Single-cell RNA Research Articles

Abstract 3795: Early seeding of Richter transformation in chronic lymphocytic leukemia

Abstract Introduction: Clonal evolution drives cancer development due to the emergence and/or selection of proliferatively advantageous subclones. Its understanding may facilitate the design of anticipation-based management strategies. Richter transformation (RT) is a paradigmatic tumor evolution in which chronic lymphocytic leukemia (CLL), an indolent neoplasia of mature B-cells, transforms into a high-grade lymphoma, usually diffuse large B-cell lymphoma (DLBCL), conferring a dismal prognosis. The evolutionary trajectories of RT and its driving (epi)genomic mechanisms remain largely unknown. Aims: To reconstruct the evolutionary history of RT and to reveal the molecular processes underlying this transformation. Methods: We characterized the whole genome (WGS), epigenome (DNA methylation, H3K27ac, ATAC-seq), and transcriptome (RNA-seq), combined with single-cell DNA and RNA sequencing analyses, of 19 CLL patients developing RT before (n=3) or after treatment with chemoimmunotherapy (n=6) and targeted therapies (BCR or BCL2 inhibitors, n=10). We analyzed 54 longitudinal samples covering up to 19 years of disease course. Results: Our WGS analyses uncovered that RT is characterized by a remarkable structural complexity. We also identified a novel treatment-independent RT-specific mutational process, which we named SBS-RT. The genetic driver landscape of RT is a compendium of alterations in genes involved in cell cycle, MYC, and NF-κB pathways, frequently targeted in single catastrophic events including chromothripsis and chromoplexy. The WGS-based phylogenic reconstruction and single-cell DNA/RNA-seq analyses identified a very early diversification of CLL leading to emergence of RT-cells carrying specific genetic drivers and transcriptomic profiles of RT already at CLL diagnosis. These small subclones were dormant for 6-19 years until rapid expansion associated with the clinical transformation. While the DNA methylome kept track of the cell of origin and proliferative history of RT cells, their chromatin configuration and transcriptional program converged into the overexpression of cell cycle regulators, Toll-like receptors, MYC, MTORC1, and OXPHOS related transcripts, as well as downregulation of BCR pathway. This phenotypic shift was related to de novo activation of key transcription factors. In vitro experiments confirmed that RT cells have a 4-fold higher oxygen consumption at routine respiration and electron transfer system capacity compared to CLL. The resistance of RT to BCR inhibition is consistent with its high OXPHOS and low BCR signaling, which mimics de novo DLBCL-OXPHOS insensitive to BCR inhibition. This OXPHOShigh-BCRlow transcriptional axis of RT can be exploited therapeutically. Conclusions: These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover therapeutic targets for the, once expanded, lethal Richter transformation. Citation Format: Ferran Nadeu, Romina Royo, Ramon Massoni-Badosa, Beatriz Garcia-Torre, Martí Duran-Ferrer, Kevin J. Dawson, Marta Kulis, Ander Diaz-Navarro, Neus Villamor, Juan L. Melero, Vicente Chapaprieta, Ana Dueso-Barroso, Julio Delgado, Riccardo Moia, Sara Ruiz-Gil, Domenica Marchese, Núria Verdaguer-Dot, Mónica Romo, Maria Rozman, Gerard Frigola, Alfredo Rivas-Delgado, Tycho Baumann, Miguel Alcoceba, Marcos González, Fina Climent, Pau Abrisqueta, Josep Castellví, Francesc Bosch, Marta Aymerich, Anna Enjuanes, Sílvia Ruiz-Gaspà, Armando López-Guillermo, Pedro Jares, Sílvia Beà, Dolors Colomer, Núria López-Bigas, Josep LlGelpí, David Torrents, Peter J. Campbell, Ivo Gut, Pablo M. Garcia-Roves, Davide Rossi, Gianluca Gaidano, Xose S. Puente, Holger Heyn, Francesco Maura, José I. Martín-Subero, Elías Campo. Early seeding of Richter transformation in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3795.

OTHR-34. Identifying mechanisms of microglia-tumor cell interactions in retinoblastoma

Abstract BACKGROUND: Treatment-related long-term sequelae and chemotherapy resistance diminish the success of retinoblastoma (RB) treatment. To unravel the mechanisms leading to tumor progression and resistance we examined the intratumoral cellular heterogeneity of RB and its interactions with cells of the tumor microenvironment (TME). METHODS: We used single-cell RNA (scRNA-seq) and ATAC sequencing (scATAC-seq) as well as spatial transcriptomics to analyze and compare RB samples from patients with or without previous chemotherapy (chemo-treated vs. naïve). In addition, we developed a 3D model by injection of RB and TME cells into retinal organoids, which mimics the heterogeneous surrounding of the tumor in a spatially and functionally organized manner. RESULTS: ScRNA-seq revealed a high intratumoral heterogeneity of tumor cells representing distinct developmental stages from progenitors to more differentiated photoreceptor cells. The predominant cell type in the TME was M2-activated microglia (MG). M2-MG exerted multiple receptor-ligand interactions with RB tumor cells which were not found in non-diseased retinas. These tumor-specific cellular interactions regulate multiple signaling pathways (e. g. VEGF-, WNT-, BMP-, PGF- signaling) known to be involved in RB progression. By comparing chemo-treated and chemo-naïve RB samples we were able to identify treatment-resistant and -sensitive subpopulations of tumor cells. Finally, data from our RB-retina in vitro model highlighted the impact of RB cells on gene expression programs of normal retinal cells. CONCLUSION: In summary, we deciphered the intratumoral heterogeneity of RB, uncovered an intricate network of microglia-tumor cell interactions that connects numerous important signaling pathways, and further identified chemotherapy-resistant/sensitive tumor cell populations. The latter observation could prove to be very helpful in the development of novel therapeutic approaches in the treatment of RB.

