title: A Tale of Two Cell Lines: Characterization of differential efficacy of small molecule drugs cediranib and NU-7441 on primary vs. metastatic colorectal cancer

status: Public on Sep 17 2025

city: DOWNERS GROVE

first: Cai

last: Roberts

state: IL

address: 555 31ST STREET, SCIENCE HALL 322-E

department: Pharmacology

institute: Midwestern University

summary: Colorectal cancer is recognized as one of the leading causes of cancer death amongst both sexes in the U.S. Despite rigorous screening, those diagnosed with colon cancer often face poor prognosis, and approximately 70% of affected patients will develop metastatic relapse. The investigation of colon carcinoma cell lines’ genetic variability and response to chemotherapy panels may aid in targeting therapies to improve outcomes. This study aims to find correlations between metastasis status, gene variability, and drug response. We used two cell lines that were isolated from the same 51-year-old male with colorectal adenocarcinoma: a primary tumor-derived line (SW480) and a secondary metastasized-derived line (SW620). Live cell imaging using time-lapse microscopy over three days exhibited differential cell death responses following treatment with multiple chemotherapeutic agents, particularly against Cediranib and Nu-7441, with SW620 demonstrating greater sensitivity. Western blots revealed changes in DNA repair machinery expression (particularly NHEJ proteins) between SW480 and SW620. RNA sequencing via Gene Ontology corroborated our findings, demonstrating upregulated DNA repair and metabolic survival genes including TGM2 in SW620 and PROM1 in SW480. Both cell lines overcame higher concentrations of Nu-7441, but an SW620 line grown in non-attachment plates and then reattached (SW620F) exhibited greater resistance against Nu-7441, expanding upon gene adaptive behavior previously seen. Correlation between drug response and gene expression crucial to cell growth and successful metastasis may reveal new biomarkers to target potential treatments.

overall_design: RNAseq study comparing biological triplicatse of each of two paris of samples. SW480 cells derived from a primary colorectal cancer vs. SW620 cells derived from a metastatic site in the same patient; and PEO4 ovarian cancer cells treated with standard growth medium (D) or medium supplemented with 10ng/mL TGFbeta (T)

first: Josephine

last: Tsang


title: Gli3R-mediated inhibition of Hedgehog signaling alters the embryonic transcriptome in zebrafish

city: Worcester

first: Summer

last: Thyme

state: MA

address: 364 Plantation St

country: USA

institute: UMass Chan Medical School

type: Expression profiling by high throughput sequencing

summary: Hedgehog signaling is a key developmental pathway required for normal patterning of the embryonic body plan. In zebrafish, disruptions to hedgehog signaling cause well-characterized defects in specific cell types including neurons and glia derived from the ventral spinal cord. We inhibited hedgehog signaling by overexpressing the Gli3 repressor (Gli3R) ubiquitously and performed bulk RNA-seq of 30 hour post-fertilization zebrafish embryos. Consistent with known roles of hedgehog signaling, we observed reduced expression of genes marking floor plate, motor neurons, Kolmer-Agduhr cells, dopaminergic neurons, slow muscle cells, and anterior pituitary. Gene set enrichment analysis using marker genes derived from the Daniocell atlas also revealed downregulation of genes expressed in ionocytes, which are located in the embryonic skin and are responsible for osmotic homeostasis. Reduced expression of ionocyte-specific transporter genes and the transcription factor foxi3a suggests that Gli activity may play a role in the development of this cell type.

overall_design: RNA-sequencing of whole Tg(ubb:zGli3R) larve at 30 hpf. A mosaic F0 male was crossed to a wildtype female, and embryos from one clutch were collected at 30 hours post-fertilization (hpf). Transgenic embryos and wildtype control siblings were sorted based on the myl7:GFP transgenesis marker. Four dechorionated embryos were pooled for each biological replicate, and four biological replicates were collected per genotype.

first: Thyme

last: Summer


title: A novel oncolytic neutrophil phenotype is induced by IL-10R inhibition [CITE-seq]

city: New York

first: Yacine

last: Marouf

state: NY

address: 413 E 69th St

department: MCC

institute: Weill Cornell Medecine

summary: While the impact of neutrophils on antitumor T cells has been an active area of study, fewer studies have focused on the potential of neutrophils to directly impact tumor cells. Here we asked whether neutrophils have the capacity to directly kill tumor cells independently of T cells. We find that anti-CD40-based therapy, when combined with interleukin 10 (IL-10) receptor blockade, initiates a Batf3-dependent pathway in which IL-12 and interferon gamma (IFNγ) secretion results in oncolytic neutrophil activity. Using a combination of microscopy, single-cell, and functional assays, we observe that killing of tumor cells by neutrophils is dependent on physical contact and degranulation. This degranulation-mediated killing is associated with an atypical dynamic invasive neutrophil phenotype. In line with our preclinical findings, our phase 1 trial of anti-CD40 shows that circulating IL-12, IFNγ, and IL-10 increase in response to anti-CD40, while our phase 1b/2 PRINCE study shows that lower circulating IL-10 is associated with favorable overall survival specifically among anti-CD40-treated patients. Finally, we find that neutrophil expansion with granulocyte colony stimulating factor (G-CSF) is associated with improved overall survival specifically in patients treated with anti-CD40, suggesting that this pathway may be amenable to therapeutic intervention in patients with advanced cancer.

overall_design: C57BL/6 wild type and Batf3 knockout mice were intradermally inoculated with B16F10 murine melanoma cells on their flanks, and eight days later, they received Isotype control, or a combination of anti-CD40/MPL/anti-IL-10R (CMI). Twelve hours post-treatment, the B16F10 tumors were harvested and processed into single-cell suspensions. Single-cell RNA sequencing (scRNA-Seq) was subsequently performed on these FACS-sorted cell suspensions.

first: Danny Nejad

last: Khalil

first: Ricardo

last: Gomez

first: Amit

last: Regev

first: Fadi

last: Samaan

first: Sadna

last: Budhu

first: Daniel

last: Hirschhorn

first: Isabell

last: Schulze

first: Jessica A.

last: Lavery

first: Nicholas

last: Ceglia

first: Samuel

last: Freeman

first: Vivien I.

last: Maltez

first: Jaynia

last: Garcia

first: Nathan

last: Suek

first: Chien-Huan (Gil)

last: Weng

first: Christopher R.

last: Cabanski

first: Sébastien

last: Monette

first: Yevgeniy

last: Romin

first: Yanyun

last: Li

first: Ronan

last: Chaligne

first: Michael

last: Yellin

first: Tibor

last: Keler

first: Deena M.

last: Maurer

first: Anne-Laure

last: Flamar

first: Lucia

last: Morgado-Palacin

first: Levi

last: Mangarin

first: James

last: Harding

first: Wungki

last: Park

first: Jaclyn P.

last: Lyman

first: Stephen

last: Maddock

first: Mark H.

last: O’Hara

first: Ghassan K.

last: Abou-Alfa

first: Robert H.

last: Vonderheide

first: Eileen M.

last: O’Reilly

first: Ronald N.

last: Germain

first: Jedd D.

last: Wolchok

first: Taha

last: Merghoub


title: A gene regulatory element modulates myosin expression and alters cellular response to stress [HiCAR]

status: Public on Sep 18 2025

city: Durham

first: Taylor

last: Anglen

state: NC

address: 101 Science Drive, CIEMAS Rm. 2323

department: Biomedical Engineering

institute: Duke University

summary: A hallmark of heart disease is gene dysregulation and reactivation of fetal gene programs. Reactivation of these fetal programs can provide cardioprotective or compensatory effects during heart failure depending on the type and stage of the underlying cardiomyopathy. Many putative cardiac gene regulatory elements have been identified that may control these programs, but their functions are largely unknown. We profile genome-wide changes to gene expression and chromatin structure in cardiomyocytes derived from human pluripotent stem cells. We identify and characterize a gene regulatory element important for the regulation of MYH6 that encodes human fetal myosin and is critical to the pathogenesis of hypertrophic and dilated cardiomyopathies. Using chromatin conformation assays in combination with epigenome editing, we find that gene regulation is mediated through direct interaction between MYH6 and the enhancer. We also find that activation of the enhancer alters cardiomyocyte response to the hypertrophy-inducing peptide endothelin-1. Collectively, these results identify regulatory mechanisms of cardiac gene expression programs that act through critical developmental factors and modulate cellular response to stress.

overall_design: Differential analysis of chromatin looping after activation of an MYH6/MYH7 enhancer and Endothelin-1 stimulus in cardiomyocytes derived from human pluripotent stem cells, assessed using HiCAR protocols.

first: Irene

middle: M

last: Kaplow

first: Baekgyu

last: Choi

first: Kevin

middle: T

last: Hagy

first: Duc

last: Tran

first: Megan

middle: E

last: Ramaker

first: Svati

last: Shah

first: Ravi

last: Karra

first: Yarui

last: Diao

first: Charles

middle: A

last: Gersbach


title: QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5’splice sites [eCLIP HEK293xT]

city: Houston

first: Eric

last: Van Nostrand

state: TX

address: 1 Baylor Plaza Rm T334

department: Therapeutic Innovation Center

institute: Baylor College of Medicine

summary: During organogenesis, the establishment of tissue architecture requires precise RNA processing. Many developmentally essential exons bear weak 5′ splice sites (5′SS) yet are spliced with high precision, implying unknown yet active splicing fidelity mechanisms. Combining transcriptome and alternative splicing profiling and with temporal eCLIP mapping of RNA interactions across development, we identify the RNA-binding protein QKI as an essential direct regulator of splicing fidelity in key cardiac transcripts. Although QKI is dispensable for cardiac lineage specification, its loss disrupts sarcomere assembly despite intact expression of sarcomere mRNAs through exon skipping and intron retention in key sarcomeric mRNAs.  QKI-dependent exons in essential cardiac genes have weak 5′SS  and frequently show poor complementarity with U6 snRNA; we show that QKI directly interacts with U6 snRNA using an overlapping interface to its traditional intronic binding activity, securing U4/U6·U5 tri-snRNP recruitment to ensure exon inclusion. Thus, QKI exemplifies how context-aware RBPs enforce splicing fidelity at structurally vulnerable splice sites by coupling local intronic cues to spliceosome engagement, thereby securing accurate exon inclusion during organogenesis.

overall_design: Human embryonic stem cells were utilzed to perform eCLIP for QKI at D0, D4, and D8 timepoints in cardiomyocyte differentiation

first: Maureen

last: Akinyi

first: Wenjie

last: Yao

first: Jakub

last: Zeman

first: Laurence

last: Heaven

first: James

last: Oo

first: Deniz

last: Bartsch

first: Kaustubh

last: Kalamkar

first: Charlotte

last: Le Roux

first: Anne

last: Starner

first: Dominik

last: Aschemeier

first: Ralf

last: Brandes

first: Fei

last: Yuan

first: Mason

last: Bartels

first: Eric L

first: Leo

last: Kurian


title: Immunogenomic Diversity of Triple-Negative Breast Cancers in Obese and Non-obese Black and White Women

city: New Orleans

first: Jovanny

last: Zabaleta

state: LA

address: 1700 Tulane Ave

department: Interdisciplinary Oncology

institute: LSUHSC

summary: Racial disparities in incidence and outcomes of triple-negative breast cancer (TNBC) have been attributed to ancestry, socioeconomic factors and/or obesity. We studied 253 TNBCs from 128 Black and 125 White women. Arms were balanced for age, AJCC stage, histological grade and molecular subtypes, and differed significantly in BMI distribution, obesity, and Area Deprivation Index. We examined survival rates, whole-transcriptome RNASeq and genetic ancestry. In our sample, Black race or obesity were not intrinsically predictive of poor outcomes. TNBC molecular portraits varied with stage and biological aggressiveness, irrespective of race.  We identified a novel group of TNBCs with a distinctive luminal-like and stem-cell-like transcriptional profile that were equally distributed among Blacks and Whites. Genetic ancestry was highly admixed. Immune deconvolution showed a higher abundance of several immune cell populations in tumors from Blacks, which may have precision therapeutic significance, warranting further investigation of potential drivers of tumor immunity.

