1/POU2F1, OCT2/POU2F2, and their co-activator OBF1/POU2AF1 maintains BCR-dependent transcription required for GC expansion. Canonical and alternative NF-κB components c-Rel and p52/RelB also promote GC maintenance. A p52–ETS1 complex has been recently shown to induce OCT1 and OBF1, thereby generating a feedforward regulatory loop.

In contrast, few signaling-induced negative regulators of the GC response that restrict clonal dominance have been identified, notably Nr4a1 (NUR77). We posited that Blimp-1 which promotes exit of GC B cells into the plasma cell (PC) pathway, could also act as a signaling-induced transient feedback gate to restrain the GC B cell response, thereby reducing clonal dominance. In keeping with this hypothesis, low and heterogenous expression of Blimp-1 has been reported in GC B cells and its loss has been shown to result in larger GCs. Furthermore, Conter et al. recently demonstrated using bone marrow chimeras that B-cell-intrinsic Blimp-1 loss results in more exuberant GC responses associated with enhanced proliferation of antigen-specific GC B cells. We note that in addition to the feedback gating model for Blimp-1 action in the GC considered by us, two other models could account for the GC phenotype: (i) increased GC seeding due to impaired plasmablast differentiation or (ii) failed GC exit due to blocked PC differentiation. We reasoned that integrated single cell (sc) transcriptional and chromatin analyses of GC B cells along with delineation of their clonal dynamics could distinguish among the three models. Furthermore, such high dimensional analyses of the perturbed and amplified GC response would yield insights into the underlying molecular mechanisms of Blimp-1 action.

Using sc transcriptome, V(D)J and chromatin profiling coupled with single nucleotide-resolution accessibility modeling of regulatory DNA sequences, we show that Prdm1-deficient B cells mount an exaggerated GC reaction characterized by larger clone sizes and enhanced affinity maturation culminating in greater clonal dominance. This phenotype is not attributable to increased GC seeding nor can it be merely accounted for by impaired exit of GC B cells into the PC pathway. Rather, the integrated genomic analyses reveal that Blimp-1 constrains expression of genes encoding components of the BCR-signaling cascade. Loss of this feedback, results in enhanced Syk, Lyn and Btk activity in GC B cells and augments chromatin engagement of signaling-inducible TFs at EICE, NF-κB and POU (OCT) motifs, promoting the G1–S transition during LZ selection and fueling DZ expansion.

overall_design: Eight to ten week old Prdm1fl/flCd19cre/+ (Prdm1 cKO) and Prdm1+/+ Cd19cre/+ (WT) mice were immunized intraperitoneally with 50 μg NP(23)-CGG (Biosearch Technologies) mixed with 50% (v/v) imject alum (Thermo Fisher Scientific) and 1 μg LPS (Sigma).

first: Godhev Kumar

last: Manakkat Vijay


title: SF3B1 Phosphorylation Prompts U2AF2 Dissociation for Widespread Control of Pre-mRNA Splicing

city: Rochester

first: Clara

last: Kielkopf

state: New York

address: 601 Elmwood Ave

department: Biochemistry & Biophysics

institute: University of Rochester

summary: Reversible pre-mRNA splicing factor phosphorylation is a well-documented feature of spliceosome assembly, activation, and disassembly, yet its functional and mechanistic roles are still emerging. SF3B1, a core spliceosome subunit, is extensively phosphorylated before the first catalytic step of pre-mRNA splicing. Intriguingly, most SF3B1 phosphorylation sites surround its binding sites for U2AF2, an early-stage pre-mRNA splicing factor. Here, we discovered that SF3B1 phosphorylation significantly decreases its association with U2AF2. We determined three crystal structures revealing electrostatic repulsion between an acidic U2AF2 alpha helix and the negatively charged phosphoryl group of SF3B1. Variants with amino acid substitutions that prevent or mimic SF3B1 phosphorylation perturbed thousands of splice sites, primarily those marked by uridine-rich splice site signals recognized by U2AF2. Collectively, our findings demonstrate a widespread but previously unrecognized role for SF3B1 phosphorylation as a gateway for U2AF2 dissociation so that pre-mRNA splicing can proceed.

overall_design: RNA-seq of SF3B1 knockdown and rescue with wildtype or TP-substituted derivatives (AP or EP) SF3B1 constructs in HEK 293T cells.

first: Christopher

middle: L

last: Kirchhoff

first: Hannah

last: Powell

first: Justin

last: Galardi

first: Sarah

last: Loerch

first: Mary

last: Pulvino

first: Jermaine

last: Jenkins

first: Paul

last: Boutz


title: Impaired CD4+ T cell help through the CD40L-CD40 pathway contributes to reduced tumor control in males

first: Elise

last: Alspach

overall_design: MB49 tumors were harvested from male and female mice 14 days following transplant.

first: Katey

last: Hunt

first: Samantha

last: Cooke

first: Lindsey

last: Kuehm

first: Tyson

last: Lobb

first: Emily

last: Ebert

first: Smirthi

last: Beeram

first: Ryan

last: Teague

first: Michelle

last: Brennan


title: Multiome Profiling Reveals Astrocyte and Neuroendocrine Targets of Prenatal Acoustic Programming in Zebra Finch Embryos

city: Clemson

first: Prakrit

last: Subba

state: SC

address: 132 Long Hall, 230 Parkway Drive

department: Biological Sciences

institute: Clemson University

summary: Prenatal sensory experience can program offspring phenotypes, yet the cell-type-specific regulatory mechanisms remain poorly understood. In zebra finches (Taeniopygia guttata), parental heat call vocalizations program offspring for thermal environments. To identify the cellular and epigenetic targets of this acoustic programming, we performed single-nucleus multiome sequencing (joint snRNA-seq and snATAC-seq) on medial hypothalamic punches from embryonic day 13 (E13) zebra finch embryos exposed to heat call or control call playback from E9–E13. We profiled 55,950 nuclei across 49 cell clusters representing 13 cell types. Differential chromatin accessibility analysis revealed a striking enrichment of chromatin remodeling in astrocytes (204 of 236 differentially accessible peaks), with Heat Call exposure predominantly increasing chromatin accessibility. chromVAR motif enrichment identified NFIC and AP-1 family transcription factors as top differentially active motifs in heat call embryos. Weighted gene co-expression network analysis identified an astrocyte module (Astro-M7) positively associated with heat call exposure, enriched for Notch signaling components. Cicero co-accessibility analysis revealed 9,502 heat call-specific chromatin loops involving 3,891 unique regulatory elements, concentrated at gliogenic regulators including NFIA, HES-5-like, and DLL1. Pseudotemporal trajectory analysis showed accelerated astrocyte developmental progression in heat call embryos. Additionally, transthyretin (TTR) showed sexually dimorphic, playback-associated expression in paraventricular nucleus glutamatergic neurons. Together, these findings reveal an epigenetic priming mechanism by which prenatal acoustic cues accelerate astrocyte maturation and reshape neuroendocrine programs in the developing hypothalamus.

