Single-cell RNA-seq Research Articles

Spatial integration of multi-omics single-cell data with SIMO

Technical limitations in spatial and single-cell omics sequencing pose challenges for capturing and describing multimodal information at the spatial scale. To address this, we develop SIMO, a computational method designed for the Spatial Integration of Multi-Omics datasets through probabilistic alignment. Unlike previous tools, SIMO not only integrates spatial transcriptomics with single-cell RNA-seq but expands beyond, enabling integration across multiple single-cell modalities, such as chromatin accessibility and DNA methylation, which have not been co-profiled spatially before. We benchmark SIMO on simulated datasets, demonstrating its high accuracy and robustness. Further application on biological datasets reveals SIMO’s ability to detect topological patterns of cells and their regulatory modes across multiple omics layers. Through comprehensive analysis of real-world data, SIMO uncovers multimodal spatial heterogeneity, offering deeper insights into the spatial organization and regulation of biological molecules. These findings position SIMO as a powerful tool for advancing spatial biology by revealing previously inaccessible multimodal insights.

FAM49B drives colorectal cancer progression by stabilizing c-Myc through NEK9 phosphorylation.

Colorectal cancer (CRC) ranks as the third most prevalent cancer globally and is the second leading cause of cancer mortality. FAM49B, a member of the FAM49 gene family, is a recently identified, evolutionarily conserved gene. Emerging studies indicate that FAM49B plays a role in various cancers, though its specific mechanism in CRC remains largely unexplored. In this study, we observed that FAM49B was abnormally expressed in CRC tissues and cell lines, with elevated expression correlating with poor patient prognosis. FAM49B knockdown markedly suppressed CRC cell proliferation by arresting the cell cycle and reducing cell migration and invasion. Single-cell RNA-seq (ScRNA-seq) analysis revealed that high FAM49B expression in malignant epithelial cell clusters was strongly linked to c-Myc oncogene activation. Further, FAM49B knockdown significantly reduced c-Myc expression by enhancing its K48 ubiquitination. We identified NEK9 as a direct interacting partner of FAM49B, with FAM49B knockdown inhibiting NEK9-Thr210 phosphorylation. Similarly, high NEK9 expression was linked to unfavorable prognosis in CRC. In FAM49B-overexpressing CRC cells, NEK9 knockdown significantly suppressed c-Myc expression, c-Myc-ser62 phosphorylation, and reduced cell proliferation, migration, and invasion. Thus, directly targeting the FAM49B/NEK9/c-Myc pathway presents a promising therapeutic approach for c-Myc positive CRC patients.

CMAP prediction and experimental validation of Forskolin as a podocyte protective and anti-proteinuric drug for nephrotoxic serum-treated mice.

Podocyte injury leads to proteinuria and glomerular diseases. Different podocyte injuries have distinct mechanisms. It is desirable to use a regimen that targets the mechanism of a given podocyte injury for a specific and improved result. However, the mechanisms of the most podocyte injuries are largely elusive, preventing optimal drug choices. Here, we test the feasibility of combining kidney single-cell RNA-seq databases and the Connectivity Map database (CMAP) to predict drugs for a specific podocyte injury. We downloaded glomerular single-cell RNA-seq dataset of nephrotoxic serum (NTS)-treated and control mice from the GEO, and compared their podocyte gene expression, resulting in identification of genes with altered expression in NTS-treated podocytes. GO and KEGG enrichment of them revealed activations of podocyte injurious NFκB, TNFα, AGE-RAGE, apoptosis, cellular senescence, MAPK, and p53 pathways, and dedifferentiation. CMAP analysis of the genes ranked Forskolin top 3. Indeed, we found that NTS-treated mice developed massive proteinuria, which was prevented by Forskolin, accompanied by pathological improvement of podocytes. In treating overdose NTS-induced severe podocyte injury, Forskolin exhibited a comparable efficacy as glucocorticoids (methylprednisolone). In vitro, Forskolin prevented NTS-induced cellular injury in cultured podocytes as shown by cell viability and cytoskeletal integrity assays. Mechanistically, Forskolin inhibited STAT3, p53, NFκB, FAK, and TGF-β pathways, while upregulated podocyte essential genes, WT1, SYNPO, and VEGFA, independently of NTS. In conclusion, Forskolin protects podocytes by directly inhibiting harmful pathways and the associated genes while enhancing podocyte essential gene expression independently of insults, resulting in an efficacy comparable with that of glucocorticoids in NTS-treated mice.