overall_design: Retrospective, two-arm observational study. The final sample set included  253 TNBCs - from 128 Black and 125 White women. The two arms were balanced with respect to age, AJCC stage, histological grade and molecular subtype. They differed significantly in BMI distribution, obesity rates and Area Deprivation Index. RNAseq was used to determine gene expression profiles associated with TNBC in African American (AA) and European American (EA), with and without obesity, from Louisiana. Gene profiles were used to determine associations with race/ethnicity, obesity stage, tumor stage, prediction of immune infiltration and survival

first: Fokhrul

last: Hossain

first: Denise

last: Danos

first: Xiao-Cheng

last: Wu

first: Luis

last: Del Valle

first: Chindo

last: Hicks

first: Jiande

first: Jerneja

last: Tomsic

first: Susan

last: Neuhausen

first: Yuan

middle: C

last: Ding

first: Kavitha

last: Mukund

first: Zahra

last: Mesrizadeh

first: Shankar

last: Subramaniam

first: Victoria

last: Seewaldt

first: Augusto

last: Ochoa

first: Lucio

last: Miele


title: QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5’splice sites [RNA-Seq]

first: Eric

last: Van Nostrand

overall_design: Human embryonic stem cells (WT and QKI KO) were utilzed to perform RNA-seq at D0, D4, and D8 timepoints in cardiomyocyte differentiation

first: Maureen

last: Akinyi

first: Wenjie

last: Yao

first: Jakub

last: Zeman

first: Laurence

last: Heaven

first: James

last: Oo

first: Deniz

last: Bartsch

first: Kaustubh

last: Kalamkar

first: Charlotte

last: Le Roux

first: Anne

last: Starner

first: Dominik

last: Aschemeier

first: Ralf

last: Brandes

first: Fei

last: Yuan

first: Mason

last: Bartels

first: Eric L

first: Leo


title: QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5’splice sites

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

overall_design: Refer to individual Series


title: BMP4 and BMP6 stimulation of adult mouse muscle stem cells activates the Notch signaling pathway [RNA-Seq II]

city: Berlin

first: Markus

last: Schuelke

state: Berlin

address: Augustenburger Platz 1

department: Neuropediatrics

country: Germany

institute: Charite

summary: Muscle stem cell (MuSC) activation, proliferation, self-renewal, and differentiation are tightly regulated processes that enable muscle development and regeneration without exhaustion of the stem cell pool. BMP signaling keeps MuSCs in a proliferative state and prevents their differentiation. We investigated the effect of bone morphogenetic protein 4 and 6 (BMP4 and BMP6) on transcriptional regulation and intracellular signaling of adult murine MuSCs using RNA-sequencing, RNA expression arrays, pathway-specific qPCR arrays, and Cleavage Under Targets and Tagmentation (CUT&Tag), which identifies the binding sites of transcription factors and the presence of specific histone marks with high spatial resolution. Freshly isolated MuSCs were grown for 42 to 72 hours, kept serum deprived for 6 hours, and then stimulated with BMP4/6. BMP4/6 addition resulted in a rapid increase in Inhibitor of DNA binding 1 (Id1)-expression, a typical BMP target gene, which peaked after one hour. As expected, stimulation with BMP4/6 resulted in a significant increase in transcription of BMP target genes such as Smad7, Nog, Id1, Klf10, and Lif, but also a strong increase in transcription of Notch pathway genes such as Hes1, Lfng, and Hey1. We showed that the important BMP pathway effectors SMAD4 and pSMAD1/5/9 bind to the promoters of these Notch targets. Our results suggest that the rapid expression of genes encoding Notch signaling components is due to direct regulation by BMP signaling.

overall_design: RNA-seq on two biological replicates of cultured adult mouse muscle stem cells stimulated with BMP6 or verhicle after serum starvation

first: Birthe

middle: K

last: Lange


title: Endothelial-enriched lncRNA Gm39822 modulates inflammation and dysfunction in non-diabetic endothelial cells

city: Boston

first: Mark

middle: W

last: Feinberg

address: 77 Avenue Louis Pasteur

department: Medicine

institute: Brigham and Women's Hospital

summary: Endothelial dysfunction underlies several vascular complications including diabetes and atherosclerosis. However, the underlying role of long non-coding RNAs (lncRNAs) remains poorly understood. This study elucidated the role of lncRNA Gm39822 in regulating endothelial dysfunction under healthy and diabetic conditions. Our data revealed that Gm39822 is enriched and upregulated in non-diabetic endothelial cells (ECs) when exposed to high glucose or inflammatory cytokines (TNF-a and IL-1b). Gm39822 overexpression promoted the expression of vascular cell adhesion molecule-1 (VCAM-1) and the adhesion of leukocytes in non-diabetic ECs but not in diabetic ECs. Conversely, Gm39822 silencing reduced VCAM1 expression and leukocyte adhesion in non-diabetic ECs and not in diabetic ECs. Gm39822 deficiency reduced the expression of inflammatory mediators (including p-P65, P65, P50, p-P38, P38. P-ERK1/2 and ERK1/2) in non-diabetic ECs. Furthermore, Gm39822 knockdown inhibited the secretion of pro-inflammatory cytokines including  TNF-a, IL-1b, and IL-6, suggesting that Gm39822 regulates EC inflammatory responses. Mechanistically, we identified C1D, a nuclear enriched corepressor, as an interacting partner of Gm39822 that could play an important role in mediating Gm39822 functions in non-diabetic ECs. Collectively, our results identify a novel lncRNA Gm39822 and provide insights into the molecular mechanisms underlying endothelial dysfunction. These findings highlight Gm39822 as a potential therapeutic target for mitigating vascular complications associated with non-diabetic endothelial dysfunction.

overall_design: Transcriptomic RNA-sequencing was performed in bEND.3 cells after ribodepletion and library construction by using standard library construction and Illumina HiSeq2500 V4 2x150 PE (Azenta). The RNA-Seq dataset comprised 4 samples each for GapmeR and Negative Control groups.

first: Amit

last: Chandra

first: Emre

last: Bektik

first: Vinay

last: Randhawa


title: Thrombospondin-1:CD47 signaling contributes to the development of T cell exhaustion in cancer II

city: NEW YORK

first: LEVI

last: MANGARIN

address: 413 E 69TH STREET

department: MEYER CANCER CENTER

institute: WEILL CORNELL MEDICINE

summary: T cell exhaustion, a hyporesponsive state of antigen-specific CD8+ T cells exhibiting elevated expression of multiple inhibitory surface receptors, is a major obstacle for the treatment of cancer. Current immune checkpoint blockade (ICB) therapies aimed at reinvigorating exhausted CD8+ T cells have demonstrated clinical effectiveness. However, not all cancer patients achieve long-term disease control due, at least in part, to the refractory nature of terminally exhausted CD8+ T cells to be functionally reinvigorated. Besides persistent antigen stimulation, the environmental sources and mechanisms that lead to CD8+ T cell exhaustion in cancer remain to be thoroughly characterized. Here, we show that the expression of CD47 [a.k.a. integrin-associated protein (IAP) and ‘‘don’t eat me’’ signal is upregulated in tumour-associated, exhausted CD8+ T cell compartments in both human and murine tumours. Our findings reveal a novel role of the extracellular matrix protein thrombospondin-1 (TSP-1) and CD47 in promoting T cell exhaustion. Engagement of TSP-1 with CD47 drives the upregulation of TOX and inhibitory immune checkpoint molecules and compromises the effector function of CD8+ T cells during tumour progression. Mechanistically, the interaction of TSP-1 with CD47 on CD+ T cells activates the calcineurin-NFAT signaling, thereby promoting TOX expression and the T cell exhaustion program in cancer.

overall_design: We performed 10X multiome for gene expression analysis, as well as chromatin assessments, from CD8+ T cells that were derived from tumors of mice treated with control or TAX2 peptides that disrupt the interaction between TSP-1 and CD47 during the course of tumor (B16 melanoma) progression. All CD8+ tumor-associated T cells were isolated for sort purification and then isolated for their nuclei for 10X multiome prep and sequencing. We mainly focus on their gene expression for comparison. Raw data are only provided for gene expression data.

first: Chien-Huan

last: Weng

first: Levi

last: Mangarin


title: Host RNA dynamics in cats with feline infectious peritonitis

city: München

first: Caroline

last: Friedel

address: Amalienstr. 17

department: Institut für Informatik

institute: Ludwig-Maximilians-Universität München

summary: Feline infectious peritonitis (FIP) is a fatal disease, if left untreated, caused by feline coronavirus (FCoV). Although the role of the immune system in the pathogenesis of FIP remains incompletely understood, an excessive and harmful immune response appears to be a key factor. In a recent study by the same authors, 18 cats with FIP were successfully cured with oral GS-441524. The present study aimed to analyze the molecular signatures of these treated cats using next-generation RNA-sequencing on full blood samples by highlighting key immune pathways involved in disease progression, treatment response, and recovery. Therefore, samples were analyzed at three stages: untreated (d0), treated with the antiviral drug (days 2, 7, 28), and fully cured (days 168, 252, 365). Additionally, gene expression profiles were compared to those of healthy FCoV-infected control cats (n=12) and uninfected healthy control cats (n=5). Multiple tests with adjusted p-value (adj.p<0.001) were performed. Within the first few days of treatment, massive changes in the RNA signature profile were observed, with approximately 75% of expressed genes significantly differentially regulated between d0 and later time points. Notably, a strong antiviral immune response, which was dominant at the onset of the disease, was significantly downregulated within two days, accompanied by a shift in blood cell composition from monocytes and neutrophils to B cells. Our findings revealed a rapid normalization of the blood RNA signature within the first week of treatment suggesting that elimination of the virus from the blood leads to a rapid down-regulation of the damaging immune response.

overall_design: RNA sequencing of feline whole blood in cats with feline infectious peritonitis before, during and after antiviral treatment

first: Katharina

last: Zwicklbauer

first: Pilar

last: Grassl

first: Martin

last: Alberer

first: Laura

last: Kolberg

first: Nina

last: Schweintzger

first: Sonja

last: Härtle

first: Kaspar

last: Matiasek

first: Regina

last: Hofmann-Lehmann

first: Katrin

last: Hartmann

first: Ulrich

last: von Both


title: Transcriptomic Profiling of L-Proline Induced Ferroptosis Reveals PPARγ-Mediated Regulatory Networks in HFpEF Progression

city: Shanghai

first: Kai

last: Huang

address: 168 Changhai Road Shanghai

country: China

institute: Changhai hospital

summary: This study investigates the molecular mechanisms by which L-proline contributes to heart failure with preserved ejection fraction (HFpEF) through ferroptosis. L-proline was found to exacerbate ferroptotic stress in cardiac tissue, partly by modulating the expression of key antioxidant and lipid metabolism genes. Notably, L-proline suppressed PPARγ signaling, a critical regulator of ferroptosis resistance and metabolic homeostasis. Downregulation of PPARγ led to activation of pro-ferroptotic mediators, including ACSL4 and TFR1, suggesting a mechanistic link between amino acid metabolism and cardiac vulnerability in HFpEF.