overall_design: Zebra finch (Taeniopygia guttata) eggs were exposed to heat call or control call playback from embryonic day 9 (E9) to embryonic day 13 (E13). At E13, medial hypothalamic punches were collected from embryos (n = 8 pools total; 4 heat call, 4 control; each pool comprised 5 hypothalamic tissue punches). Nuclei were isolated and processed using the 10x Chromium Single Cell Multiome ATAC + Gene Expression kit to obtain joint chromatin accessibility and gene expression profiles from the same single nuclei. Libraries were sequenced and aligned to the zebra finch reference genome (bTaeGut1.4.pri) using Cellranger-arc (v2.0.2). After quality control, 57,388 of 60,138 nuclei were retained for RNA-seq analysis and 70,720 of 76,730 nuclei were retained for ATAC-seq analysis. Joint multimodal clustering using Weighted Nearest Neighbor (WNN) analysis in Seurat/Signac identified 49 clusters representing 13 cell types across 55,950 nuclei.

first: Vijay

last: Shankar

first: Kaitlyn

last: Williams

first: Trudy

last: Mackay

first: Patrick

last: Freymuth

first: Natalie

last: Shay

first: Allison

last: Rees

first: Shannon

last: Liedl

first: Jonathan

last: Roberts

first: David

last: Clayton

first: Julia

last: George


title: A Glutamine Antagonist Prodrug Reprograms Dendritic Cells to Enhance Colorectal Cancer Immunotherapy

city: xi'an

first: Wei

last: Gaofei

state: Shaanxi

address: beilin

institute: Northwestern Polytechnical University

summary: The tumor microenvironment (TME) imposes profound metabolic and immunosuppressive barriers that compromise the functionality of immune cells and limit effective anti-tumor responses. A hallmark of many tumors is their heightened dependency on glutamine as a metabolic substrate, which leads to glutamine depletion within the TME. This glutamine scarcity impairs the function of dendritic cells (DCs), resulting in defective antigen presentation and diminished activation of CD8⁺ cytotoxic T cells. Here, we report T26, a bifunctional immunometabolic prodrug composed of JHU083 (a glutamine antagonist) and MSA-2 (a STING agonist), designed to restore DC functionality and enhance anti-tumor immunity. T26 effectively suppresses tumor growth in colorectal cancer (CRC) models by reprogramming the TME toward a more immunostimulatory state. Mechanistically, T26 promotes DC maturation, enhances antigen presentation, and facilitates robust CD8⁺ T cell responses, leading to durable tumor suppression. Importantly, DC depletion experiments demonstrate that the anti-tumor efficacy of T26 is critically dependent on DC activation. Moreover, T26 reprograms tumor-derived extracellular vesicles (EVs), enhancing their capacity to transmit immunostimulatory signals and support CD8⁺ T cell–mediated cytotoxicity. In addition, T26 exhibits strong synergistic effects when combined with standard chemotherapy, immune checkpoint blockade, and anti-angiogenic therapy, further potentiating therapeutic efficacy. Collectively, these findings position T26 as a promising candidate for combination cancer immunotherapy, offering a dual-acting strategy that overcomes metabolic suppression and immune evasion in the TME to improve treatment outcomes.

overall_design: We designed T26, a novel bifunctional immunometabolic agent that links JHU083 with MSA-2, a STING agonist, via a cleavable amide bond. This design allows for the synchronized delivery of glutamine antagonism and STING activation directly within the TME. Using murine CRC models, we demonstrated that T26 not only inhibits tumor cell proliferation but also remodels the metabolic landscape, activating innate immune signaling, promoting DC maturation, and enhancing CD8+ T cell infiltration and cytotoxic function. Additionally, we found that T26 reprograms tumor-derived extracellular vesicles (EVs) to enhance antigen presentation and immune activation, providing a novel mechanism of intercellular communication that drives immune responses. Importantly, T26 also exhibited significant synergy with chemotherapies, immunotherapies, and targeted therapies, enhancing tumor control without inducing systemic toxicity.

first: Gaofei

last: Wei

first: Bingjie

last: Zhang

first: Renming

last: Fan


title: Developmental control of DNA damage responses in α- and β-cells shapes the selective beta-cell susceptibility in diabetes

first: Sangeeta

last: Dhawan


title: RNA-Seq of T26-treated and untreated MC38 cell

summary: This study investigates the transcriptional changes in murine colon adenocarcinoma MC38 cells in response to treatment with T26, a novel compound designed to modulate glutamine metabolism and immune-related pathways. RNA sequencing (RNA-Seq) was performed to compare the transcriptomic profiles of MC38 cells treated with T26 versus untreated controls. Differentially expressed genes were analyzed to elucidate the molecular mechanisms underlying T26-mediated anti-tumor activity. This dataset provides a resource for understanding how T26 impacts tumor cell-intrinsic gene expression and may inform its potential role in cancer immunotherapy.

overall_design: MC38 murine colon cancer cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO₂. Cells were treated with T26 at a final concentration of 5 μM for 24 hours. Untreated cells cultured under identical conditions served as controls. Total RNA was extracted from three biological replicates per group using trizol reagent.


title: Impact of LDOC1 depletion on transcriptome of human EGFR mutated NSCLC PC9 cell line

city: Zhunan Miaoli County

first: Chia

middle: Huei

last: Lee

address: 35, Keyan Rd.

department: National Institute of Cancer Research

country: Taiwan

institute: Taiwan National Health Research Institute

summary: Transcriptome profiling of NSCLC PC9 cells with (shLDOC1) or without (shCtrl) LDOC1 depletion revealed widespread alterations in gene expression, suggesting that LDOC1 broadly influences transcriptional programs. One possibility is that LDOC1 exerts its impact on the transcriptome through epigenetic mechanisms

overall_design: Total RNA was harvested from PC9-shLDOC1 and PC9-shCtrl cell lines cultured under standard growth conditions.

first: Chia-Huei

first: Pin-Feng

last: Hung


title: Multiomics immune profiling of a patient-relevant orthotopic lung cancer model using SEPARATE-Seq.

city: Brussels

first: Daliya

last: Kancheva

address: Laarbeeklaan 103, Building E

country: Belgium

institute: Vrije Universiteit Brussel - VIB

summary: Relevant pre-clinical models are essential for driving progress in cancer therapy research. Here, we developed a pre-clinical study framework using an injectable orthotopic lung adenocarcinoma (LUAD) model (ORTHO) that replicates key features of human LUAD patients and is dissectible into tumoural and non-tumoural adjacent tissue, in analogy with patient samples. We also present SEPARATE-Seq, a technique enabling the partitioning of vascular and intratissue immune cells along with scRNA-Seq. By applying both SEPARATE-Seq and spatial transcriptomics to our dissectible ORTHO model, we confirmed that our model replicates key immune features of human LUAD patients. Similarly to these patients, we observed NK-cell dysfunction and neutrophil dichotomy, and uncovered that these are driven by their vascular/intratissue or tumour/adjacent location, highlighting the need for these spatial distinctions. Additionally, we reveal that several immune populations are restricted to specialised, local niches within the tumour, including a ring of lipid-associated TAMs lining the tumour edge and hubs of interferon-stimulated cells. Overall, our resource, available through an interactive tool, provides a comprehensive multiomics immune characterisation of a reproducible pre-clinical LUAD mouse model.