Characterization of SARS-CoV-2 Entry Genes in Skeletal Muscle and Impacts of InVitro Versus InVivo Infection.

COVID-19 has been associated with both respiratory (diaphragm) and non-respiratory (limb) muscle atrophy. It is unclear if SARS-CoV-2 infection of skeletal muscle plays a role in these changes. This study sought to: 1) determine if cells comprising skeletal muscle tissue, particularly myofibres, express the molecular components required for SARS-CoV-2 infection; 2) assess the capacity for direct SARS-CoV-2 infection and its impact on atrophy pathway genes in myogenic cells; and 3) in an animal model of COVID-19, examine the relationship between viral infection of skeletal muscle and myofibre atrophy within the diaphragm and limb muscles. We used in silico bioinformatics analysis of published human single cell RNA-seq datasets, as well as direct qPCR examination of human myotubes and diaphragm biopsies, to assess expression of key genes involved in SARS-CoV-2 cellular entry. InVitro, we determined the ability of SARS-CoV-2 to directly infect myogenic cells and employed qPCR to assess the impact on muscle atrophy pathway genes (ubiquitin-proteasome, autophagy). Invivo, the diaphragm and quadriceps of Roborovski hamsters with SARS-CoV-2 respiratory infection were examined at day 3 post-inoculation to evaluate the relationship between atrophy pathway and SARS-CoV-2 transcripts by qPCR, as well as histological measurements of myofibre morphology. Angiotensin converting enzyme 2 (ACE2), the primary receptor for SARS-CoV-2, as well as cooperating proteases (furin, cathepsins B and L), are expressed by myofibres. ACE2 expression was increased 5-fold (p = 0.01) in the diaphragms of mechanically ventilated human subjects compared to controls. InVitro, a time-dependent increase of SARS-CoV-2 transcript levels was observed in myotubes directly exposed to the virus (p = 0.002). This was associated with downregulation of the ubiquitin ligase MuRF1 (by 64%, p = 0.002) and the autophagy gene LC3B (by 31%, p = 0.009). In contrast, invivo infection led to upregulation of MuRF1 in quadriceps (23-fold, p = 0.0007) and autophagy genes in both quadriceps (5.2-fold for Gabarapl1, p = 0.03; 7-fold for p62, p = 0.0002) and diaphragm (2.2-fold for Gabarapl1, p = 0.03; 2.3-fold for p62, p = 0.057). In infected hamsters the diaphragm lacked viral transcripts but exhibited atrophy (48% decrease in myofibre area; p = 0.02), whereas the quadriceps lacked myofibre atrophy despite elevated viral transcripts in the muscle. Although myogenic cells express the genes required for SARS-CoV-2 entry and can be directly infected, there was no evident relationship between viral transcript levels and manifestations of atrophy, either invitro or invivo. Our results do not support direct myofibre infection by SARS-CoV-2 as a likely cause of atrophy in COVID-19.

MAP3K4 signaling regulates HDAC6 and TRAF4 coexpression and stabilization in trophoblast stem cells.

Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) promotes fetal and placental growth and development, with MAP3K4 kinase inactivation resulting in placental insufficiency and fetal growth restriction. MAP3K4 promotes key signaling pathways including JNK, p38, and PI3K/Akt, leading to activation of CREB-binding protein. MAP3K4 kinase inactivation results in loss of these pathways and gain of histone deacetylase 6 (HDAC6) expression and activity. Tumor necrosis factor receptor-associated factor 4 (TRAF4) binds MAP3K4 and promotes MAP3K4 activation of downstream pathways in the embryo; however, the role of TRAF4 and its association with MAP3K4 in the placenta is unknown. Our analyses of murine placenta single-cell RNA-Seq data showed that Traf4 is coexpressed with Map3k4 in trophoblast stem (TS) cells and labyrinth progenitors, whereas Hdac6 expression is higher in differentiated trophoblasts. We demonstrate that, like HDAC6, TRAF4 expression is increased in MAP3K4 kinase-inactive TS (TSKI) cells and upon inhibition of MAP3K4-dependent pathways in WT TS cells. Moreover, Hdac6 shRNA knockdown in TSKI cells reduces TRAF4 protein expression. We found that HDAC6 forms a protein complex with TRAF4 in TS cells and promotes TRAF4 expression in the absence of HDAC6 deacetylase activity. Finally, we examine the relationships among MAP3K4, TRAF4, and HDAC6 in the developing placenta, finding a previously unknown switch in the coexpression of Traf4 with Map3k4 versus Traf4 with Hdac6 during differentiation of the placental labyrinth. Together, our findings identify previously unknown mechanisms of MAP3K4 and HDAC6 coregulation of TRAF4 in TS cells and highlight these MAP3K4, TRAF4, and HDAC6 associations during placental development.