overall_design: A murine HFpEF model was induced via high-fat diet and L-NAME. Mice were divided into control and L-proline treated groups. After 8 weeks, left ventricular tissue was harvested for total RNA extraction. Differentially expressed genes associated with L-proline–induced ferroptosis and PPARγ signaling were identified by RNA sequencing using Illumina NovaSeq 6000. Bioinformatic analysis included:

first: Lin

last: Han


title: Interactions between gamma-delta T lymphocytes and M-MDSC cells in the pathogenesis of chronic lymphocytic leukemia

city: Lublin

first: Michal

last: Zarobkiewicz

address: Chodzki 4a

department: Clinical Immunology

country: Poland

institute: Medical University of Lublin

summary: RNA-seq of zoledronate-expanded Vdelta2 gamma-delta T cells from healthy donors, cultured alone or co-incubated with myeloid-derived suppressor cells (M-MDSC) or vitamin C. The study investigates how M-MDSC influence gamma-delta T-cell function and differentiation, and the potential modulatory effects of vitamin C and vitamin D. These data provide insight into immune regulation in CLL and mechanisms relevant for immunotherapy development.

overall_design: This study investigated the effect of myeloid-derived suppressor cells (M-MDSC) and vitamin C on the transcriptomic profile of zoledronate-expanded Vδ2 γδ T cells from healthy human donors. Peripheral blood γδ T cells were expanded in vitro with zoledronate and IL-2, then either cultured alone (control), co-incubated with autologous M-MDSC, or treated with vitamin C. Total RNA was extracted and subjected to paired-end RNA sequencing (150 bp) using the DNBSEQ-T7 platform (MGI Tech). Differential expression analysis was performed to compare gene expression between experimental conditions.


title: A Ternary-code DNA Methylome Atlas of Mouse Tissues

city: Philadelphia

first: Rahul

middle: M.

last: Kohli

state: Pennsylvania

address: 3610 Hamilton Walk, 502B Johnson Pavilion

department: Department of Medicine

institute: University of Pennsylvania

type: Methylation profiling by high throughput sequencing

summary: DNA cytosine modifications, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are key epigenetic regulators with distinct functions. Dissecting the ternary code (C, 5mC, 5hmC) across tissues and cell types remains a critical priority due to the limitations of traditional profiling methods based on bisulfite conversion. Here, we leverage the combined bisulfite and enzymatic (bACE) conversion with the Mouse Methylation BeadChip to generate 265 base-resolution ternary-code modification maps of 5mC and 5hmC across 29 mouse tissue types spanning 8-76 weeks of age and both sexes. Our atlas reveals a complex grammar of 5hmC distribution, jointly shaped by cell mitotic activity, chromatin states, and interplay with 5mC at the same and neighboring CpG sites. Of note, we demonstrate that 5hmC significantly complements 5mC-based biomarkers in delineating cell identity in both brain and non-brain tissues. Each modification state, including 5hmC alone, accurately discriminates tissue types, enabling high-precision machine learning classification of epigenetic identity. Furthermore, the ternary methylome variations extensively implicate gene transcriptional variation, with age-related changes correlated with gene expression in a tissue-dependent manner. Our work examining the interplay of tissue type, sex, and age on methylome dynamics deepens our understanding of cytosine modification biology, augments the scope of DNA modification biomarker discovery, and provides a reference atlas to explore epigenetic dynamics in development and disease.

overall_design: ACE-Seq was performed on gDNA isolated from mouse cerebellum, heart, kidney, and blood tissues to quantify levels of 5hmC by next generation sequencing-based approaches as means to validate and compare with array-based methods.

first: Sol Moe

last: Lee

first: David

last: Goldberg

first: Cameron

last: Cloud

first: Jared

middle: B

last: Parker

first: Christopher

last: Krapp

first: Christian

last: Loo

first: Elliot

last: Kim

first: Ivan

last: Zhao

first: Chengcheng

last: Jin

first: Rishi

last: Porecha

first: Marisa

middle: S

last: Bartolomei

first: Wanding

last: Zhou


title: Thrombospondin-1:CD47 signaling contributes to the development of T cell exhaustion in cancer.

first: LEVI

last: MANGARIN

overall_design: For the analysis of scRNA-seq of tumor-associated T cells of interests, we performed adoptive transfer of naïve-sorted CD8+ T cells from Pmel-1 CD47+/+ and Pmel-1 CD47+/- mice and combined them in 50% to 50% for adoptive transfer into B16 melanoma implanted mice. After 14 days of transfer, tumors from each mouse were dissected to get all the tumor-associated T cells for sorting. We then sorted live, CD8+ T cells that were transferred to perform scRNA-seq analysis separately.

first: Chien-Huan

last: Weng

first: Levi

last: Mangarin


title: Essentiality and dynamic expression of the human tRNA pool during viral infection [IndividualKO_tRNAseq]

city: Rehovot

first: Noa

last: Aharon-Hefetz

address: 234 Herzl Street

country: Israel

institute: Weizmann Institute of Science

summary: Human viruses rely on host translation resources, including the cellular tRNA pool, because they lack tRNA genes. Using tRNA sequencing, we profiled mature tRNAs during infections with human cytomegalovirus (HCMV) and SARS-CoV-2. HCMV-induced alterations in mature tRNA levels were predominantly virus-driven, with minimal influence from the cellular immune response. Certain post-transcriptional modifications correlated with tRNA stability and were actively manipulated by HCMV. By contrast, SARS-CoV-2 caused minimal changes in mature tRNA levels or modifications. Comparing viral codon usage with proliferation- versus differentiation-associated codon-usage signatures in human genes revealed striking divergence. HCMV genes aligned with differentiation codon usage, whereas SARS-CoV-2 genes matched proliferation codon usage. Structural and gene-expression genes in both viruses showed strong adaptation to host tRNA pools. Finally, a systematic CRISPR screen of human tRNA genes and tRNA-modifying enzymes identified specific tRNAs and enzymes that either enhanced or restricted HCMV infectivity and influenced cellular growth. Together, these data define a dynamic interplay between the host tRNA landscape and viral infection and illuminate mechanisms governing host–virus interactions.

overall_design: tRNA-seq profiling of individual-tRNA knockout HFF or non-targeting HFF sample

first: Michal

last: Schwartz

first: Noam

last: Stern-Ginossar

first: Orna

last: Dahan

first: Yitzhak

last: Pilpel


title: P300 Modulates Endothelial Mechanotransduction of Fluid Shear Stress

city: Chapel Hill

first: William

last: Polacheck

state: North Carolina

address: 10010B Mary Ellen Jones Bldg CB 7575

institute: UNC

summary: P300 is a lysine acetyltransferase that plays a significant role in regulating transcription and the nuclear acetylome. While P300 has been shown to be required for the transcription of certain early flow responsive genes, relatively little is known about its role in the endothelial response to hemodynamic fluid stress. Here we sought to define the role of P300 in mechanotransduction of fluid shear stress in the vascular endothelium.

overall_design: Bulk RNA sequencing of human umbilical vein endothelial cells exposed to static culture conditions or fluid shear stress (15dyn/cm2) for 24 hours in the presence or absence of P300 inibitor A485.

first: Chloe

middle: P

last: Whitworth

middle: J


title: Identification of GREM-1 and GAS6 as specific biomarkers for cancer-associated fibroblasts derived from patients with non-small cell lung cancer

city: Seoul

first: Sang-Won

last: Um

address: 81 Irwon-ro

department: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center

country: South Korea

institute: Sungkyunkwan University School of Medicine

summary: Cancer-associated fibroblasts (CAFs) play a pivotal role in the tumor microenvironment. We conducted an analysis using RNA sequencing to identify specific markers for CAFs compared to normal fibroblasts (NFs) in non-small cell carcinoma (NSCLC) under various condition. We analyzed 22 CAF samples and 11 NF samples. The 22 CAF samples consisted of 12 adenocarcinomas and 10 squamous cell carcinomas (SqCC), with 16 samples from the lungs and 6 samples from the lymph nodes. Notably, COL11A1, GREM1, CD36, and GAS6 showed higher expression in CAFs than in NFs, whereas TNC and CXCL2 were more highly expressed in NFs. CD36 levels were elevated in CAFs from lymph nodes (LN-CAFs) compared with those from lung specimens (Lung-CAFs) and NFs. COL11A1 levels in Lung-CAFs surpassed those in LN-CAFs and NFs. Both GREM1 and GAS6 showed strong expression in Lung-CAFs and LN-CAFs relative to NFs. CAFs exhibited features of the myofibroblast CAF subpopulation, whereas NFs displayed traits of the antigen-presenting CAF subtype. In NSCLC, GREM1 and GAS6 can be valuable diagnostic and therapeutic targets for CAFs from primary tumors and metastatic sites; they warrant further study.

overall_design:
RNA-seq profiling of CAF (cancer-associated fibroblast) and NF (normal fibroblast) from 22 patients with NSCLC

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Bo-Guen

first: Kyunghee

last: Park

first: Mina

last: Hwang

first: Hyewon

last: Lee

first: Kyung-Mi

first: Junsu

last: Choe

first: Sun Hye

last: Shin

first: Byeong-Ho

last: Jeong

first: Kyungjong

first: Junghee

first: Yeong Jeong

last: Jeon

first: Jong Ho

last: Cho

first: Hong Kwan

first: Woong-Yang


title: PRDM16 Regulates Prostate Cancer Cell Dormancy and Prevents Bone Metastatic Outgrowth

city: Tampa

first: Conor

last: Lynch

state: Florida

address: 12902 USF Magnolia Drive

institute: H.Lee MOFFITT Cancer Center

summary: Understanding dormancy in prostate cancer (PCa) is challenging due to model availability. Here, using murine and human PCa cell lines, we generated a stress-induced model of dormancy in vitro and demonstrated that the phenotype could be sustained upon intrailiac artery delivery into the bone marrow microenvironment. RNA-Seq analysis revealed that the transcription factor PR domain containing 16 (PRDM16) was commonly upregulated in dormant PCa cells compared to controls. Further, bone marrow disseminated tumor PCa cells from primary orthotopic tumors were largely positive for PRDM16. Genetic ablation and forced ectopic expression supported a role for PRDM16 in maintaining PCa dormancy in vitro and in vivo. Clinically, PRDM16 negatively correlated with disease recurrence, and PRDM16 negatively correlated with the E2F cell cycle program in disseminated tumor cells derived from PCa patient bone marrow. Gene enrichment and characterization studies implicated PRDM16 as a regulator of metabolic and cell cycle pathways. ChIP-qPCR further revealed that PRDM16 binds upstream of the promoter of RB1, a potent repressor of E2F activity. Overall, this study developed a straightforward method for inducing cancer cell dormancy and applied this approach to find that PRDM16 governs an intrinsic dormancy program in PCa.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNASeq analysis of different PCa cell lines in active, dormant, and reawakened conditions after short in vitro culture 48 hours in dormancy and 24 hours in reawakened conditions.

first: Mostafa

last: Nasr


title: Solithromycin mitigates Prevotella intermedia–induced exacerbation of MRSA ventilator-associated pneumonia by modulating hyperinflammatory responses and enhancing alveolar macrophage function

city: Nagasaki

first: Naoki

last: Iwanaga

address: Sakamoto, 1-7-1

country: Japan

institute: Nagasaki University

summary: Solithromycin protects by activating alveolar macrophages and promoting TNF-related responses, suggesting a novel immunomodulatory role for solithromycin in host defense against exacerbation of MRSA-VAP by P. int. sup.