overall_design: Cell line scRNA-Seq: In vitro cultured cells from LLC-Thy1.1 cell lines: (i) not passaged (P0) or (ii) passaged twice in vivo (P2). P0 and P2 samples were multiplexed using both cell multiplexing oligonucleotide (CMO) and hashtag oligos (HTO). Cells were sorted for 7-AAD- singlets.    ORTHO/SUBCUT CITE-Seq: 7-AAD-CD45+Thy1.1- sorted immune cells, isolated from (i and ii) tumours or adjacent tissue of the ORTHO LLC-Thy1.1 P2 mouse model, (iii) lung tissue from HBSS mock-inoculated mice, or (IV) from subcutaneous LLC-Thy1.1 P2 mouse tumors. Individual murine samples were multiplexed using HTO hashing (4 mice per sample type), and the vascular cells of each murine sample were labeled using STREP-tags (binding to IV injected CD45-biotin antibody).

first: Bardet

last: Pauline

first: Allonsius

last: Lize

first: Laoui

last: Damya


title: Astrocyte-mediated neuroimmune signaling and extracellular matrix regulation in depression and antidepressant response

city: Atlanta

first: Benjamin

last: Siciliano

state: GA

address: 615 Michael Street

institute: Emory University

summary: Major depressive disorder (MDD) is a debilitating condition that imposes a significant burden on global health, with many patients developing treatment-resistant depression (TRD) despite available interventions. Personalized medicine, which tailors treatment based on individual molecular profiles, offers a promising strategy to address this challenge. The primary objective of this study was to elucidate astrocyte-specific mechanisms underlying differential responses to selective serotonin reuptake inhibitors (SSRIs) in MDD and to identify potential therapeutic targets for overcoming treatment resistance. Astrocytes, key regulators of neurotransmitter metabolism, synaptic activity, and neuroimmune responses, have increasingly been implicated in the pathophysiology of depression. To investigate these processes, we employed human-induced pluripotent stem cell (hiPSC) technology to generate astrocytes from SSRI-responsive patients, SSRI-nonresponsive patients, and healthy controls, thereby capturing patient-specific genetic and epigenetic variations in an in vitro platform. Comprehensive transcriptomic profiling, differential gene expression, and coexpression network analyses revealed distinct patterns in immune signaling, extracellular matrix remodeling, and cell cycle regulation between responders and nonresponders, while transcription factor assessments further indicated altered unfolded protein response signaling in nonresponders. Complementary LINCS-based computational screening identified FDA-approved antidepressants and novel repurposing candidates that counteract the pathological astrocyte states observed in nonresponders. Collectively, these results lay the groundwork for leveraging patient-specific astrocyte models to develop innovative, astrocyte-focused interventions for TRD, although further in vivo validation and larger-scale studies are warranted.

overall_design: RNA-seq profiling of hiPSC-derived astrocytes from SSRI responsive and non-responsive MDD patients, as well as age- and sex-matched healthy controls.

first: Youssef

last: Zohdy

first: Chongchong

last: Xu

first: Ming-Fen

last: Ho

first: Richard

last: Weinshilboum

first: Zhexing

last: Wen


title: Development and validation of a 5mC-based prognostic model for lung adenocarcinoma survival: a multicentre, retrospective study

city: Nanjing

first: Yifan

last: Wu

state: Jiangsu

address: Xianlin Road

department: School of Lif Sciences

institute: Nanjing University

type: Methylation profiling by genome tiling array

summary: Lung adenocarcinoma (LUAD) exhibits marked prognostic heterogeneity. Existing methylation-based prognostic models lack robustness, accuracy, or external validation, limiting their clinical utility. We develop MethPro-LUAD, a prognostic model based on eight 5-methylcytosine features, using TCGA-LUAD data (training, N=269; testing, N=180), and validate the model across two independent hospital cohorts (Beijing, N=195; Nanjing, N=88) and two external GEO datasets (N=82 and N=155) to ensure its generalizability and effectiveness. MethPro-LUAD stratifies patients into high- and low-risk groups, with high-risk individuals consistently showing significantly shorter overall survival across all cohorts. In addition, the model’s predictive performance remains robust in subgroups defined by age, sex, stage, and EGFR mutation, and in disease-free survival analyses. Compared with other reported LUAD prognostic models, MethPro-LUAD demonstrates better performance across cohorts, which offers valuable support for personalized postoperative treatment and follow-up, and warrants further prospective evaluation, highlighting its strong potential for clinical translation through feasible targeted assays.

overall_design: We conducted a multicentre, retrospective trial to establish independent validation cohorts at two hospitals in northern and eastern China (Peking Union Medical College Hospital and Nanjing Drum Tower Hospital). Patients with clinically diagnosed LUAD who underwent surgical resection and had well-preserved , paraffin-embedded tumor tissues were included. Exclusion criteria were evidence of distant metastases or local recurrence at baseline; any serious uncontrolled diseases; any other malignancies. All samples were histologically confirmed as lung adenocarcinoma, and their pathologic information was based on AJCC 8th edition criteria and confirmed by at least two pathologists. For LUAD patients who met the enrollment criteria, surgically excised, paraffin-embedded tumor tissues were retrieved and sectioned into multiple 10 µm slices. One section was subjected to H&E staining, and a pathologist delineated the tumor area for subsequent collection. DNA was extracted from FFPE samples using the QIAamp DNA FFPE Tissue Kit (Qiagen, #56404) according to the manufacturer's instructions. Rigorous quality control was performed post-extraction, including DNA quantification using Nanodrop One and Qubit 3.0, as well as assessment of DNA degradation via agarose gel electrophoresis, to ensure data quality and reliability of subsequent analysis. A total of 500 ng of genomic DNA (gDNA) was used for bisulfite conversion with the EZ DNA Methylation Kit (Zymo Research, D5001). Methylation assays were conducted according to the instructions for the Infinium MethylationEPIC BeadChip Kit (Illumina, WG-317-1002) by Sinotech Genomics and Genergy using the Illumina iScan System.