Ionizing radiation-induced disruption of Rela-Bclaf1-spliceosome regulatory axis in primary spermatocytes causing spermatogenesis dysfunction

IntroductionIonizing radiation (IR) poses a significant threat to male fertility by inducing substantial changes in the testis, yet the mechanisms underlying IR-induced spermatogenesis disorders remain poorly understood, necessitating the development of more effective radioprotective agents.MethodsWe employed Bulk RNA-seq and single-cell RNA-seq (scRNA-seq) on Balb/c mice testes models following IR exposure to assess cellular and transcriptional alterations. Histological examination, sperm concentration and motility analysis, Western blotting (WB), and reverse transcription quantitative PCR (RT-qPCR) were used to evaluate testicular injury. The therapeutic potential of NF-κB agonists was investigated in an IR-induced spermatogenesis disorder model.ResultsA 6 Gy IR dose induced spermatogenesis disorder and suppressed the spliceosome pathway, predominantly affecting the cell abundance of spermatogonia and primary spermatocytes. Bioinformatics analysis revealed that IR induced splicing disorders in differentiation-related genes, thereby impairing the differentiation ability of primary spermatocytes. Mechanistically, This IR-induced disruption was linked to IR-induced inhibition of NF-κB/Rela and Bclaf1 activity. Notably, NF-κB agonists were found to ameliorate this damage via upregulating Bclaf1 and spliceosome-related genes expression, thereby normalizing splicing patterns and rescuing IR-induced spermatogenesis disorders.ConclusionThis study reveals a novel IR-mediated Rela-Bclaf1-spliceosome regulatory axis in primary spermatocytes and propose Rela as a potential drug target for mitigating IR-induced spermatogenesis disorders. This study not only provides new insights for further research into IR-induced damage and spermatogenic disorders caused by other factors, but also offers potential therapeutic strategies for developing radioprotective agents in cancer radiotherapy.

A high-resolution view of the immune and stromal cell response to Haemophilus ducreyi infection in human volunteers.

Haemophilus ducreyi causes the genital ulcer disease chancroid and cutaneous ulcers in children. To study its pathogenesis, we developed a human challenge model in which we infect the skin on the upper arm of human volunteers with H. ducreyi to the pustular stage of disease. The model has been used to define lesional architecture, describe the immune infiltrate into the infected sites using flow cytometry, and explore the molecular basis of the immune response using bulk RNA-seq. Here, we used single cell RNA-seq (scRNA-seq) and spatial transcriptomics to simultaneously characterize multiple cell types within infected human skin and determine the cellular origin of differentially expressed transcripts that we had previously identified by bulk RNA-seq. We obtained paired biopsies of pustules and wounded (mock infected) sites from five volunteers for scRNA-seq. We identified 13 major cell types, including T- and NK-like cells, macrophages, dendritic cells, as well as other cell types typically found in the skin. Immune cell types were enriched in pustules, and some subtypes within the major cell types were exclusive to pustules. Sufficient tissue specimens for spatial transcriptomics were available from four of the volunteers. T- and NK-like cells were highly associated with multiple antigen presentation cell types. In pustules, type I interferon stimulation was high in areas that were high in antigen presentation-especially in macrophages near the abscess-compared to wounds. Together, our data provide a high-resolution view of the cellular immune response to the infection of the skin with a human pathogen.IMPORTANCEA high-resolution view of the immune infiltrate due to infection with an extracellular bacterial pathogen in human skin has not yet been defined. Here, we used the human skin pathogen Haemophilus ducreyi in a human challenge model to identify on a single cell level the types of cells that are present in volunteers who fail to spontaneously clear infection and form pustules. We identified 13 major cell types. Immune cells and immune-activated stromal cells were enriched in pustules compared to wounded (mock infected) sites. Pustules formed despite the expression of multiple pro-inflammatory cytokines, such as IL-1β and type I interferon. Interferon stimulation was most evident in macrophages, which were proximal to the abscess. The pro-inflammatory response within the pustule may be tempered by regulatory T cells and cells that express indoleamine 2,3-dioxygenase, leading to failure of the immune system to clear H. ducreyi.