overall_design: In vitro, total RNA was extracted from the lungs infected with MRSA in the presence of P. int. sup. or P. int. sup. (SOL) at 12 h post-infection in mice

first: Koki

last: Fukushima


title: The α/β hydrolase DWARF14-Like 2 regulates growth and development in Arabidopsis thaliana

city: Beijing

first: Nie Xiaofan

last: NieXiaofan

address: 北京市怀柔区怀北镇怀北庄380号

institute: University of Chinese Academy of Sciences

summary: To understand the role of DLK2 in regulating plant growth and development, we performed the comparative transcriptome profiling WT and DLK2-OE1 seedlings.

overall_design: 11-day-old WT and DLK2-OE1 seedlings were transferred from 1/2 Murashige and Skoog (MS) plates to soil and grown for 13 additional day. The rosette leaves of this 24-day-old seedlings were  frozen in liquid nitrogen immediately. Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversly transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB#7530,New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments were end repaired. A base added, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1.0X).And polymerase chain reaction (PCR) amplified. The resulting cDNA library was sequenced using Illumina Novaseq6000.

first: Xiaofan

last: Nie

first: Liangliang

last: Li

first: Chenbo

last: Zhu

first: Kaixin

last: Yang

first: Jiahe

last: Fu

first: Wenbo

last: Jia

first: Wei

last: Xie

first: Weiqiang


title: QKI ensures splicing fidelity during cardiogenesis by engaging the U6 tri-snRNP to activate splicing at weak 5’splice sites [eCLIP Hues6]

first: Eric

last: Van Nostrand

overall_design: Human embryonic stem cells were utilzed to perform eCLIP for QKI at D0, D4, and D8 timepoints in cardiomyocyte differentiation

first: Maureen

last: Akinyi

first: Wenjie

last: Yao

first: Jakub

last: Zeman

first: Laurence

last: Heaven

first: James

last: Oo

first: Deniz

last: Bartsch

first: Kaustubh

last: Kalamkar

first: Charlotte

last: Le Roux

first: Anne

last: Starner

first: Dominik

last: Aschemeier

first: Ralf

last: Brandes

first: Fei

last: Yuan

first: Mason

last: Bartels

first: Eric L

first: Leo

last: Kurian


title: Macrophage-derived Chemokine (C-C motif) ligand 24 (CCL24) promotes cardiac fibrosis

city: Minneapolis

first: Preethy

last: Parthiban

email: parth034@umn.edu, xrevelo@umn.edu

state: MN

address: 2231 6th St SE

department: Integrative Biology and Physiology

institute: University of Minnesota

summary: Cardiac resident macrophages (CRMs) have key roles in progression and prevention of cardiac fibrosis. We have previously shown CRMs are a genetically diverse and heterogenous cell population. Using single-cell RNA sequencing we identified a CRM subset with high expression of Chemokine (C-C motif) ligand 24 (CCL24). In this study, we uncover a profibrotic role for CCL24 through fibroblast activation and regulation of ECM pathways.

overall_design: Wildtype (WT) and CCL24 knockout (CCL24 KO) mice were subjected to sham (n = 2,2) and transversal aorta constriction (TAC; n = 6,4) surgeries, followed by RNA extraction from cardiac ventricular tissue 1 week later.

first: Fanta

last: Barrow

first: Huy

last: Nguyen

first: Micah

last: Draxler

first: Haiguang

last: Wang

first: Upendra

last: Chalise

first: Natalia

last: Araujo

first: Fernando

last: Souza-Neto

first: Jean

last: Pallais

first: Dogacan

last: Yucel

first: Hong

last: Liu

first: Erin

last: Ciske

first: Patrick

last: Fehrenbach

first: Sang

middle: H

last: Lee

first: Adam

last: Herman

first: Marc

last: Rothenberg

first: Samuel

last: Dudley

first: Jop

last: van Berlo

first: Xavier

last: Revelo


title: Enhanced Angiogenic Potential of Electrically Stimulated Human Adipose-Derived MSCs for Ischemic Tissue Regeneration

city: Gwangju

first: Seungjun

address: 123 cheomgan gwagiro

department: MSE

institute: Gwangju institute of science and technology

summary: Effective treatment of ischemic disease requires the reconstruction of blood vessels through the delivery of angiogenic factors, such as chemicals, proteins, and cells. In particular, substantial efforts have focused on enhancing the therapeutic potential of mesenchymal stem cells (MSCs) for treating ischemic diseases. In this study, we investigated the use of electrical stimulation (ES) to potentiate the pro-angiogenic properties of human adipose-derived MSCs (hAD-MSCs). Electrically potentiated MSCs (epMSCs) were generated by applying optimized ES parameters (0.3 V, 100 Hz). EpMSCs exhibited significantly enhanced angiogenic potential, including upregulated expression of pro-angiogenic factors (e.g., VEGF-A and HGF) and improved endothelial cell migration and tube formation in vitro. Transcriptomic and proteomic analyses revealed activation of key angiogenic pathways, particularly VEGFA-VEGFR2 signaling, which plays a critical role in enhancing the functionality of epMSCs. In vivo studies using a murine hindlimb ischemia model demonstrated that epMSCs enhanced blood flow recovery, induced angiogenesis, and reduced muscle atrophy more effectively than unstimulated MSCs. Overall, these findings suggest that electrical potentiation of MSCs is a promising strategy for effectively enhancing their angiogenic capabilities for treating ischemic diseases.

overall_design: Total RNA was extracted from 1 × 106 cells and subjected to Quantitative RNA sequencing (Quant-Seq) analysis (Ebiogen, Seoul, Republic of Korea). Raw read counts were normalized using the Trimmed Mean of M-values method and transformed to count-per-million. The results were analyzed using ExDEGA software (Ebiogen, Seoul, Republic of Korea). DEGs (fold-changes ≥ 1.5, p ≤ 0.05) were visualized as a volcano plot. Pathway enrichment analysis was performed using the DAVID (http://david.abcc.ncifcrf.gov/) and QuickGO (https://www.ebi.ac.uk/QuickGO/) databases. Ninety-nine genes were represented as a heatmap using the MultiExperiment Viewer software (J. Craig Venter Institute, Rockville, MD, USA, version 4.9.0). Ten key GO biological processes were identified. Protein-protein interactions among DEGs were analyzed using the STRING v12.0, web tool (http://string-db.org/), with k-means clustering (three clusters).

first: Jongdarm

last: Yi

first: Chisoen

last: Ryu

first: Gaeun

last: Kim

first: Junghyun

first: Jae Young


title: BMP4 and BMP6 stimulation of adult mouse muscle stem cells activates the Notch signaling pathway [CUT&Tag]

first: Markus

last: Schuelke

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: CUT&Tag-seq for H3K27ac, SMAD4 and pSMAD1/5/9 on two biological replicates of cultured adult mouse muscle stem cells stimulated with BMP6 or verhicle after serum starvation

first: Birthe

last: Lange

first: Viktoriia

last: Huryn


title: CXCR4-modification boosts CAR-T cell efficacy by improving tumor tracking and bone marrow homing in B cell malignancies [In vitro]

city: chengdu

first: pei

last: shu

address: guoxue lane

institute: west chia hospital, sichuan university

summary: Hematological malignancies of B-cell origin are characterized by frequent expressions of CXCR4 and CXCL12. The CXCR4-CXCL12 axis facilitates the metastasis of B-cell lymphoma and multiple myeloma (MM). It is also a pivotal regulator in the migration and bone marrow homing of T-cells. Herein, we hypothesized that engineering CAR-T cells to overexpress CXCR4 could utilize CXCR4-CXCL12 axis to enhance their therapeutic efficacy by increasing tumor tracking and bone marrow accumulation.

overall_design: In this study, we found that lentiviral transduction caused significant CXCR4 downregulation on T-cells, leading to impaired migration to CXCL12. The CXCR4 receptor-overexpressed (CXCR4hi) CD19 CAR-T and BCMA CAR-T cells showed superior tumor tracking and clearance capacities in localized and systemically disseminated models of B-cell lymphoma and MM, respectively. Notably, CXCR4-modification facilitated homing of CAR-T cells and accumulation in bone marrow, which further promoted memory T-cell differentiation, persistence and prolonged anti-tumor activity. Building on these findings, an investigator-initiated clinical trial (IIT) evaluating CXCR4hi CD19 CAR-T cells in patients with relapsed/refractory B-cell malignancies (NCT04684472) achieved encouraging efficacy: the low-dose cohort yielded 3 complete responses (CR) and 1 partial response (PR) within one-month post-infusion. These findings support CXCR4 modification as a strategy to improve CAR-T cell efficacy in hematologic B-cell malignancies, warranting further clinical investigation.

first: yongsheng

last: wang

first: dan

last: li

first: fuchun

last: guo

first: diyuan

last: qin

first: liqun

last: zou


title: CXCR4-modification boosts CAR-T cell efficacy by improving tumor tracking and bone marrow homing in B cell malignancies [In vivo]


title: Evolutionarily conserved but mechanistically diverse piRNA defense against gypsy in insect ovaries

city: Acton

first: Rippei

last: Hayashi

state: Australian Capital Territory

address: 131 Garran Road

department: John Curtin School of Medical Research

country: Australia

institute: The Australian National University

type: Non-coding RNA profiling by high throughput sequencing

summary: Organisms possess highly adaptive defence systems to silence rapidly evolving mobile genetic elements such as transposons. While individual transposons may appear and disappear over time, the evolutionary stability of the defence pathways targeting them remains unclear. In the ovarian somatic cells of Drosophila, envelope-carrying gypsy LTR retrotransposons are silenced by the piRNA pathway, which relies solely on Piwi - an evolutionary derivative of an Aubergine/Piwi gene duplication specific to flies. Recent studies, however, have revealed that envelope-carrying gypsy elements are widespread across metazoans and particularly abundant in insect genomes. This prompted us to investigate whether and how the piRNA pathway targets ovarian somatic gypsy elements in other insects. We found that Aedes and Anopheles mosquitoes, as well as stingless bees and crickets, all express piRNAs targeting gypsy elements in ovarian somatic cells, indicating a continuous arms race for more than 400 million years of insect evolution. Interestingly, we found that Aedes aegypti activate the same set of piRNA clusters in ovarian somatic cells as in other somatic tissues where they are known to target RNA viruses. This suggests that piRNA-based antiviral defence in mosquitoes may have originated from the ovarian somatic piRNA pathway. Moreover, we discovered that the mechanisms of piRNA biogenesis in ovarian somatic cells differ among insects: slicing-independent phasing dominates in dipterans, ping-pong amplification in bees, and slicing-dependent phasing in crickets. These findings indicate that distinct piRNA pathways have independently evolved at different timepoints to silence the same class of retrotransposons in insect evolution.

sample_organism:
Acheta domesticus
Aedes aegypti
Anopheles stephensi
Bactrocera tryoni
Teleogryllus oceanicus
Tetragonula carbonaria
Eristalis tenax

overall_design: The study investigates Piwi-interacting RNAs (piRNAs) expressed in the somatic cells of female ovaries in a variety of insect species with the aim to understand the evolutionary conflict between the host and gypsy LTR retrotransposons and other mobile elements that are active in this cell niche. We generated oxidised small RNA sequencing libraries from freshly dissected ovaries and eggs, and sequenced them by single-read 150bp mode on Illumina Novaseq.