title: Metasasis-associated wound repair promotes reciprocal activation of the lung epithelium and breast cancer metastases during metastatic outgrowth

city: Aurora

first: Andrew

last: Goodspeed

state: CO

address: 12801 E 17th Ave, Mail Stop 8303

institute: University of Colorado Anschutz Medical Campus

summary: When tumor cells colonize distant organs during metastasis, they interact extensively with surrounding cells. These interactions often change the behavior of surrounding cell populations which collectively induce a pro-tumor microenvironment that permits tumor cell outgrowth into overt, clinically detectable metastatic disease. The lung is one of the most common sites of breast cancer (BC) metastasis, and alveolar epithelial cells are the most common cell type in the lung. A gap in our ability to treat patients with established lung metastases lies in our lack of knowledge regarding how the lung microenvironment is remodeled during metastatic outgrowth and how this contributes to disease progression. Immunocompetent preclinical mouse models of BC metastasis were used to study late-stage lung metastatic outgrowth, the stage at which most patients are diagnosed with metastatic disease. A custom imaging panel was developed to quantify lung wound repair and inflammation, and single-cell RNA-sequencing (scRNAseq) was performed on mouse lungs with high or low metastatic burden to identify cell-type specific genes altered during metastatic outgrowth. No-contact co-culture experiments were used to examine reciprocal paracrine interactions between lung type II alveolar epithelial (AT2) cells and BC cells. A chronic wound repair-related phenotype developed within the lung microenvironment during metastatic outgrowth, which was characterized by an increased number and activation of AT2 cells surrounding growing metastases. scRNAseq of lungs with a high versus low metastatic burden showed that metastatic outgrowth significantly changed AT2 gene expression, resulting in a modified secretome. No-contact co-culture experiments indicated that BC-derived secreted factors alter AT2 gene expression, while AT2-derived secreted factors promoted TNBC growth. We investigated the possible mechanism(s) responsible for these effects and found that AT2 secreted factor genes, regulated by the cAMP response element-binding protein (CREB), contribute to BC proliferation. Targeting CREB signaling with the phosphodiesterase 4 (PDE4) inhibitor roflumilast reduced AT2-BC reciprocal interactions in vitro and metastatic outgrowth in vivo. Our studies demonstrate the potential for targeting metastasis-associated wound repair and lung epithelial cell activation with PDE4 inhibitors. This strategy may be an effective way to treat and manage metastatic BC progression in established, clinically detectable disease.

overall_design: Induced pluripotent stem cells (iPSC) cells were differentiated into lung type II alveolar epithelial cells (iAT2) following the protocol outlined in Tamò et al. 2018. Transwell plates with 0.4µm pores were used to investigate paracrine interactions between cell lines (no contact co-culture). iAT2 cells were no contact co-cultured with TNBC cells (SUM159PT or MDA-MB-453) for 5 days. Control iAT2 cells were cultured alone.

first: Jessica

last: Christenson

first: Nicole

last: Spoelstra

first: Michelle

last: Williams

first: Kathleen

last: O'Neill

first: David

last: Orlicky

first: Jennifer

last: Wagner

first: Li Wei

last: Kuo

first: Lyndsey

last: Crump

last: Richer


title: Autocrine Activity of Engineered IL-33 mRNA Enhances Adoptive T Cell Therapy for Peritoneal Carcinomatosis and Synergizes with IL-12 mRNA

city: Pamplona

last: Ruiz Guillamón

state: Navarra

address: Av. de Pío XII, 55

department: Programa de Inmunología e Inmunoterapia

country: Spain

institute: Centro de Investigación de Medicina Aplicada (CIMA) - Clínica Universidad de Navarra

summary: Peritoneal carcinomatosis (PC) remains a significant clinical challenge, with limited therapeutic options. Adoptive cell therapy (ACT) using tumor-specific T cells has emerged as a promising strategy; however, its efficacy is often hindered by the immunosuppressive tumor microenvironment (TME). Interleukin-33 (IL-33), a member of the IL-1 family, plays a dual role in immunity and inflammation, with the potential to enhance antitumor responses. Here, we investigated the impact of IL-33 mRNA-engineered T cells on ACT efficacy in murine models of PC and explored the potential synergy with IL-12 mRNA.

overall_design: OT.I and PMEL-1 T cells were electroporated with mRNA encoding IL-33, IL-12, or an IL-33 mutein variant and assessed for cytokine production and cytotoxic function in vitro. RNA sequencing (RNA-seq) was performed to characterize gene expression profiles following IL-33 mRNA electroporation.

first: Leire

last: Arrizabalaga

last: Ruiz-Guillamón

first: José

last: González-Gomariz

first: Fernando

last: Aranda

first: Celia

last: Gomar

first: Nuria

last: Ardaiz

first: Aline

last: Risson

first: Pedro

last: Berraondo

first: Claudia Augusta

last: Di Trani

first: Virginia

last: Belsue


title: Gene expression profile at single cell level of intestinal epithelial cells from the distal colon

city: Washington

first: Anelia

last: Horvath

state: DC

address: 2300 I St NW

department: Biochemistry and Molecular Medicine

institute: George Washington University

summary: In our inital data, we found AOM-treated AND-1 mutant mice (T819A ) showed increased colorectal cancer (CRC) incidence. Since common fragile sites (CFSs) instability were identified to be associated with CRC. We used colon as a model to perform single cell RNA sequencing to broadly characterize CFSs stability and CFSs-associate gene expression across cell types.

overall_design: Female C57BL/6J  mice were used to generate all samples. Mice were treated with azoxymethane (AOM)  to genereate tumor and treated with Hydroxyurea (HU) to induce DNA replication stress.Tumor adjent normal distal colon tissues was harvested from mice in wild type and AND-1 T819A muatnt groups to perform single cell sequencing.

first: Zhuqing

last: Li

first: Yihan

last: Peng

first: Siera

last: Martinez

first: Wenge

last: Zhu


title: In vivo CRISPR screens of Toxoplasma gondii targeting proteins of the apical complex and the pellicle.

city: Suita

first: Yuta

last: Tachibana

state: Osaka

address: 3-1, Yamadaoka

department: Research Institute for Microbial Diseases (RIMD)

country: Japan

institute: Osaka University

summary: We performed in vivo CRISPR screens of Toxoplasma targeting proteins of the apical complex and the pellicle during mouse infection.

overall_design: This apical-pellicle library targeting 309 genes which consisted 3090 gRNAs targeting proteins of the apical complex and the pellicle together with dispensable and essential control genes.

first: Masahiro

last: Yamamoto


title: BCL-2 disruption during CAR T cell production augments potency through non-apoptotic adaptive changes [scRNA-seq]

city: Chicago

first: James

last: LaBelle

state: IL

address: 900 East 57th Street, KCBD 5122

institute: University of Chicago

summary: BH3 mimetics were initially designed to induce cancer cell death through targeting key anti-apoptotic members within the BCL-2 family of proteins. Venetoclax, the only FDA-approved BCL-2-specific BH3 mimetic, has demonstrated robust clinical efficacy in killing acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) cells attributed to its direct apoptotic tumor effect. However, little is known regarding the direct effect of venetoclax on tumor infiltrating T cells. Recent studies have reported apoptosis-independent immunomodulatory activities of venetoclax that still are not fully understood. Given the vital role of BCL-2 family proteins in the dynamic processes of T cell activation, differentiation to effector or memory phenotype and contraction, it becomes imperative to dissect the direct effect of BH3 mimetics on T cell functions. We previously demonstrated that murine T cells expanded for 5 days in vitro in the presence of venetoclax acquire adaptive-like reprograming leading to cell death resistance. Further, long-term in vivo treatment with venetoclax results in transcriptional alterations of signaling pathways involved in T cell activation and exhaustion resistance. Herein, we investigate whether this venetoclax-induced T cell reprograming can be employed to improve the efficacy of adoptive T cell therapy. Using murine and human CD19-targeted chimeric antigen receptor (CAR) T cell therapy (CD19CART) as a model, we show that venetoclax treatment during expansion of CD19CARTs prepared from T cells isolated from healthy donors and patients exhibit enhanced antitumor efficacy in vitro and in vivo using pre-clinical xenograft models of lymphoma and leukemia. Mechanistically, bulk and single-cell RNA sequencing analyses reveal that venetoclax treatment during CART manufacturing transcriptionally reprograms CART cells at key T cell signaling (JAK/STAT5, PI3K/AKT, NFKB) and metabolic (PGC1α, OXPHOS, glycolysis) pathways to enhance CAR T cell fitness and durability, mechanisms dependent upon BCL-2 affinity to venetoclax. Our study highlights a possible novel therapeutic approach to induce adaptive anti-apoptotic reprograming to enhance the efficacy of CAR T cell therapy and supports the continued examination of BH3 mimetics as immune modulators.