Nintedanib improves bleomycin-induced pulmonary fibrosis by inhibiting the Clec7a/SPP1 pathway in interstitial macrophages.

Idiopathic pulmonary fibrosis (IPF) is a terminal lung disease with high mortality rate. Although Nintedanib (Nin) is an effective treatment for IPF, its precise mechanism of action remains unclear. In this study, we performed an integrated analysis of single-cell sequencing and RNA-seq data from lung tissues of both fibrotic and Nin-treated fibrotic mice to uncover new therapeutic mechanisms of Nin in IPF. Our results revealed an increase in interstitial macrophages following bleomycin (BLM) treatment. We used Monocle2, Cellchat, and in vivo experiments to demonstrate that Nin can inhibit Clec7a in interstitial macrophages, thereby suppressing the SPP1-mediated profibrotic pathway. Additionally, we utilized Scenic to predict transcription factors and identified NFκB as a major transcription factor in interstitial macrophages. In the in vitro experiments, we found that inhibiting Clec7a improved the secretion of SPP1 by M2 macrophages through the NFκB pathway. In subsequent in vivo experiments, we found that inhibiting of Clec7a improves pulmonary fibrosis through the NFκB/SPP1 pathway, and Nin alleviated BLM-induced pulmonary fibrosis by inhibiting Clec7a in interstitial macrophages. In summary, our study indicates that interstitial macrophages are upregulated in pulmonary fibrosis, and Nin reduces fibrosis by inhibiting Clec7a in interstitial macrophages, which in turn diminishes the NFκB /SPP1 pathway. These findings provided a new perspective on the mechanism of action of Nin in treating pulmonary fibrosis.

Glucocorticoid receptor suppresses GATA6-mediated RNA polymerase II pause release to modulate classical subtype identity in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer with a 5-year survival rate of 12%. It has two major molecular subtypes: classical and basal, regulated by the master transcription...

Deciphering the complex clonal heterogeneity of polycythemia vera and the response to interferon alpha.

Interferon alpha (IFNa) is approved for the therapy of patients (pts) with polycythemia vera (PV), a subtype of myeloproliferative neoplasms (MPN). Some pts achieve molecular responses (MR), but clonal factors sensitizing for MR remain elusive. We integrated colony formation and differentiation assays with single-cell RNA seq and genotyping in PV-derived cells vs. healthy controls (HC) to dissect how IFNa targets diseased clones during erythroid differentiation. IFNa significantly decreased colony growth in MPN and HC, with variable transcriptional responses observed in individual colonies. scRNAseq of colonies demonstrated more mature erythroid PV-derived colonies compared with HC. JAK2V617F-mutant cells exhibited upregulated STAT5A, heme, and G2M checkpoint pathways compared to JAK2WT cells from the same pts. Subgroup analysis revealed that IFNa significantly decreased immature erythrocytic cells in PV (basophilic erythroblasts p<0.05; polychromatic erythroblasts p<0.05) but not in HC. CD71-/CD235a+ cells from HC (p<0.05) but not from PV pts were inhibited by IFNa, and number of reticulocytes was less affected in PV. Robust IFNa responses persisted throughout differentiation, leading to significant apoptosis in PV. Apoptotic cells displayed downregulation of ribosomal genes. This link between apoptosis and ribosomal genes was corroborated through analysis of mitochondrial variants demonstrating IFNa-induced eradication of specific clones, characterized by elevated expression of ribosomal genes. Our findings indicate that PV-derived clones either undergo apoptosis or pass through differentiation, overall reducing cycling mutant cells over long-term treatment. Further, the significance of ribosomal genes and clonal prerequisites in IFNa's therapeutic mechanism is underscored, shedding light on the intricate dynamics of IFNa treatment in PV.