first: Shashank

last: Chary


title: Quantifying HLA transcripts by genotype in chimeric mixtures at single-cell resolution [scrHLAtyping_MAS-seq]

city: Seattle

first: Sami

last: Kanaan

address: 1100 FAIRVIEW AVE N

department: TST

institute: FRED HUTCHINSON CANCER CENTER

summary: Gene products from the highly variable major histocompatibility locus, including HLA, are essential for self-recognition and immune surveillance of malignancy. Following allogeneic hematopoietic cell transplantation (alloHCT), genetic and epigenetic alterations in HLA can drive  disease recurrence, making precise HLA assessment critical for determination of future therapy. However, current methods lack the sensitivity to quantify HLA transcripts at the single cell level, limiting their clinical utility. We introduce scrHLA-typing, a novel method that accurately identifies and quantifies HLA transcripts in single cells using hybridization capture and long-read sequencing. When applied to samples from patients with post-transplant relapse, scrHLA-typing successfully detected allele-specific expression of MHC gene products at clinically actionable levels. By characterizing allele expression in residual leukemia cells, our assay identified differences in expression patterns among patients. This capability highlights scrHLA-typing’s potential to improve risk stratification and guide the selection of appropriate salvage therapies, enhancing personalized treatment strategies after post-transplant relapse.

overall_design: Mononuclear cells from bone marrow aspirates from patients with relapsing AML post-alloHCT were density-gradient collected and magnetically enriched for cells expressing on their surface the CD34 progenitor marker (in one patient, the CD117 marker was used instead). From both unsorted and enriched cells, full transcriptome single-cell libraries (10x Genomics) were generated using 5-prime chemistry capture (by default, unless otherwise indicated, i.e., prefix '3p' tagged to the sample name) and short-read sequenced to quantify global RNA expression. For most patients, cells were also stained prior to droplet capture with oligonucleotide-barcoded antibodies specific to cell surface/principal lineage antigens, and antibody-derived tags (ADT) were short-read sequenced and quantified. From the leftover transcriptomes, single-cell barcoded HLA full transcripts were magnetically enriched with biotinylated hybridization probes HLA-specific and streptavidin magnetic beads, afterwhich they were PCR-amplified and long-read sequenced. The HLA probe design incorporated all known HLA alleles available from the IMGT/HLA reference. In a few cases, long-read sequencing of HLA transcripts was augmented using ‘multiplexed arrays isoform sequencing’ (MAS-seq). The molecular method was supplemented with a computational pipeline we designed to iteratively align reads, predict genotypes, filter errors, and summarize HLA counts. Short-read and long-read sequencing were conducted on Illumina and PacBio platforms, respectively.


title: Inhibition of ACLY enhances tumor immunogenicity and resolves MASH-driven HCC

city: Hamilton

first: Gregory

last: Steinberg

state: Ontario

address: 1280 Main Street

country: Canada

institute: McMaster University

summary: Immunosuppressive tumor microenvironments are common in cancers such as metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC). While immune cell metabolism influences effector function, the impact of tumor metabolism on immunogenicity is less understood. ATP citrate lyase (ACLY), a key enzyme linking catabolic and anabolic processes, supports lipid metabolism and gene regulation. Although ACLY inhibition shows anti-proliferative effects in various tumors, clinical translation has been limited by challenges in inhibitor development and compensatory metabolic pathways. Using a mouse model of MASH-driven HCC that mirrors human disease, genetic inhibition of ACLY in hepatocytes and tumors reduced neoplastic lesions by over 70%. To evaluate the therapeutic potential of this pathway, a novel small-molecule ACLY inhibitor, EVT0185 (6-[4-(5-carboxy-5-methyl-hexyl)-phenyl]-2,2-dimethylhexanoic acid), was identified via phenotypic screening. EVT0185 is converted to a CoA thioester in the liver by SLC27A2 with cryo-EM structural analysis revealing EVT0185-CoA directly interacts with ACLY’s CoA binding site. Oral delivery of EVT0185 in four mouse models of MASH-HCC dramatically reduces tumor burden as monotherapy and enhances efficacy of current standards of care including tyrosine kinase inhibitors and immunotherapies. Transcriptomic and spatial profiling in mice and humans linked reduced tumor ACLY with increases in CXCL13, tumor infiltrating B-cells and tertiary lymphoid structures. Remarkably, the depletion of B cells blocked the anti-tumor effects of ACLY depletion. Together, these findings illustrate how targeting tumor metabolism can rewire immune function and suppress cancer progression in MASH-HCC.

overall_design: RNA-seq profiling of liver tumor tissues derived from C57BL-6 Acly floxed mice with and without ACLY deletion by AAV8-YFP or AAV8-TTR-Cre injection. Tissues were obtained at either 4 weeks or 8 weeks post AAV injection.

first: Jaya

last: Gautam

first: Jianhan

last: Wu

first: James

last: Lally


title: Quantifying HLA transcripts by genotype in chimeric mixtures at single-cell resolution [scrHLAtyping]


title: Inhibition of ACLY enhances tumor immunogenicity and resolves MASH-driven HCC [scRNA-seq]

first: DONGDONG

last: WANG

address: 1200 Main Street West

summary: Immunosuppressive tumor microenvironments are common in cancers such as metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC). While immune cell metabolism influences effector function, the impact of tumor metabolism on immunogenicity is less understood. ATP citrate lyase (ACLY), a key enzyme linking catabolic and anabolic processes, supports lipid metabolism and gene regulation. Although ACLY inhibition shows anti-proliferative effects in various tumors, clinical translation has been limited by challenges in inhibitor development and compensatory metabolic pathways. Using a mouse model of MASH-driven HCC that mirrors human disease, genetic inhibition of ACLY in hepatocytes and tumors reduced neoplastic lesions by over 70%. To evaluate the therapeutic potential of this pathway, a novel small-molecule ACLY inhibitor, EVT0185 (6-[4-(5-carboxy-5-methyl-hexyl)-phenyl]-2,2-dimethylhexanoic acid), was identified via phenotypic screening. EVT0185 is converted to a CoA thioester in the liver by SLC27A2 with cryo-EM structural analysis revealing EVT0185-CoA directly interacts with ACLY’s CoA binding site.  Oral delivery of EVT0185 in four mouse models of MASH-HCC dramatically reduces tumor burden as monotherapy and enhances efficacy of current standards of care including tyrosine kinase inhibitors and immunotherapies. Transcriptomic and spatial profiling in mice and humans linked reduced tumor ACLY with increases in CXCL13, tumor infiltrating B-cells and tertiary lymphoid structures. Remarkably, the depletion of B cells blocked the anti-tumor effects of ACLY depletion. Together, these findings illustrate how targeting tumor metabolism can rewire immune function and suppress cancer progression in MASH-HCC.

overall_design: To investigate the transcriptional effects of EVT0185, single-cell RNA sequencing was performed on snap-frozen livers from two mouse models of MASH-HCC: the WD-CCl4 and WD-DEN models. Each of the four experimental conditions (WD-CCl4 + vehicle, WD-CCl4 + EVT0185, WD-DEN + vehicle, WD-DEN + EVT0185) included 3 biological replicates, for a total of 12 samples. To enable high-throughput processing, these 12 samples were multiplexed across four pooled libraries, with each pool comprising cells from multiple samples. While 16 samples were processed and sequenced in total, only 12 are relevant to the present study; the remaining 4 were part of the same sequencing run but are not analyzed or discussed here. Sample-specific count matrices are provided for the 12 relevant samples.

middle: R

first: Russta

last: Fayyazi


title: single cell RNA-seq of samples from Cdh5-creERt2/Shb flox/flox mice

city: Uppsala

first: L.

last: He

address: Dag Hammarskjölds Väg 20

country: Sweden

institute: Uppsala University

summary: E0771.lmb tumors were grown after tamoxifen injection as previously described in wild-type and Cdh5-creERt2/Shb flox/flox mice. Mice were sacrificed on day 14-16 after tumor cell injection. The tumors were digested, CD45+-cells isolated by MACS microbeads (Miltenyi, Bergich Gladbach, Germany)

overall_design: single cell RNAseq was performed on 6 wild type and 6 KO samples in three separate experiments.

first: Michael

last: Welsh


title: Trajectories and Influencing Factors of Psychological Distress in Nasopharyngeal Carcinoma Patients Receiving Radiotherapy (Incorporating Genetic Factors): A Multicenter Longitudinal Study

city: Nanning

first: Pingping

last: Zeng

state: Guangxi

address: Guangxi Medical University, No. 8, Shuangyong Road, Nanning, Guangxi Zhuang Autonomous Region, China.

institute: Guangxi Medical University

summary:
Background: This multicenter longitudinal study seeks to investigate the dynamic changes of psychological distress (PD) in nasopharyngeal carcinoma (NPC) patients during radiotherapy, and reveal the expression profiles and regulatory networks of circRNAs in these NPC patients.
Methods: 282 newly diagnosed NPC patients from three hospitals in China were included. Participants completed questionnaires and provided blood samples. PD trajectories were identified via a latent class growth model (LCGM). Moreover, the factors that influence the PD trajectories were explored. Whole transcriptome sequencing was performed to investigate genetic factors. The real-time quantitative PCR was applied to validate circRNAs. We predicted the target mirnas, target Mrnas and target RNA-binding proteins (RBPs) of the top 10 malregulated circrnas. Subsequently, circRNA-miRNA-mRNA (ceRNA) and circRNA-RBP networks were constructed. In addition, the role of circRNA and target mRNA parent genes was predicted by KEGG and GO analysis.
Results: LCGM identified two of the most important PD trajectories during radiotherapy in NPC patients: Class 1 “decline distress group” (11.0%) and Class 4 “rise distress group” (20.3%). Household monthly per capita income, coping strategies, and perceived social support emerged as important predictors of PD trajectories. Regarding genetic factors, 600 circRNAs and 123 miRNAs were identified as being significantly differentially expressed. Notably, hsa_circ_0004277 demonstrated significant differences between patients in the rise and decline distress groups (P < 0.01). ceRNA and RBP networks may influence the pathophysiology of PD in NPC patients undergoing radiotherapy.
Conclusion: This study unraveled that PD trajectories in NPC patients during radiotherapy were heterogeneous, indicating the need for screening and timely interventions within this population. Furthermore, the expression patterns of ceRNA and circRNA–RBP networks and pathways related to these networks suggested a potential role of circRNAs in developing PD among NPC patients receiving radiotherapy.

overall_design:
Our experimental design involves collecting questionnaire data and blood samples from patients at admission and after radiotherapy through a multi-center longitudinal approach. Then, based on the latent categorization model (a statistical method), they were divided into the rise distress group and the decline distress group. Then, randomly select the blood of 3 patients from each of the rise and decline  distress groups for whole transcriptome sequencing (a total of 12 blood samples at two time points, with D1-3 being the blood of patients in the rise psychological distress group at admission; TD1-3 is the blood of patients in the group with a rise distress after radiotherapy. R1-3 represents the blood of patients in the group with decline distress upon admission. TR1-3 is the blood of patients in the group with decline distress after radiotherapy. Among them, the single-end (e.g. D1.fastq.gz) is the raw data of miRNA, and the paired end (e.g. D1_R1.fastq.gz) is the raw data of IncRNA, mRNA, and circRNA.
Peripheral blood (5 mL) was collected from participants who provided informed consent at T1 and T4. Using the PAXgene Blood RNA Kit (BD, USA), the total RNAs were extracted from samples of the rise distress group (Class 4) and decline distress group (Class 1) divided by LCGM following the manufacturer’s instructions. A Nanodrop ND-1000 (Thermo Fisher, USA) was applied to measure the concentration and quality of total RNAs. The sequencing library was built using around 1–2 μg of total RNAs per sample. RNA sequencing services were provided by CloudSeq Inc. (Shanghai, China). Trizol (Invitrogen, USA) was utilized to extract Total RNA, and rRNA was removed via the GenSeq® rRNA Removal Kit (GenSeq, Inc., Shanghai, China). Directional RNA libraries were built via the GenSeq® Directional RNA Library Prep Kit. RNA was fragmented to ~300 nt in length. Reverse transcriptase and random hexamers were utilized to synthesize the first-strand cDNA. Moreover, the dUTP Mix was applied to synthesize the second-strand cDNA. The double-stranded cDNA fragments underwent end repair, adapter ligation, and dA-tailing. The cDNA, ligated with adapters, was PCR-amplified and purified to generate the final sequencing libraries. Furthermore, the libraries were sequenced in paired-end 150 bp mode.