overall_design: Single-cell suspensions of vehicle- or venetoclax-treated CARTs of three healthy donors were prepared and stained with Zombie NIR Dye to sort live cells. Sorted cells were counted and 10000 live cells were partitioned into droplets for single-cell omics assays via Chromium Next GEM Single-Cell 5’Kit v2 (10x Genomics, 1000263). Sample were analyzed using scRNAseq.

first: Nada

last: Aboelella

first: Ryan

last: Park

first: Erting

last: Tang


title: BCL-2 disruption during CAR T cell production augments potency through non-apoptotic adaptive changes

overall_design: T cells were isolated from PBMCs of three healthy donor on day 0 and activated overnight with human anti-CD3/CD28 beads, followed by transduction with lentivirus designed to express CD19CAR on day 1. On day 2, cells were either treated with vehicle (DMSO) or with venetoclax daily until day 13. Single-cell suspensions of vehicle or venetoclax-treated CD19CARTs of the three healthy donors were prepared and stained with Zombie NIR Dye, CD4, CD8, CAR+ GFP to sort CD4+ CAR +, CD8+ CAR + live T cells. Sample were analyzed using Bulk-RNAseq


title: Effect of PA200 depletion on gene expression in lung cancer cell lines A549 and NCI-H1299

city: Borstel

first: Silke

last: Meiners

address: Parkallee 1-40

department: Leibniz Lung Center

country: Germany

institute: Research Center Borstel

summary: Proteasome activator 200 (PA200; also known as PSME4) is upregulated in non-small cell lung cancer (NSCLC) and linked to poor prognosis. We have previously demonstrated that the overexpression of PA200 in NSCLC is associated with immune evasion and reduced responsiveness to immune checkpoint inhibitors. The cell autonomous function of PA200 in cancer growth, however, has not been solved. We here demonstrate that deletion of PA200 in two distinct lung cancer cell lines induced cell-specific alterations in proteasome composition and activities with a minor direct impact on overall proteasome activity. Deficiency of PA200 in lung cancer cells did not consistently alter tumor cell growth in vitro and in vivo. However, we observed concerted inhibition of tumor cell migration and invasion with conserved downregulation of the integrin ITGB3 and transcriptional dysregulation of multiple cell adhesion and extracellular matrix regulators. Our transcriptome profiling revealed a striking disparity in the transcriptional response to PA200 deletion in the two lung cancer cell lines. Together with our PA200 interactome analysis that uncovered an unexpected cell-dependent profile of PA200-interacting proteins, our data indicate that the function of PA200 is cell specific and depends on the cellular context. In conclusion, we here demonstrate that PA200 cell-autonomously regulates the invasive capacities of tumor cells thereby potentially promoting lung cancer spread and metastasis formation. This mechanism might add to PA200-related immune evasion and contribute to the observed poor prognosis of individuals with PA200-overexpressing lung cancer.

overall_design: RNA-sequencing of the WT and PA200 knock-out A549 and NCI-H1299 cell lines generated via CRISPR/Cas9

first: Ayse

last: Yazgili

first: Georgia

last: Giotopoulou

first: Sabine

last: Behrend

first: Georgios

last: Stathopoulos


title: Chemogenomic dissection of the role of Class I Histone Deacetylases as therapeutically relevant dependencies in H3K27M-glioma

city: Cleveland

first: Yaw

last: Asante

state: OH

address: 10900 Euclid Ave

department: Department of Nutrition

institute: Case Western Reserve University

summary: Diffuse midline glioma (DMG), Histone 3 K27-altered, is a deadly pediatric brain tumor. Disease pathogenesis is driven by epigenetic dysregulation which can be exploited using histone deacetylase (HDAC) inhibitors. However, the precise mechanism of efficacy for these agents in DMG has not been fully explored, and their translational potential has been limited by off-target effects and poor central nervous system (CNS) penetration of clinically available drugs. We here confirm genetic and chemical dependencies of nuclear-localized HDAC class I isoforms, particularly HDAC2, in patient-derived DMG cell lines using CRISPR/Cas9 screening and chemical validation. Moreover, we identify an HDAC inhibitor termed “Compound 26” with HDAC1/2 isoform selectivity and excellent CNS penetration. Using RNA-sequencing and HiChIP, we show that Compound 26 induces immediate transcriptional rewiring of DMG cells to restore physiologic pontine histone marks and induce cell cycle arrest. Most importantly, Compound 26 potently inhibits DMG growth in-vitro and in an aggressive orthotopic mouse model. This study comprehensively outlines the reliance of DMG cells on HDAC class I isoforms, elucidates novel mechanisms for this dependency, and highlights a compelling new agent to bypass prior translational pitfalls.

overall_design: H3K27ac and H3K27me3 HiChIP in DMG cells treated with DMSO or C26

first: Berkley

middle: E

last: Gryder

first: Mariella

last: Filbin

first: Jun

last: Qi


title: Gestational diabetes mellitus impairs metabolic homeostasis and cardiac development of offsprings by epigenetic regulation of H3K27 methylation to key pro-fibrosis transcription factor MEOX1

city: 成都

first: Lu

last: He

address: WUHAN

institute: TONGJI

summary: As one of the most perinatal period metabolic disorders, gestational diabetes mellitus (GDM) has negative influence to the metabolic health as well as organ development of offsprings. The underlying mechanism(s) is not fully revealed.

overall_design: Methods: by using STZ-induced pregnant female mice, we established the GDM animal model. We monitored the growth, biochemistry indexes as well as tolerance of insulin and glucose of offspring to reveal their metabolic state. High fat diet was also applied to cause metabolic stimulation. The heart of offspring was harvested and examined for morphology, protein and transcriptome analysis. Epigenetic markers were screened in neonatal heart by western blot. Primary neonatal fibroblast was separated and cultured in vitro. The enrichment of transcription factor MEOX1 or epigenetic marker H3K27 methylation was investigated by chromatin immunoprecipitation.