Abstract B016: Targeting the angiotensin receptor in cancer-associated fibroblasts improves tumour perfusion, oxygenation, and radiotherapy response

Abstract The solid tumor microenvironment is a key determinant of successful cancer therapy, with hypoxic tumor cells known to contribute to poor therapeutic outcome after radiation therapy, chemotherapy, and surgery. Several targeted therapies have been developed against hypoxic tumor cells that have shown promise in combination with radiation therapy in pre-clinical models and early phase clinical trials, although the difficulty inherent in targeting hypoxic cells has limited the overall efficacy of this approach. Our recent data suggest that hypoxia can develop in solid tumors due to the activity of sub-populations of cancer-associated fibroblasts (CAFs), and that therapeutically targeting CAF activation can restrict the development of tumor hypoxia and prime tumors to respond to radiation therapy. Angiotensin II type 1 receptor (AT1R) blockers (ARBs) are anti-hypertensive agents that inhibit the activity of the signaling peptide angiotensin II derived from the renin-angiotensin system. ARBs can also exhibit anti-fibrotic activity by blocking the AT1R expressed on myofibroblasts in various tissues. We recently found that the ARB telmisartan inhibits the deposition of collagen I in solid tumors, increasing net tumor perfusion and stabilizing microregional tumor blood flow. Collagen I can be produced by CAFs, although recent clinical data have identified multiple, functionally-diverse CAF subsets in breast and ovarian tumors. To understand how ARBs affect the solid tumor microenvironment, we wanted to determine how the ARB telmisartan influences the activity of CAF subsets and whether telmisartan-associated changes in perfusion impact hypoxia and radiation response in solid tumors. We identified several murine CAF subsets that could be discriminated by cell surface marker expression across a panel of seven tumor xenograft lines. Using single cell RNAseq of cells isolated from tumors with or without ARB treatment, we found that telmisartan altered the transcriptome of CAF subsets and inhibited CAF activation. In tumors containing myofibroblast-like CAFs, telmisartan decreased collagen deposition and eliminated ‘acute’ hypoxia that can be caused by unstable microregional perfusion. Telmisartan-treated tumors also had improved response to ionizing radiation therapy. Taken together, our data indicate that CAFs contribute to tumor hypoxia and radiation resistance of solid tumors in an AT1R-dependent manner. Using a retrospective cohort of over 1,100 oropharyngeal cancer patients, we also found that patients taking ARBs for hypertension had dramatically improved therapeutic outcomes after radiation therapy. Our work indicates that CAFs contribute to the development of hypoxia in solid tumors and suggests that angiotensin receptor blockers represent a novel targeted therapy to inhibit CAF activity, modify the solid tumor microenvironment, reduce tumor hypoxia, and improve radiation response. Citation Format: Che-Min Lee, Brennan J. Wadsworth, Rachel A. Cederberg, Zi Han Li, Kiersten N. Thomas, Meredith Clark, Rocky Shi, Michael Hall, Nikita Telkar, Ryan Urban, Wan L. Lam, Sarah N. Hamilton, Kevin L. Bennewith. Targeting the angiotensin receptor in cancer-associated fibroblasts improves tumour perfusion, oxygenation, and radiotherapy response. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B016.

Pro-cumulin addition in a biphasic in vitro oocyte maturation system modulates human oocyte and cumulus cell transcriptomes.

Biphasic in vitro oocyte maturation (IVM) can be offered as a patient-friendly alternative to conventional ovarian stimulation in in vitro fertilization (IVF) patients predicted to be hyper-responsive to ovarian stimulation. However, cumulative live birth rates after IVM per cycle are lower than after conventional ovarian stimulation for IVF. In different animal species, supplementation of IVM media with oocyte-secreted factors (OSFs) improves oocyte developmental competence through the expression of pro-ovulatory genes in cumulus cells. Whether the addition of OSFs in human biphasic IVM culture impacts the transcriptome of oocytes and cumulus cells retrieved from small antral follicles in minimally stimulated non-hCG-triggered IVM cycles remains to be elucidated. To answer this, human cumulus oocyte complexes (COCs) that were fully surrounded by cumulus cells or partially denuded at the time of retrieval were cultured in a biphasic IVM system either without or with the addition of pro-cumulin, a GDF9: BMP15 heterodimer. Oocytes and their accompanying cumulus cells were collected separately, and single-cell RNA-seq libraries were generated. The transcriptomic profile of cumulus cells revealed that pro-cumulin upregulated the expression of genes involved in cumulus cell expansion and proliferation while downregulating steroidogenesis, luteinization and apoptosis pathways. Moreover, pro-cumulin modulated the immature oocyte transcriptome during the prematuration step, including regulating translation, apoptosis and mitochondria remodeling pathways in the growing germinal vesicle (GV) oocytes. The addition of pro-cumulin also restored the transcriptomic profile of matured metaphase II (MII) oocytes that were partially denuded at collection. These results suggest that cumulus cell and oocyte transcriptome regulation by pro-cumulin may increase the number of developmentally competent oocytes after biphasic IVM treatment. Future studies should assess the effects of pro-cumulin addition in human biphasic IVM at the proteomic level and the embryological outcomes, particularly its potential to enhance outcomes of oocytes that are partially denuded at COC collection.