first: Jiajie

last: Ning

first: Jianhua

last: Huang

first: Yan

last: Liao

first: Jianxiong

last: Long


title: RNA-seq of endothelial cells in wild-type and Shb iECKO mice

summary: E0771.lmb tumors were grown after tamoxifen injection as previously described in wild-type and Shb iECKO (Shb flox/flox; Cdh5-CreERt2) mice, alternatively Shb ICKO (Shb flox/flox; Vav1-cre) mice. Mice were sacrificed on day 14-16 after tumor cell injection. The tumors were digested, CD45+-cells isolated by MACS microbeads (Miltenyi, Bergich Gladbach, Germany)  and subsequently CD31+-cells isolated by fluorescence-activated cell sorting (FACS) after staining for CD31. RNA from EC CD31+-cells were subjected to bulk RNAseq.

overall_design: Bulk EC RNAseq was performed on 11 wild type (from nine tumors) and 10 Shb iECKO (from eight tumors) samples in four separate experiments.


title: Quantifying HLA transcripts by genotype in chimeric mixtures at single-cell resolution [10x_RNA_ADT-seq]


title: Harnessing distinct tissue-resident immune niches via S100A9/TLR4 improves metabolic imbalance

city: Geneva

first: Roberto

last: Coppari

address: rue Michel servet 1

country: Switzerland

institute: University of Geneva

summary: The interplay between immune cells and metabolic pathways in health and disease is only partially understood. Yet, therapeutic strategies to harness immune cells for improving metabolic diseases are lacking. Here, we focused on insulin deficiency (ID), a metabolic disorder affecting millions worldwide and for which better treatment is needed. Our murine data reveal that ID rearranges the hepatic immune landscape whereby T cell presence lowers, while the Kupffer cell (KC) component increases. This is accompanied by a shift in the transcriptional signature and polarization of the latter towards the KC2 phenotype. By generating ID mice re-expressing Toll-Like Receptor 4 (TLR4) only in KCs, we demonstrate that KC TLR4 is required for the hyperketonemia- and hypertriglyceridemia lowering actions of S100A9 in ID. This effect is accompanied by a TLR4-dependent reshaping of the KC proteome and S100A9-mediated improvements in KC transcriptional aberrancies and polarization defects caused by ID. Moreover, we uncovered immune-niche-specific metabolic effects of S100A9 in ID. While TLR4 in KCs underlies the dyslipidemia improving action of S100A9, TLR4 in bone-marrow derived immune cells underpins the hyperglycemia-improving effects of S100A9 in skeletal muscle in ID. In summary, our findings pinpoint the S100A9-TLR4 axis as a new tool to harness immune cells for improving ID-related metabolic dysfunction.

overall_design: RNA seq analysis of GFP-tagged nuclei isolated from the Kupffer cells of healthy, diabetic and S100A9-treated diabetic mice expressing GFP in KC nuclei only.

first: Giulia

last: Lucibello

first: Gloria

last: Ursino

first: Pryscila

middle: D

last: S Teixeira

first: Szabolcs

last: Zahoran

first: Francesca

last: Fanuele

first: Marinos

last: Kallikourdis

first: Florian

last: Visentin

first: Christelle

last: Veyrat-Durebex

first: Ariane

last: Widmer

first: Yibo

first: Marco

last: Cremonesi

first: Claes

last: Wollheim

first: Perrine

last: Castets

first: Giorgio

last: Ramadori


title: Analyze the transcriptional signature of primary human renal progenitor cells (hRPCs)

city: Firenze

last: Carangelo

address: Viale Pieraccini 6

country: Italy

institute: University of Florence

summary: Podocytes and podocyte progenitors are interdependent components of the kidney's glomerular structure, with podocytes forming the glomerular filtration barrier and progenitors being key players in podocyte regeneration during pathophysiological processes. Both cell types are subjected to constant mechanical forces, whose alterations can initiate podocytopathy and worsen glomerular injury.

overall_design: Transcriptional signature of primary human renal progenitor cells

first: Maria Elena

last: Melica

first: Laura

last: Lasagni

first: Paola

last: Romagnani


title: Growth hormone releasing hormone analogue protects against diabetic renal injury via inhibiting oxidative stress and ferroptosis

city: Shenyang

first: Rong

last: Fu

address: Beiling Street, Yuhong District, Shenyang City, Liaoning Province

institute: Shenyang Medical College

summary: MR409 is a synthetic growth hormone-releasing hormone (GHRH) analogue. MR409 has been shown to enhance the survival rate and efficacy of islet cell transplantation in streptozotocin (STZ)-induced diabetes mice and exerts beneficial effects on cardiovascular diseases. The present study investigated the protective effects of MR409 on DB/DB and STZ-induced diabetic mice and to explore the underlying mechanisms that related to oxidative and ferroptosis. DB/DB mice or STZ combined with high fat diet were used to establish the T2D models. MR409 (15 μg/mouse/day) was subcutaneously administrated for 8 weeks. Treatment with MR409 significantly improved renal function and decreased the renal injury and fibrosis in DB/DB mice and STZ-induced mice. MR409 increased the expression of renal GHRH receptor without affecting the growth hormone level. MR409 attenuated oxidative stress, as indicating by decreasing the expressions of NADPH oxidase subunits p22phox, gp91phox, DHE oxidative fluorescence intensity, MDA and 4-HNE expression in the kidney of DB/DB mice. Meanwhile, MR409 inhibited ferroptosis in DB/DB mice, as indicated by upregulating the expressions of GPX4, Nrf2, FTH and downregulating TFR expression. Treatment with MR409 also increased the level of GSH in the kidney of DB/DB mice. Notably, MR409 activated the PPARγ and its downstream targeted gene Klotho in the kidney of the DB/DB mice. Therefore, the present study demonstrated that MR409 alleviates diabetic nephropathy (DN) via attenuating oxidative stress and ferroptosis, which provides a novel insight into the treatment for DN.

overall_design: To further elucidate the mechanism by which MR409 alleviates renal injury in DB/DB mice, we conducted a broad-spectrum gene screening in kidney tissues using RNA-sequencing analysis.

first: Yueyang

last: Liu

first: Qi

last: Tang

first: Yaoxia

last: Zhang

first: Ruiping

last: Cai

first: Junjia

last: Gao

first: Ming-Sheng

last: Zhou


title: Effect of Fak1/Ptk2 knockout in a mouse model of NF2-related schwannomatosis

city: Indianapolis

first: Steven

last: Angus

state: IN

address: 1044 W Walnut St

department: Pediatrics

institute: Indiana University School of Medicine

summary: NF2-related schwannomatosis (NF2-SWN) is a cancer predisposition syndrome characterized by the development of bilateral vestibular (VS) and spinal schwannomas. While benign, these tumors can cause significant morbidity and effective pharmacological treatments remain limited. Here we demonstrate that genetic ablation of focal adhesion kinase 1 (FAK1/PTK2) impairs tumor formation and preserves hearing in a murine model of NF2. Mechanistically, we show that Fak1 deletion decreases macrophage infiltration, attenuates NLRP3 inflammasome activation and suppresses the HGF-MET axis. Pharmacological inhibition of FAK with single agent VS-4718 did not significantly reduce macroscopic tumor volume, however, its use in combination with the MEK inhibitor selumetinib resulted in both a significant reduction tumor volume and the preservation of dorsal root ganglion (DRG) architecture. Our findings establish a critical role for FAK in schwannoma development and provide rationale for evaluation of combination FAK plus MEK inhibition in future clinical trials for NF2-associated schwannomas.

overall_design: Dorsal root ganglia (8 per mouse) tissue was microdissected from 10-month old euthanized Postn-Cre(+);Nf2 flox/flox animals (n=6, 3 male, 3 female) and from Postn-Cre(+);Nf2 flox/flox;Fak1 flox/flox (double knockout, DKO) animals(n=6, 3 male, 3 female), flash frozen and sent to Azenta/GenWiz for mRNA sequencing.

first: Dana

last: Mitchell


title: Targeting a Galectin-3/EGR1 transcriptional complex inhibits the pro-metastatic signature in TRIM49-deficient gastric adenocarcinoma II

city: Chong Qing

first: Xianfeng

last: Li

address: Da ping

department: Department of Gastroenterology, Daping Hospital

institute: Army Medical University (Third Military Medical University)

summary: Distal metastasis, driven by intracellular signaling rewiring, is a leading cause of cancer mortality. The role of post-translational modifications in metastasis remains unclear. A CRISPR screen in a gastric adenocarcinoma (GAC) mouse model identified TRIM49 as a metastasis suppressor. TRIM49 is downregulated in most advanced GACs, correlating with poor prognosis. Galectin-3, stabilized in TRIM49-deficient cells due to impaired degradation, forms a complex with EGR1, enhancing pro-metastatic gene expression. Disrupting this complex with GB1107 inhibits metastasis in mouse models. The Galectin-3/EGR1 complex in TRIM49-deficient GAC is a key driver of metastasis and a potential therapeutic target.

overall_design: The in vivo CRISPR-Cas9 screen experiment with whole genome CRISPR knockout pooled library (hGeCKOv2.0) was performed by Shanghai Genechem Co.,Ltd. Firstly, the GC cells infected with pooled hGeCKOv2.0 lentivirus library at a multiplicity of infection of 0.3 to ensure most cells received only one stably integrated RNA guide. Then, the infected GC cells were orthotopically injected to stomach. Further, the in situ and metastatic focuses were harvested. To guarantee optimal utilization of data, each specimen was divided into two sub-libraries, and genomic DNA was isolated for the amplification of sgRNAs, which were then sequenced on an Illumina HiSeq X instrument.

first: Zhongyi

last: Qin

first: Linrong

last: Che

first: Bin

last: Wang


title: Effect of STING inhibitor IFM-004490-7 (STINGX) on ethanol induced fibrogenic gene expression in LX2 cells

city: New Haven

first: Suyavaran

last: Arumugam

state: Connecticut

address: 300 Cedar Street

department: Internal Medicine

institute: Yale University

summary:
The LX2 cells were subjected to 50mM ethanol stimulus with or without  STING inhibitor IFM-004490-7 10μM (STINGX) pretreatment.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 different groups (Control, ETOH, ETOH-STX, STX)

overall_design: Comparative gene expression profiling analysis of RNA-seq data for LX2 cells untreated or subjected to TGFβ with or without STINGX pretreatment.

first: Wajahat

last: Mehal

first: Frhaan

last: Zahrawi


title: Low-level tragus stimulation alleviates sepsis-associated acute kidney injury by promoting renal M2 macrophage polarization.