title: Mll5 haploinsufficiency attenuates microglial phagocytosis through dysregulated TREM2-SGK3-GSK3β signaling and recapitulates ASD-like behaviors in mice

city: Beijing

first: Ya Bin

last: Wei

state: Beijing

address: 38 Xueyuan load

institute: Peking University

summary: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by persistent deficits in social communication and repetitive behaviors. Recent studies indicated that heterozygous mutations in the mixed lineage leukemia 5 (MLL5) gene are implicated in the ASD susceptibility and associated with neurodevelopmental abnormalities. However, the detailed mechanisms remain unclear. Here, we demonstrate that Mll5 haploinsufficiency in mice impairs microglial phagocytosis, drives neuronal hyperexcitability, and recapitulates core ASD-like behaviors. We also show that Mll5 acts as an epigenetic regulator, modulating microglial phagocytosis via the TREM2-SGK3-GSK3β signaling axis, which is associated with deficient glucose metabolism. Furthermore, ASD individual-derived microglia exhibit parallel reductions in MLL5 expression and phagocytic function. By targeting this pathway, lithium chloride, a GSK3β inhibitor, rescues both microglial phagocytosis deficits and behavioral abnormalities in Mll5 haploinsufficiency mice. Our findings highlight MLL5’s critical role in ASD and its potential as a therapeutic target.

overall_design: The microglia were sorted as CD11b+CD45low by the common standard from the hippocampus of three WT and three Mll5+/- P17-21 mice and analyzed using RNA-seq.

first: Shumin

last: Gao


title: Transcriptomic effect of LDOC1 and H2Bub1 in NSCLC

first: Chia

last: Lee

summary: Despite declining incidence and improved survival rate, lung cancer therapeutic outcomes remain unsatisfactory and still is a leading cause of cancer death. Metastasis and treatment resistance hampers the survival of lung cancer patients. Lung adenocarcinoma (LUAD) is the main subtype of lung cancer. Our studies showed that LDOC1 strongly inhibits human LUAD A549 cells invasiveness and LDOC1 knockdown (KD) sensitized A549 cells to cisplatin. The mechanisms have not been fully elucidated.Coimmunoprecipitation-western blot and confocal microscopic analysis demonstrated the interaction between LDOC1 and the E3 ubiquitin ligase RNF40. RNF40 and its paralog RNF20 forms a heterodimer to monoubiquitylate histone H2B on lysine 120. H2B monoubiquitination (H2Bub1) is a prominent epigenetic mark involved in a variety of cellular processes mainly by regulating the transcription of specific gene sets. Our results showed that LDOC1 negatively regulates H2Bub1 in cell lines and specimens of LUAD. The regulatory mechanism may involve the proteasome degradation of H2B and nuclear translocation of RNF40 mediated by LDOC1. Microarray analysis revealed LDOC1 KD caused a global change in gene expression. SLC7A11, which encodes a cystine-glutamate antiporter, is a key component of ferroptosis epigenetically regulated by H2Bub1 and is significantly upregulated in LDOC1-deficient A549 cells, supporting that the increased H2Bub1 level is the cause of transcriptome reprogram triggered by LDOC1 KD. The IPA results and GEPIA database suggested nine genes upregulated by LDOC1 KD were lethal metastasis gene (LM) candidates with their high expression is associated with shorter overall survival of LUAD patients. Genes involved in inhibition of idiopathic pulmonary fibrosis signaling and upregulated in LDOC1 KD cells will be considered as cisplatin-responsive (CS) gene candidates regulated by LDOC1/H2Bub1 axis.We hypothesized that LDOC1 epigenetically affects tumor metastasis and chemotherapy efficacy of LUAD via regulation of H2Bub1. We aimed to meta-analysis the gene expression array data and chromatin immunoprecipitation (ChIP)-sequencing data to identify lethal-metastasis (LM) and cisplatin-responsive (CR) genes that regulated by LDOC1/H2Bub1 axis in LUAD.

overall_design: (1) Identification of LM and CR genes in LUAD regulated by the LDOC / H2Bub1 axis. (2)Investigation of the molecular mechanisms that LDOC1 regulates the levels of H2Bub1.

first: Chia-Huei

first: Hsien-Neng

last: Huang


title: Fine-tuning m6A and METTL3 levels have profound impact on cellular proliferation and protein synthesis

city: Aarhus C

first: Samir

last: Watson

state: Midtjylland

address: Universitetsbyen 83

department: Institute of Molecular Biology and Genetics

country: Denmark

institute: Aarhus University

summary:
This study investigates how partial loss of the m6A RNA methyltransferase METTL3 alters the transcriptomic landscape and translational regulation in Triple Negative Breast Cancer cells. m6A is the most abundant internal RNA modification in eukaryotic mRNAs and long non-coding RNAs and plays important roles in RNA metabolism, translation, and gene expression regulation. Dysregulation of the m6A pathway has been implicated in multiple cancers, including triple negative breast cancer (TNBC), where reduced METTL3 expression has been associated with poor prognosis and increased metastatic potential.

To investigate how reduced METTL3 dosage affects RNA methylation and downstream cellular processes, we generated CRISPR/Cas9-engineered METTL3 heterozygous knockout (HKO) cell lines in the human TNBC cell line MDA-MB-468. These cells exhibit reduced METTL3 expression and modest global decreases in m6A levels, allowing investigation of the effects of partial m6A depletion, which may better reflect physiologically relevant perturbations observed in cancer.

Using Oxford Nanopore Direct RNA Sequencing, we performed transcriptome-wide profiling of m6A modifications in wild-type and METTL3 heterozygous knockout cells. Computational approaches were applied to identify differential m6A sites and estimate modification stoichiometry across transcripts. These data were integrated with functional analyses including proliferation assays, quantitative proteomics, phosphoproteomics, and global protein synthesis measurements to assess the biological consequences of altered m6A levels.

The study aims to determine how partial reductions in METTL3 activity reshape the m6A landscape and influence gene expression programs, particularly those related to translational control, growth signalling, and cancer cell proliferation. Our findings demonstrate that modest decreases in global m6A preferentially affect transcripts involved in translational regulation and are associated with increased translational output and proliferative capacity, whereas stronger pharmacological inhibition of METTL3 suppresses translation and cell growth.

Overall, this work provides insight into the dose-dependent effects of METTL3 and m6A RNA methylation on gene regulation, highlighting how partial disruption of the m6A machinery can reprogram translational pathways and contribute to tumour progression.

overall_design:
Wild-type (WT) and METTL3 heterozygous knockout (HKO) MDA-MB-468 cells were used to investigate the effects of partial loss of METTL3 on transcriptome-wide RNA methylation patterns. The HKO cell line was generated using CRISPR/Cas9-mediated genome editing targeting exon 1 of METTL3, resulting in reduced METTL3 expression and partial depletion of global m6A levels.

Total RNA was extracted from WT and METTL3 HKO cells grown under standard culture conditions. Polyadenylated RNA was isolated and subjected to Oxford Nanopore Direct RNA Sequencing to enable direct detection of RNA modifications without reverse transcription or amplification. Sequencing libraries were prepared according to the Oxford Nanopore Technologies direct RNA sequencing protocol and sequenced on Nanopore flow cells.