Telomemore enables single-cell analysis of cell cycle and chromatin condensation.

Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC+RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.

Single-cell RNA-seq analysis reveals microenvironmental infiltration of myeloid cells and pancreatic prognostic markers in PDAC

BackgroundPancreatic ductal adenocarcinoma (PDAC) has a heterogeneous make-up of myeloid cells that influences the therapeutic response and prognosis. However, understanding the myeloid cell at both a genetic and cellular level remains a significant challenge.MethodsSingle-cell RNA sequencing (scRNA-seq) data were downloaded from t the Tumor Immune Single-cell Hub and gene expression data were retrieved from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database. Gene set variation analysis (GSVA) was used to estimate the relative proportions of each cell type based on the signatures identified by scRNA-seq or previous literature. Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) was performed to evaluate the abundance of immune infiltrating cells. For further analysis, LASSO and Cox analyses were used to construct a risk model using univariate Cox regression.ResultsUsing the scRNA-seq dataset, we identified 7 clusters of myeloid cells, and these clusters were assigned a cell type based on their marker genes. In addition, the results of the CellChat analysis and SCENIC analysis indicate that TAM-spp1 cells may promote the migration of pancreatic tumor cells on different levels. Moreover, the TAM-spp1 cell is most closely related to poor prognoses. An 8-gene risk model was constructed by using univariate Cox and LASSO analyses. In the GEO cohorts, this risk model demonstrated excellent predictive abilities for prognosis. Further, patients with high-risk scores had a lower likelihood of benefiting from immunotherapy.ConclusionUsing bulk RNA-seq and single-cell RNA-seq, we analyzed myeloid heterogeneity at the single-cell level, and we developed an 8-gene model that predicts survival outcomes and immunotherapy response in PADC.

CD34hi subset of synovial fibroblasts contributes to fibrotic phenotype of human knee osteoarthritis.

Osteoarthritis (OA) shows various clinical manifestations depending on the status of its joint components. We aimed to identify the synovial cell subsets responsible for OA pathophysiology by comprehensive analyses of human synovium samples in single-cell resolution. Two distinct OA synovial tissue groups were classified by gene expression profiles in RNA-Seq: inflammatory and fibrotic. The inflammatory group exhibited high expression of inflammatory cytokines, histologically inflammatory infiltrate, and a more severe pain score. The fibrotic group showed higher expression of fibroblast growth factor (FGFs) and bone morphogenetic proteins (BMPs), showed histologically perivascular fibrosis, and showed a lower pain score. In single-cell RNA-Seq (scRNA-Seq) of synovial cells, MERTKloCD206lo macrophages and CD34hi fibroblasts were associated with the inflammatory and fibrotic groups, respectively. Among the 3 fibroblast subsets, CD34loTHY1lo and CD34loTHY1hi fibroblasts were influenced by synovial immune cells, whereas CD34hi fibroblasts were influenced by mural and endothelial cells. Particularly, in CD34hi fibroblast subsets, CD34hiCD70hi fibroblasts promoted proliferation of Tregs, potentially suppressing synovitis and protecting articular cartilage. Elucidation of the mechanisms underlying the regulation of these synovial cell subsets may lead to novel strategies for OA therapeutics.

A Functional Variant rs2122031 in ATG7 Is Associated with the Risk of Radiation Pneumonitis.