city: Changsha, Hunan

first: Yufan

last: Yang

state: China

address: No. 86 Ziyuan Road

institute: Hunan Children's Hospital

summary: Background: Low-level tragus stimulation (LL-TS) can reduce acute renal injury in sepsis and improve survival rates. However, the underlying molecular mechanisms remains unknown. Renal macrophages that contributed to AKI in sepsis under low-level tragus stimulation were characterized. Methods: Mice were randomly assigned to five groups following diverse treatments: Control, Sham, Sepsis, LL-TS + sepsis, and LL-TS + sepsis + Methyllycaconitine citrate (MLA). Blood creatinine testing and hematoxylin and eosin (HE) staining were used to assess kidney injury. Single-cell RNA sequencing were used to profile renal macrophages from CD45+ cells in mouse kidney. Western blot, immunofluorescence and HE staining were performed to verify the level of the alpha7 nicotinic acetylcholine receptor (α7nAChR) or interleukin-1β expression in kidney. Results: HE staining and blood creatinine testing of renal tissue indicated that LL-TS alleviated renal injury in mice with sepsis-associated acute kidney injury (AKI). A map of the renal cellular landscape was generated to identify macrophages that contributed to AKI in sepsis under LL-TS. Pseudo-timing analysis showed that LL-TS played an anti-inflammatory role by promoting renal monocyte conversion into M2 macrophages. Moreover, LL-TS significantly increased α7nAChR expression and decreased interleukin-1β expression in renal tissues. Conclusions: LL-TS alleviated sepsis-associated AKI by increasing α7nAChR expression and M2 macrophage polarization which promotes the anti-inflammatory process in sepsis. The results suggests that LL-TS may serve as a potential therapeutic strategy for sepsis-induced kidney dysfunction, ultimately improving patient outcomes.

overall_design: Mouse kidneys were collected 24 hours after CLP. In this study, kidney samples were obtained from one mouse in each of the following groups: the control group, the sepsis group, and the LL-TS combined with sepsis group. Each kidney specimen was precisely sectioned into two equal parts, which were subsequently subjected to enzymatic digestion for further analysis. Immune cells were sorted using CD45 magnetic beads. CD45-sorted positive cells were mixed with non-sorted cells after enzymatic digestion of the same tissue block in a 1:2 ratio. The 10X Genomics GemCode technology was used to encapsulate single cells in droplets, which were then processed by OE Biotech. Co., Ltd. (Shanghai, China). A unique molecular identifier was used to barcode every cell and transcript, and a single-cell 30 Reagent Kit v2 (10X Genomics) was used to generate cDNA for Illumina sequencing.

first: Zhijuan

last: Kang

first: Yinghui

last: Peng

first: Hengyun

last: He

first: Jiaotian

last: Huang

first: Xinping

first: Liang

first: Zhenghui

last: Xiao

first: Zhihui


title: Transcriptome response to FAK1 (PTK2) targeting in a murine Nf2 null schwannoma cell line model

first: Steven

overall_design: An immortalized Schwann cell line derived from the Nf2 flox/flox mouse model was previously infected with Cre to generate the Nf2-/- MS03 cell line. The MS03 cell line was treated with DMSO, 1 micromolar defactinib, 1 micromolar VS4718, 1 micromolar GSK2256098, 1 micromolar IN10018, or the PROTAC FAK degraders: 1 micromolar PROTAC FAK degrader 1 (PROTAC-3, HY-119932), 0.1 micromolar GSK-215, or 0.1 micromolar BSJ-04-146 for 24 hours. Cells were harvested, RNA extracted for RNA sequencing.

first: Dana


title: miRNAseq of U251 cell line

city: Besthesda

first: Charlotte

last: Degorre

state: Maryland

address: 10 Center drive

department: NCI

institute: NIH

summary: miRNAseq analysis of U251 cell line to check for miRNA expression profile

overall_design: miRNA expression profile in U-251 cells (4 samples per group)

first: Taqveema

last: Ali

first: Philip

last: Tofilon


title: Single-cell transcriptome analysis deciphers stem cell-like epithelial cells as metastatic phenotype and immune characteristics of tumor deposition positive in Colorectal Cancer

city: Xi’an

first: Shuang

last: Xie

address: No. 569, Xinsi Road, Baqiao District, Xi’an, Shaanxi 710038, China

institute: Department of General Surgery, The Second Affiliated Hospital of Air Force Medical University

summary: Background: Intratumoral heterogeneity (ITH) limits the accurate diagnosis and treatment of colorectal cancer (CRC). Currently, diagnoses, treatment decisions, and prognoses for CRC are determined through multiple superficial endoscopic biopsies. However, there is still a lack of understanding regarding the immune microenvironment in samples obtained from multiple biopsy sites. Additionally, CRC cases with positive tumor deposition (TD) are associated with a higher risk of distant metastasis and a worse prognosis. Methods: A total of 11 tissue specimens from three TD-positive CRC patients were annotated to identify the primary tumor (T), the mucosal side (Ti) of primary loci, the serosal side (To) of primary loci, TD, and lymph nodes (LN). For each subregion, we analyzed the transcriptome profile using single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing, Hotspot gene module analysis, and validated our findings with the NanoString ‘PanCancer Progression Panel’. Immunohistochemistry (IHC), Western blotting, Transwell functional experiments, and data from public cohort databases were employed to verify. Results: Cancercells 1/2/4/5, mainly composed of stem-like cells, had a higher risk of metastasis than CancerCells 6/8. CancerCells 6/8 with weak metastatic ability were located only in the Ti side, while CancerCells 1/2/4/5 tumor cells with strong metastatic ability were located in To and metastasis. Bulk sequencing revealed that immune and metastasis-related genes such as SPP1, CD44, and FXYD5 were significantly upregulated in To and/or metastasis than Ti side. Notably, the immune suppression characteristics are more pronounced in To than Ti, which is reflected in the exhaustion of T cell function, more recruitment of tumor-associated macrophages, and reduction of plasma cells. The TD sample had fewer T cells than T and LNm, which present an "immune desert" state. Thus, multiple endoscopic superficial biopsies are limited, and endoscopists and pathologists must consider investigating deeper sites of T, TD, or metastatic LN for the assessment of patient treatment. Furthermore, considering that FXYD5 promotes the expression/stability of the membrane protein CD44, we identified that CancerCells 1/2/4/5 co-expressing FXYD5 and CD44 were more likely to metastasize. Conclusions: This study provides new insights and references for the understanding of the heterogeneity of CRC, as well as for the clinical value of precise diagnosis and future precise treatment provided by in-depth sampling.

overall_design: To investigate the transcriptomic heterogeneities and cellular diversities in CRC, we conducted scRNA-seq on primary tumors and paired metastatic lesions from three tumor-derived positive CRC patients.

first: Xi'e

last: Hu

first: Gang

first: Zuyin

last: Wu

first: Ning

last: Liang

first: Qian

first: Yunhu

last: Bai

first: Jixin

last: Zhang

first: Chunxu

first: Xiaolong

last: Wei

first: Qingrui

last: Hou

first: Guoqiang

last: Bao

first: Xianli


title: A Scheduler for Rhythmic Gene Expression

city: Basel

first: Dimos

last: Gaidatzis

address: Maulbeerstrasse 66

department: Computational Biology

institute: Friedrich Miescher Institute for Biomedical Research

summary: Genetic oscillators drive precisely timed gene expression, crucial for development and physiology. Using the C. elegans molting clock as a model, we investigate how oscillators can schedule the orderly expression of thousands of genes. Single cell RNA sequencing reveals a broad peak phase dispersion in individual issues, mirrored by rhythmic changes in chromatin accessibility at thousands of regulatory elements identified by time-resolved ATAC-seq. We develop a linear model to predict chromatin dynamics based on the binding of >200 transcription factors. This identifies nine key regulators acting additively to determine the peak phase and amplitude of each regulatory element. Strikingly, these factors can also generate constitutive, non-rhythmic activity through destructive interference. The model accurately predicts the impact of GRH-1/Grainyhead perturbation on both chromatin and transcript dynamics, validating its power. This work provides a powerful framework for understanding how combinatorial, non-cooperative transcription factor binding schedules complex gene expression patterns in development and other dynamic biological processes.

overall_design: Refer to individual Series


title: mRNA-seq time course (in parallel to ATAC-seq time course), of wildtype C. elegans larvae sampled from 14h to 30h at 25oC [RNAseq_N2]

summary: We collected timecourse samples for RNA-seq, in parallel to ATAC-seq, in order to have matching samples for comparisson. The overall goal was to link oscillating gene expression (RNA-seq) to oscillating changes in chromatin accessibility (ATAC-seq), in order to identify enhancer elements that are critical to oscillatory regulation.

overall_design:
mRNA-seq of RNA isolated from larval C. elegans (N2 strain) in a time course spanning 16 hours of development. Synchronized L1 animals were seeded (2000 animals per plate) on multiple 10 cm NG 2% plates coated with OP50 bacteria, and then 1 plate per timepoint (2000 animals) was harvested every hour starting 14 hours after seeding. RNA was isolated with Trizol and the Direct-zol RNA microprep kit (R2062, Zymo Research) including DNase treatment. Libraries were prepared according to the TruSeq Illumina mRNA-seq (stranded - high input) protocol. Library was sequenced using Hiseq 50 Cycle Single-end reads.
In these experiments, samples were collected in parallel for mRNA-seq and ATAC-seq. And two independent experimental replicates were performed.

first: Gaidatzis

last: Dimos

first: Graf-Landua

last: Maike

first: Methot

last: Stephen P

first: Großhans

last: Helge


title: mRNA-seq of wildtype C. elegans L3 larvae sampled at molt entry and molt exit [RNAseq_MoltEntry-MoltExit_N2]

summary: In order to determine the gene expression profile of worms at molt entry and exit in more detail, we collected animals specifically at these points based on signal from a luciferase-based developmental assay (Olmedo et al., 2015, Genetics). This approach allowed us to pool sufficient animals to perform mRNA sequencing in order to specifically determine the transcriptome of animals at these developmental stages. Using these transcriptome profiles,we can re-align time course RNA-seq datasets to the beginning of a larval stage (molt exit).

overall_design: Synchronized L1 animals, expressing luciferase, were grown while measuring developmental trajectories using a luminometer. Assay was stopped when the majority of animals were at L3 molt entry and L3 molt exit respectively. Based on luciferase traces, individual animals of the appropriate developmental timepoint were collected from their wells and pooled for RNA isolation and library preparation.

first: Nahar

last: Smita

first: Hauser

last: Yannick

first: Meeuse

last: Milou


title: Whole-organism C. elegans L2/L3 NHR-23 ChIP-seq [NHR23_ChIPseq]

summary: We performed ChIP-seq to determine chromatin regions bound by transcription factor NHR-23, and thereby potential binding motifs and sites, in C. elegans (larval stage 2) whole organisms

overall_design: One sample was used as "input", ie non-specific pulldown.  Two NHR-23-specific samples were generated by collecting after 20 hrs at 25°C to cover the L2-L3 stage.