Three independent biological replicates were generated for each condition (WT and METTL3 HKO). Sequencing reads were basecalled and aligned to the human reference transcriptome to obtain transcript-level coverage. Computational analysis was performed to detect and quantify m6A modifications and identify differentially modified sites between WT and HKO samples.

Multiple nanopore-based analysis pipelines were applied, including m6Anet to estimate global m6A modification ratios at DRACH consensus motifs and differential modification analysis using xPore and Nanocompore. Selected high-confidence sites were further analysed using a modified nanoRMS pipeline to estimate m6A stoichiometry based on nanopore signal features.

This experimental design enabled transcriptome-wide comparison of RNA modification patterns between WT and METTL3 heterozygous knockout cells, allowing identification of both global and site-specific changes in m6A methylation associated with partial loss of METTL3.

first: Johanna

last: Luige

first: Sarah

last: N'Da Konan

first: Peter

last: Alling Dam

first: Alessandro

last: Brambilla

first: Helene

last: Overvad Pederson

first: Ulf

last: Andersson Vang Ørom


title: Transient high fat diet causes persistent susceptibility to ferroptosis in CD8 T cells [Ex_vivo_RNAseq]

city: Kyoto

first: Masaki

last: Tajima

address: Yoshidakonoe cho

institute: Kyoto University

summary: Chronic metabolic stress impacts the immune cell function, increasing the incidence of autoimmune diseases and cancers. Whether past history of a high fat diet (HFD) leads to immune dysfunction is less understood. Moreover, whether factors other than persistent epigenetic changes would impact on the immune function remain unknown. We find that even a transient exposure to HFD causes persistent metabolome changes and long-term vulnerability of CD8 T cells to oxidative stress. The past HFD signature of CD8 T cells includes enrichment of lipid species highly susceptible to ferroptosis, and depletion of metabolites with anti-oxidant function. We find that xanthine, through the salvage purine pathway fuels the GTP, which is subsequently converted to tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2). Xanthine-derived biopterins serve as ROS scavengers, rescuing CD8 T cells from activation-induced ferroptosis. Administration of xanthine or BH2 markedly reduces tumor growth in mice transiently fed HFD. Our data uncover that metabolic stress remains tattooed on membrane lipids, generating vulnerability to oxidative stress and ferroptosis, that can be mitigated by purine salvage pathway through the replenishment of guanines and biopterins pools.

overall_design: To explore the global transcriptome landscape of CD8 T cells, CD4 T cells, and  B cells from mice treated with normal diet (12 weeks), high fat diet (12 weeks), and switched diet (high fat diet for 6 weeks and then normal diet for 6 weeks) , RNA were isolated from FACS-sorted cells and processed for RNA sequencing.

first: Baihao

last: Zhang

first: Yuhsien

last: Lin

first: Ryoma

last: Kato

first: Sidonia

last: Fagarasan


title: Next Generation Sequencing Facilitates Quantitative Analysis of  Macrophage and Leukemia Cell Transcriptomes  in Mouse Iron-overload  MLL-AF9 Acute Myeloid Leukemia

first: feifei

last: yang

address: Changle Road 68

institute: Nanjing first hospital,Nanjing medical university

summary: The goals of this study aim to reveal functional and phenotypic diversity of  Macrophage and Leukemia Cell in response to the Iron-overloaded microenvironmental cues  in mouse acute myeloid leukemia

overall_design: Anasizy the transcriptomes of Macrophage and Leukemia cell in Mouse Iron-overload acute myeloid leukemia

first: Feifei

last: Yang


title: Effect of depletion of HNRNPC on multiple myeloma cells

summary: The goals of this study aim to reveal functional changes mediated by HNRNPC in multiple myeloma cells

overall_design: Anasizy the transcriptomes of multiple myeloma cells


title: Co-regulated Transcription Termination by SMN, FUS, TDP-43, and arginine dimethylation of the RNA Polymerase II CTD

city: Toronto

first: Ulrich

last: Braunschweig

state: Ontario

address: 160 College Street

department: Donnelly Centre

institute: University of Toronto

summary: The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. We previously found that symmetrical demethylation (me2s) of a CTD Arginine residue (R1810 in human) causes recruitment of the Tudor domain of SMN, which interacts with Senataxin.  SMN is mutated in spinal muscular atrophy (SMA), and Senataxin is sometimes mutated in Amyotrophic Lateral Sclerosis (ALS).  R1810me2s and SMN, like Senataxin, are important for resolving R-loops (DNA:RNA hybrids) at transcription terminators.  FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are sometimes mutated in ALS and FTD (Frontotemporal dementia).  Here we show that TDP-43 and, to some extent, FUS are recruited by the R1810me2s-SMN pathway.  Defects in FUS and TDP-43 recruitment influence RNAPII termination and R-loop accumulation, leading to elevated DNA damage at terminators that may contribute to neurodegenerative disorders like ALS and FTD.

overall_design:
ChIP-Seq of RNA polymerase II in control, FUS and TDP-43 knockout HEK293 cells
Authors state that the 'processed data' consist of a plot only since the analyses (including the plots as supplementary files) were done directly from BAM files.

first: Dorothy

middle: Y

last: Zhao

first: Zuyao

last: Ni

first: Shuye

last: Pu

first: Guoqing

last: Zhong

first: Frank

last: Schmitges

first: Benjamin

middle: J

last: Blencowe

first: Jack

last: Greenblatt


title: Cross-Species Transcriptomic Integration Reveals a Conserved, MIRO1-Mediated Macrophage-to-T Cell Signaling Axis Driving Immunosuppression in Glioma [RNA-Seq]

status: Public on Mar 05 2026

city: Palo Alto

first: Xinnan

last: Wang

state: CA

address: 1201 Welch Road, MSLS Building, Rm P151

department: Neurosurgery

institute: Stanford University

summary: We generated bulk RNA sequencing (RNA-seq) data from human glioma surgical resections treated ex vivo with a MIRO1-binding compound (MR3) or vehicle control. Fresh tumor specimens were processed and cultured under controlled conditions prior to RNA extraction and high-throughput transcriptomic profiling to characterize treatment-associated gene expression changes within the tumor microenvironment (TME). The dataset captures global transcriptional programs from tumor tissue, reflecting contributions from malignant cells as well as stromal and immune components present in the resections. These data enable analysis of MIRO1-dependent transcriptional regulation in human glioma and facilitate cross-species integration with murine single-nucleus RNA-seq datasets. This resource supports investigation of mitochondrial-associated transcriptional programs and their impact on immune microenvironment remodeling in human glioma.

overall_design: Bulk RNA-seq profiling of human glioma tumor core specimens treated ex vivo with 10 μM MIRO1-binding compound (MR3) or vehicle control (DMSO) for 24 hours, with snap-frozen tissues collected for transcriptomic analysis.