Radiation pneumonitis (RP) is characterized by inflammation and is associated with autophagy. However, the relationship between functional genetic variants of autophagy-related genes and radiation pneumonitis remains unknow. In this study we aimed to investigate whether genetic variants of genes involved in autophagy are associated with radiation pneumonitis. Genotyping was conducted on a total of 301 patients for thirteen single nucleotide polymorphisms (SNPs) of 5 genes in the autophagy pathway using MassArray and Sanger sequencing. Two radiation oncologists independently measured the degree of RP by chest X-ray or CT. The multivariate Cox hazard analysis and multiple testing showed that ATG7: rs2122031 GA/GG significantly decreased the risk of RP ≥ grade 3 (HR=0.369, 95% CI: 0.189-0.720, P=0.003, Pc=0.039). Furthermore, qRT-PCR and immunohistochemical analysis demonstrated that the ATG7: rs2122031 AA genotypes were related to decreased expression of ATG7. Loss of autophagy by deletion of ATG7 in fibroblasts or conditional ATG7 knockout mice was proven to increase radiation pneumonitis. Single-cell RNA-seq revealed regulation of autophagy related genes enriched after irradiation stress in conditional ATG7 knockout mice. Our findings indicated that genetic variants of ATG7 were associated with RP and may therefore be used to predict RP before radiotherapy. Loss of ATG7 was also shown to promote RP, which suggested that ATG7 may be an intervention target for RP.

Shiba: A versatile computational method for systematic identification of differential RNA splicing across platforms.

Alternative pre-mRNA splicing (AS) is a fundamental regulatory process that generates transcript diversity and cell type variation. We developed Shiba, a comprehensive method that integrates transcript assembly, splicing event identification, read counting, and differential splicing analysis across RNA-seq platforms. Shiba excels in capturing annotated and unannotated AS events with superior accuracy, sensitivity, and reproducibility. It addresses the often-overlooked issue of junction read imbalance, significantly reducing false positives to aid target prioritization and downstream analyses. Unlike other tools that require large numbers of biological replicates or resulting in low sensitivity and high false positives, Shiba's statistics framework is agnostic to sample size, as demonstrated by simulated data and its effective application to real n =1 RNA-seq datasets. To extend its utility to single-cell RNA-seq, we developed scShiba, which applies Shiba's pseudobulk approach to analyze splicing at the cluster level. scShiba successfully revealed AS regulation in developmental dopaminergic neurons and differences between excitatory and inhibitory neurons. Both Shiba and scShiba are available in Docker/Singularity containers and Snakemake pipelines, ensuring reproducibility. With their comprehensive capabilities, Shiba and scShiba enable systematic quantification of alternative splicing events across various platforms, laying a solid foundation for mechanistic exploration of the functional complexity in RNA splicing.

Inhibition of vascular smooth muscle cell PERK/ATF4 ER stress signaling protects against abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAA) are a life-threatening cardiovascular disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by vascular smooth muscle cell (VSMC) dysfunction and apoptosis, for which the mechanisms regulating loss of VSMCs within the aortic wall remain poorly defined. Using single-cell RNA-Seq of human AAA tissues, we identified increased activation of the endoplasmic reticulum stress response pathway, PERK/eIF2α/ATF4, in aortic VSMCs resulting in upregulation of an apoptotic cellular response. Mechanistically, we reported that aberrant TNF-α activity within the aortic wall induces VSMC ATF4 activation through the PERK endoplasmic reticulum stress response, resulting in progressive apoptosis. In vivo targeted inhibition of the PERK pathway, with VSMC-specific genetic depletion (Eif2ak3fl/fl Myh11-CreERT2) or pharmacological inhibition in the elastase and angiotensin II-induced AAA model preserved VSMC function, decreased elastin fragmentation, attenuated VSMC apoptosis, and markedly reduced AAA expansion. Together, our findings suggest that cell-specific pharmacologic therapy targeting the PERK/eIF2α/ATF4 pathway in VSMCs may be an effective intervention to prevent AAA expansion.