first: Woelk

last: Michaela

first: van der Does

last: Marit


title: Reprogramming the neuroblastoma tumor immune microenvironment to enhance GPC2 CAR T cells

city: Philadelphia

first: Evan

last: Cresswell-Clay

state: PA

address: 3501 Civic Center Blvd

department: DBHi

institute: CHOP

summary: Poor tumor trafficking and the immunosuppressive tumor microenvironment (TME) limit chimeric antigen receptor (CAR) T cell efficacy in solid tumors, such as neuroblastoma. We previously optimized GPC2 CARs in human neuroblastoma xenografts leading to clinical translation, however, there have not been preclinical studies using immunocompetent models. Thus, here we generated murine GPC2 CAR T cells using the D3-GPC2-targeting single-chain variable fragment being utilized clinically (NCT05650749) and tested them in neuroblastoma syngeneic allografts. Immune profiling of GPC2 CAR T cell-treated tumors revealed significant reprogramming of the TME, most notably poor intra-tumor CAR T cell persistence being associated with increased recruitment of myeloid-derived suppressor cells (MDSCs), along with MDSC-recruiting CXCL1/2 chemokines. These tumor-infiltrating MDSCs directly inhibited GPC2 CAR T cell activation and proliferation ex vivo. To both capitalize on this chemokine gradient and mitigate MDSC-tumor trafficking, we engineered GPC2 CAR T cells to express the CXCL1/2 receptor, CXCR2. CXCR2-armored GPC2 CAR T cells migrated towards CXCL1/2 gradients, enhanced anti-neuroblastoma efficacy, and reduced the level of MDSCs in the TME. Together, these findings suggest CAR T cell studies in immunocompetent models are imperative to define mechanisms of solid tumor immune escape and rationally design armoring strategies that will lead to durable clinical efficacy.

overall_design: RNA-seq profiling of neuroblastoma TH-MYCN allograft-derived tumors from C57BL/6 mice treated with murine GPC2 CAR T cells or control tCD19 T cells at day 5 after treatment (n=4 per group. 8 samples in total)

first: Anna

last: Giudice

first: Sydney

middle: L

last: Roth

first: Stephanie

last: Matlaga

last: Cresswell-clay

first: Pamela

last: Mishra

first: Patrick

last: Schürch

first: Kwame Attah

last: Boateng-Antwi

first: Minu

last: Samanta

first: Vincent

last: Zecchino

first: Guillem

last: Pascual-Pasto

first: Timothy

last: Spear

first: Brendan

last: McIntyre

first: Neil

last: Chada

first: Tingting

last: Wang

first: Lingling

last: Liu

first: Ruoning

first: John

last: Wilson

first: Adam

last: Wolpaw

first: Kristopher

last: Bosse


title: Genome-wide mapping of H3K27ac by Cut&Tag in CD4+ and CD8+ immune cells of astronauts following prolonged orbital space flight.

city: Stuttgart

first: Philipp

last: Rathert

state: Germany

address: Allmandring 31

department: Biochemistry

institute: University Stuttgart

summary: Cut&Tag for H3K27ac was performed in isolated CD4+ and CD8+ immune cells of astronauts undergoing prolonged orbital space flight and in control subjects.

overall_design: Blood samples for astronauts were collected at the following time points: 55 – 185 days before flight (pre-flight, PF), within the first 24 h after landing (R+1) and 20 – 50 days after landing (R+35±15). Blood draw for control subjects was performed at the following time points: at the beginning of the study (PF), after 251 days (R+1) and 20-50 days after the R+1 blood draw (R+35±15). CD8+ and CD4+ immune cells were isolated from blood of astronauts and control subjects and subjected to Cut&Tag for H3K27ac.

first: Tabea L

last: Fullstone

first: Lukas

last: Fischer


title: Age-related transcriptional diversity in multiple skeletal muscles of mice

city: Chengdu

first: dongbin

last: zheng

state: 四川Sichuan

address: No. 211 Huimin Road, Wenjiang District

department: College of Animal Science and Technology

institute: Sichuan Agricultural university

summary: Aging leads to a gradual decline in muscle function, causing diseases such as sarcopenia, with different skeletal muscles responding to aging through distinct processes due to extensive transcriptional diversity. In this study, we constructed transcriptional maps of 11 skeletal muscles from young (3 month) and old (24 month) mice

overall_design: Female C57BL/6J mice were maintained under a 12-hour light/dark cycle with ad libitum access to standard laboratory chow. All experimental mice were of specific pathogen-free (SPF) grade. Mice were sacrificed at 3 months and 24 months of age, and 11 tissues were collected, including Ventricle (Ven), Diaphragm (Dia), Extensor Digitorum Longus (EDL), Masseter (Mas), Soleus (Sol), Gastrocnemius (Gas), Quadriceps (Qua), Tibialis Anterior (TA), Longissimus dorsi muscle (LDM), Psoas Major Muscle (PMM), and Semitendinosus (ST). The tissues were immediately flash-frozen in liquid nitrogen. We optimized the sample collection process to minimize variation.

first: Can


title: single cell RNA-seq of samples from Vav-cre/Shb flox/flox mice

first: L.

last: He

summary: E0771.lmb tumors were grown as previously described in wild-type and Vav-cre/Shb flox/flox mice (KO). Mice were sacrificed on day 14-16 after tumor cell injection.

overall_design: single cell RNAseq was performed on 4 wild type and 4 KO samples in two separate experiments.

first: Michael

last: Welsh


title: scRNAseq timecourse from wildtype C. elegans larvae sampled from 18 to 24 hours at 25oC, with FACS sorting cells using ubiquitous markers.

first: Dimos

last: Gaidatzis

summary: We collected a timecourse scRNA-seq dataset from wildtype C. elegans larvae, using ubiquitous flurophores to FACS sort cells in order to collect as many tissues as possible. The data deposited were collected from one single experiment with pools A,B,C representing different 10X runs for a given sample. The overall goal of the experiment was to identify the tissues in which oscillating gene expression occurs, and to understand tissue-specific differences in oscillatory output. We were able to identify 18 different tissues, 7 of which show clear oscillatory expression patterns.

overall_design: HW3125 worms, expressing transgenes for ubiquitous GFP and mCherry expression, were synchrnoized as hatched L1 larvae. 200,000 worms per plate were seeded onto 16 peptone-rich XL plates coated with 10x concentrated OP50 and grown at 25oC. 600,000 worms (3 XL plates) were collected at each timepoint, 18h, 20h, 22h and 24h after seeding. At each timepoint, worms were immediately processed to isolate cells, FACS sort, and perform 10x scRNA-seq protocol. To dissociate single cells, we followed the protocol of Cao et al., 2017, Science) with minor adjustments. To remove animal debry, cells were then sorted on a BD FACSAria using the GFP and mCherry markers. Following sorting, cells were diluted and libraries prepared using the 10x Genomics protocol with the Chromium Controller (10x 3'RNAv3). Sequencing was then performed using NextSeq HIGH-OUT 75 cycle paired-end sequencing.

first: Gaidatzis

last: Dimos

first: Graf-Landua

last: Maike

first: Methot

last: Stephen P.

first: Brancati

last: Giovanna

first: Braun

last: Kathrin

first: Aluri

last: Sirisha

first: Kohler

last: Hubertus

first: Smallwood

last: Sebastien

first: Großhans

last: Helge


title: Progestogens and androgens influence root morphology of angiosperms in a brassinosteroid-independent way - C21- and C19-steroids a novel class of phytohormones?

city: Jena

first: Emanuel

last: Barth

address: Leutragraben 1

department: Bioinformatics Core Facility FSU Jena

institute: Friedrich Schiller University Jena

summary: Progestogens and androgens are steroids that have been found in a broad range of plants, but little is known about their physiological function. Within this study we sowed seeds of angiosperms on progestogen- and androgen-containing medium and analyzed the morphological effects. Moreover, we analyzed the effects of progesterone and testosterone on brassinosteroid profiles and gene expression of A. thaliana. Additionally, we analyzed the effects of progesterone and testosterone on A. thaliana plants that overexpress the steroid 5α-reductase DET2. We found that progestogens and androgens have strong negative effects on root length particularly in Brassicaceae species. Moreover, these steroids led to uncoordinated cell growth and enhanced lateral root formation. We could not detect an influence on brassinosteroid profiles and gene expression of brassinosteroid-regulated genes. The overexpression of DET2 led to enhanced root growth, but the effects of progesterone and testosterone were not reduced. We conclude that progestogens and androgens act in a brassinosteroid-independent way. We see this as a hint that progestogens and androgens might be a novel class of steroidal phytohormones in plants.

overall_design:
7 days old A. thaliana plants were transferred to PNM medium. After 2 days of adaption to the new medium, plants were treated with 10 µM PO (dissolved in 0.12% DMSO) or 10 µM TO, while 0.12% DMSO was used as a mock treatment. Leaves were harvested after 1 h and immediately frozen in liquid nitrogen.
Comparative gene expression profiling analysis using data obtained from RNA-seq of A. thaliana leaves under different conditions.

first: Karl

last: Körber

first: Sudip

last: Paul

first: Jana

last: Oklešťková

first: Felix

last: Feistel

first: Henk

last: Oppermann

first: Ceren

last: Oktay

first: Wolfgang

last: Kreis

first: Miroslav

last: Strnad

first: Jennifer

last: Munkert

first: Alexandra

last: Furch

first: Jan

last: Klein


title: ATAC-seq time course (in parallel to mRNA-seq time course), of wildtype C. elegans larvae sampled from 14h to 30h at 25oC [ATACseq_N2]

first: Dimos

last: Gaidatzis

summary: We collected timecourse samples for ATAC-seq, in parallel to mRNA-seq, in order to have matching samples for comparisson. The overall goal was to link oscillating gene expression (RNA-seq) to oscillating changes in chromatin accessibility (ATAC-seq), in order to identify enhancer elements that are critical to oscillatory regulation.

overall_design:
ATAC-seq was performed on nuclei isolated from larval C. elegans (N2 strain) in a time course spanning 16 hours of development. Synchronized L1 animals were seeded (2000 animals per plate) on multiple 10 cm NG 2% plates coated with OP50 bacteria, and then 7 plates per timepoint (14000 animals) were harvested every hour starting 14 hours after seeding. Nuclei were prepared from frozen animal pellets by douncing similar to a previosuly established protocol (Daugherty et al., 2017, Genome Res.). Library was sequenced using the NextSeq HIGH-OUT 75 Cycle Paired-end protocol.
In these experiments, samples were collected in parallel for mRNA-seq and ATAC-seq. And two independent experimental replicates were performed.

first: Gaidatzis

last: Dimos

first: Graf-Landua

last: Maike

first: Methot

last: Stephen P


title: Mechanism of Radiatiom resistance in BRAFV400E  pediatric gliomas

city: San Antonio

first: Hanzhou

last: Wang

state: TX

address: 8403 Floyd Curl Drive, MSC 7784

institute: UT Health at San Antonio

summary: Inhibitors of the MAPK pathway (MAPKi) have shown significant activity in treatment of childhood BRAF-activated brain tumors. However, for tumors harboring BRAFV600E mutations, the drugs are rarely curative, and patients can become refractory to treatment. Combining MAPKi with low dose X-ray therapy (XRT) has the potential to improve the cure rate, but development of XRT resistance poses a major challenge.  To understand the mechanisms of radiation resistance in BRAFV600E type pediatric low-grade gliomas, radiation resistance models were developed by using IC365 \ pediatric low-grade glioma (PLGG) PDX model harboring BRAFV600E mutation. RNA sequencing was performed to determine the gene expression profile of primary tumors and resistant tumors.  Each tumor tissue was transplanted to 3 scid mice respectively. When tumor volume reach ~0.3~0.6 cm3, tumor tissue (from three mice, triplicates) were collected  and were snap-frozen in liquid nitrogen. RNA was isolated and applied to subsequent 100bp paired read sequencing run with Illumina NovaSeq 6000 platform at 100 bp paired-end module.

overall_design: Each tumor tissue was transplanted to 6-8 weeks old female scid mice.  Tumor tissue were harvested when tumor size reach 0.3~0.6cm^3, RNA was isolated and applied to subsequent 100bp paired read sequencing run with Illumina NovaSeq 6000 platform at 100 bp paired-end module.

first: Fuyang

last: Li

first: Kathy

last: Bondra

first: Zhao

last: Lai

first: Peter

last: Houghton

first: Yidong

last: Chen