first: Menghan

last: Li

first: Zehui

last: Du


title: Differential cell signaling testing for cell-cell communication inference from single-cell data by dominoSignal

city: Portland

first: Lisa

last: Coussens

state: OR

address: 3181 SW Sam Jackson Park Rd

institute: Oregon Health and Science University

summary: Motivation: Algorithms for ligand-receptor network inference have emerged as commonly used tools to estimate cell-cell communication from reference single-cell data. Many studies employ these algorithms to compare signaling between conditions and lack methods to statistically identify signals that are significantly different. We previously developed the cell communication inference algorithm Domino, which considers ligand and receptor gene expression in association with downstream transcription factor activity scoring. We developed the dominoSignal software to innovate upon Domino and extend its functionality to test statistically differential cellular signaling. Results: This new functionality includes the compilation of active signals as linkages from multiple subjects in a single-cell data set and testing condition-dependent signaling linkage. The software is applicable for analysis of single-cell data sets with multiple subjects as biological replicates as well as with bootstrapped replicates from data sets with few or pooled subjects. We use simulation studies to benchmark the number of subjects in compared groups and cells within an annotated cell type sufficient to accurately identify differential linkages. We demonstrate the application of the Differential Cell Signaling Test (DCST) in the dominoSignal software to investigate consequences of cancer cell phenotypes and immunotherapy on cell-cell communication in tumor microenvironments. These applications in cancer studies demonstrate the ability of differential cell signaling analysis to infer changes to cell communication networks from therapeutic or experimental perturbations, which is broadly applicable across biological systems.

overall_design: scRNAseq from MMTV-PyMT tumors with no treatment or treated with Entinostat

first: Jacob

last: Mitchell

first: Orian

last: Stapleton

first: Kavita

last: Krishnan

first: Sushma

last: Nagaraj

first: Dmitrijs

last: Lvovs

first: Christopher

last: Cherry

first: Amanda

last: Poissonnier

first: Wes

last: Horton

first: Andrew

last: Adey

first: Varun

last: Rao

last: Huff

first: Jacquelyn

last: Zimmerman

first: Luciane

last: Kagohara

first: Neeha

last: Zaidi

first: Elizabeth

last: Jaffee

first: Jeniffer

last: Elisseeff

first: Elana

last: Fertig


title: Identification and biosynthesis of a novel thymidine hypermodification Na-dapT in Acinetobacter baumannii phage SH-Ab 15599

city: Shanghai

first: Haoyu

last: Ma

state: Shanghai

address: Zhongke Road, Pudong

department: iHuman institute

institute: ShanghaiTech University

summary: Bacteriophage genomes exhibit exceptional diversity in nucleobase modifications, which primarily function to counteract host immunity and reshape DNA physicochemical properties. Recent discoveries of aGPT-Pplase2-catalyzed novel thymidine hypermodifications reveal a broader enzymatic and chemical diversity in phage DNA modification systems. However, the diversity of thymidine hypermodification in other bacteriophages remains largely unexplored. Here we discovered a novel thymidine hypermodification, Na-dapT, in the Acinetobacter baumannii phage SH-Ab 15599, and elucidated its biosynthetic pathway, including the key diamine DNA transferase (DADT, formerly aGPT-Pplase2). DADT utilizes the abundant metabolite 1,3-diaminopropane of host to modify 5hmdU-DNA, exhibiting broad in vitro substrate specificity but a strong in vivo preference for 1,3-diaminopropane. Structural and mutagenesis analyses revealed the molecular basis for substrate recognition and catalysis. The Na-dapT modification occurs specifically at TG dinucleotides and confers resistance to multiple host restriction enzymes, enabling phage escape from the host restriction-modification system. Furthermore, RNA-seq analysis showed that phage infection reprograms host metabolism, upregulating genes for 1,3-diaminopropane synthesis to supply the modification precursor. Our study identified SH-Ab 15599 DADT as a versatile enzyme responsible for thymidine hypermodification, and comprehensively describes a viral strategy of exploiting host metabolites for DNA modification to evade bacterial defense.

overall_design: Samples were taken from Acinetobacter baumannii lysates infected with phage SH-Ab 15599 at seven internal time(0,5,10,15,20,30,40 min). Three independent experiments were performed.

first: Wenjing

last: Li

first: Suwen

last: Zhao


title: Fibro-Adipogenic Progenitor cells from murine SMA muscles are intrinsically adipogenic

city: Gainesville

first: Yangyi

middle: Edward

last: Luo

state: FL

address: 1864 Stadium Road

institute: University of Florida

summary: Spinal muscular atrophy is a neurodegenerative disorder caused by mutations in the SMN1 gene. Although classically viewed as a neurogenic disease, SMA patients exhibit poor skeletal muscle regeneration and increased fatty-fibrotic infiltration. Fibro-Adipogenic progenitors are mesenchymal precursor cells that contribute to muscle remodeling and underly fat and fibrosis formation. Because FAPs transiently express Smn1 during regeneration, FAPs were examined in muscles from adult  SMA and control mice to determine if reduced Smn activity altered their properties. We performed a non-biased screen of FAPs following BaCl2-induced injury using an in-situ cell surface proteomic strategy that probed the cellular membrane and environment of FAPs in early regeneration. Proteomic profiling revealed early adipogenic priming in SMA tissues, with increased levels of perilipin-4 and adipocyte lipid-binding proteins. Significantly more adipocytes accumulated in  SMA muscles after glycerol injection versus controls. Further, SMA FAPs produced more fat than control FAPs when transplanted into glycerol injured muscles lacking FAPs. RNA sequencing of FAPs isolated after BaCl2 or glycerol injury identified transcriptional enrichment of lipid biosynthesis and dysregulated lipid metabolism in SMA FAPs. Primary FAPs isolated from SMA muscles mirrored heightened adipocyte formation, which was normalized by increasing Smn activity with Risdiplam. Conversely, adipogenesis of primary FAPs from control muscles was enhanced when subjected to siRNA Smn1 knockdown. Together, these findings demonstrate that reduced Smn activity potentiates intrinsic adipogenic bias in FAPs that may contribute to pathological fat deposition in SMA muscle.

overall_design: Primary FAPs isolated using MACS at 3dpi after either BaCl2 or Glycerol injury

first: Yangyi E.

first: Elisabeth R.

last: Barton


title: Gene expression during primary Hepatic Stellate Cells activation

city: Daejeon

first: Thi Nga

last: Ha

address: 282 Munhwa-ro Jung-gu

department: Medical Science

country: South Korea

institute: Chungnam National University

summary: RNA-seq was conducted to investigate transcriptional dynamics during in vitro activation of primary hepatic stellate cells (HSCs). Cells were collected at Day 1, Day 4, and Day 7 of culture to represent progressive activation stages. Differential expression analysis identified time-dependent changes in genes associated with extracellular matrix remodeling, proliferation, inflammatory signaling, metabolic and fibrogenic pathways. These data provide insight into the molecular mechanisms governing HSC activation.

overall_design: RNA-seq profiling of primary Hepatic Stellate Cells Day 1, Day 4, and Day 7

first: Hyon-Seung

last: Yi