P0087 Calprotectin contributes to refractory Crohn’s disease fistula through dysregulation of epithelial wound healing responses

Abstract Background Refractory perianal fistula in Crohn’s disease (CD) still present an unmet clinical need with a high disease burden. Despite increasing interest, pathophysiology leading to the refractory phenotype remains unclear, with only epithelial-to-mesenchymal-transitioning (EMT) commonly mentioned as a contributing factor. Using a systematic approach comparing therapy refractory CD fistula, therapy responsive non-IBD fistula and normal rectum, we aimed to differentiate between general healing responses and dysregulation thereof in refractory CD fistula. Methods Total RNASeq (n=51), single cell RNASeq (n=31) and spatial transcriptomic (n=8) analysis were performed on the internal opening and tract of CD fistula and idiopathic fistula. Results were validated using immunohistochemistry. Functional studies were performed in human adult organoid cultures and intestinal cell lines. Results In the fistula tracts we identified development of a squamous epithelium characterized by expression of WFDC2, keratin 5 and keratin 13. Although some EMT markers were expressed in the squamous epithelium or its intermediates, no activation of a complete EMT program could be detected in any of the tissue types, regardless of the underlying diagnosis. Upstream analysis indicated TNFα, IL6 and TGFβ as inducers of WFDC2+ epithelium, and human adult colon organoids stimulated with these cytokines (but not TGFβ alone) indeed showed a transciptional profile similar to the fistula epithelium, confirming redifferentiation potential. While WFDC2+ epithelium did occur in CD fistula, abundance was strongly decreased compared to non-IBD fistula. In addition, those WFDC2+ cells present had a more pro-inflammatory profile and were morphologically disorganized. Surprisingly, pseudotime analysis indicated epithelial calprotectin (S100A8/9) as a key factor in the aberrant development of WFDC2+ tissue in CD. Normal intestinal epithelium does not express calprotectin, but a mix of cytokines present in fistula induced clear intracellular expression of S100A8/9 in both epithelial cell lines and primary colon organoids. In CD fistula, calprotectin expression was particularly intranuclear, suggesting a transcriptional role. CHIP analysis indeed showed binding of calprotectin to various inflammatory genes and knockdown of calprotectin abrogated expression of these genes supporting a crucial regulatory role. Conclusion These data identify redifferentiation of rectal mucosa to a squamous phenotype as a normal damage response during fistula formation. However, in CD patients, transcriptional effects of calprotectin derail this process, resulting in disorganized inflammatory tissue not amenable for surgical closure and contributing to refractory disease.

P0057 METTL3 potentiates aged CD4+T cell response via programming effector differentiation in elderly ulcerative colitis

Abstract Background The burden of IBD in the elderly is increasing globally. However, the mechanisms underlying the age-related IBD susceptibility remain unclear. Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to inflammatory and autoimmune disease. As a fundamental mRNA modification, N6-methyladenosine (m6A) participates in various pathological processes. However, the role of METTL3-mediated m6A RNA modification in elderly IBD remains unclear. Herein, we sought to determine the role of immunosenescence in age-dependent colitis and to explore the underlying mechanisms. Methods Young (2-3 months), middle-aged (10-12 months), and aged (20-24 months) mice were established models of colitis by 2% dextran sulfate sodium salt (DSS) treatment. We detected the senescence-associated-β-galactosidase (SA-β-Gal) activity in lymphocytes isolated from colon of mice. Single-cell and bulk RNA-seq of colonic immune cells in mice with different ages were performed to investigate the precise molecular mechanisms. CD4+CD25-CD45RBhi T cell adoptive transfer model was used to further analyze the role of senescent CD4+T cells in colitis. Mettl3fl/lfCd4Cre mice were obtained to explicit role of METTL3 in regulating CD4+T cell senescence. Results Ageing increased the severity of DSS-induced colitis. CD4+T cell senescence was implicated in the progression of aging-related experimental colitis, exhibiting an increase of SA-β-Gal and accumulation of CD4+ effector memory T (TEM) cells in colon. Aged colonic CD4+ T cells generate higher levels of IFN-γ and IL-17 comparing to young colonic CD4+ T cells. Single-cell analysis revealed that aging increased colonic CD4+ TEM cells, which were associated with T cell-mediated cytotoxicity and cytokine-mediated signaling pathway. Adoptive transfer of aged CD4+CD25-CD45RBhi T cells to Rag-/- mice induced more severe colitis compared with young CD4+CD25-CD45RBhi T cells transfer. Aged CD4+T potentiates Th1/Th17/Tfh differentiation and exacerbates the progression of colitis. METTL3 was increased in aged colonic CD4+T cells. Inhibiting METTL3 in CD4+T cell protects from cellular senescence, effector differentiation and colitis. Conclusion These results provide a significant insight into the contribution of senescent CD4+ T cells to age-dependent colitis and provide an attractive possibility that targeting T cell specifically might be a potential strategy to treat elderly IBD patients.