9-OR: ADA Presidents' Select Abstract: m6A mRNA Methylation Regulates the Innate Immune Response in Human Beta Cells

Innate immunity is directly associated with Type 1 diabetes (T1D) . Recently, N6-methyladenosine (m6A) has been shown to modulate human β-cell biology and innate immunity. Here, we focused on the contribution of m6A in modulating T1D development. RNA-seq. in β-cells revealed downregulation of the m6A writers (Mettl3, Mettl14, and Wtap) in pre-diabetic NOD mice. Consistently, pancreases from female NOD mice and human T1D showed downregulation of METTL3 protein levels in insulin-positive cells as the disease progressed. m6A-sequencing in human T1D versus control islets and intersection with differentially expressed genes (DEGs) in T1D β-cells from a single-cell RNA seq. dataset, revealed enriched pathways associated with ER function, T1D, and apoptosis. Pro-inflammatory cytokines are directly linked to β-cell autoimmunity and are considered a useful tool to study T1D onset. We applied m6A-sequencing in cytokine-treated human islet preparations, and in parallel studies, treated EndoC-βH1 cells with cytokines followed by RNA-sequencing. Intersection of m6A-decorated and common DEGs in human islets and EndoC-βH1 cells revealed enrichment in pathways associated with the innate immune response. METTL3 silencing in EndoC-βH1 impacted several innate immune mediators upon cytokine challenge. Incubation of human islets from multiple donors with ER-stress inducers, ROS donors, NO donors, and ROS or NO scavengers revealed that pre-existing ER stress and ROS decrease METTL3, while physiological levels of NO upregulate METTL3. We conclude that ROS drives METTL3 downregulation associated with T1D. Exploring therapeutic relevance by AAV-8 mediated gene delivery showed that β-cell specific upregulation of Mettl3 leads to a decrease in hyperglycemia during early stages of T1D in female NOD mice. Together, these results demonstrate a novel layer of regulation in human β-cells in the context of T1D and point to targeting METTL3 as a therapeutic approach to promote β-cell survival and improve glycemia. Disclosure D.F.De jesus: n/a. D.L.Eizirik: None. R.Kulkarni: Advisory Panel; Biomea, Novo Nordisk, REDD Pharma, Consultant; Relay Therapeutics, Research Support; Inversago, SerPlus. Z.Zhang: Research Support; SinoVac. N.K.Brown: None. G.Basile: None. L.Xiao: None. S.Kahraman: Employee; Boehringer Ingelheim International GmbH. C.E.Mathews: None. A.C.Powers: None. M.A.Atkinson: None. Funding American Diabetes Association (7-21-PDF-140) ; R01 DK067536R01 DK103215R01 DK117639

Glycan Profiling by Sequencing to Uncover Multicellular Communication: Launching Glycobiology in Single Cells and Microbiomes.

Glycans are essential building blocks of life that are located at the outermost surface of all cells from mammals to bacteria and even viruses. Cell surface glycans mediate multicellular communication in diverse biological processes and are useful as “surface markers” to identify cells. Various single-cell sequencing technologies have already emerged that enable the high-throughput analysis of omics information, such as transcriptome and genome profiling on a cell-by-cell basis, which has advanced our understanding of complex multicellular interactions. However, there has been no robust technology to analyze the glycome in single cells, mainly because glycans with branched and heterogeneous structures cannot be readily amplified by polymerase chain reactions like nucleic acids. We hypothesized that the generation of lectins conjugated with DNA barcodes (DNA-barcoded lectins) would enable the conversion of glycan information to gene information, which may be amplified and measured using DNA sequencers. This technology will enable the simultaneous analysis of glycan and RNA in single cells. Based on this concept, we developed a technology to analyze glycans and RNA in single cells, which was referred to as scGR-seq. Using scGR-seq, we acquired glycan and gene expression profiles of individual cells constituting heterogeneous cell populations, such as tissues. We further extended Glycan-seq to the profiling of the surface glycans of bacteria and even gut microbiota. Glycan-seq and scGR-seq are new technologies that enable us to elucidate the function of glycans in cell–cell and cell–microorganism communication, which extends glycobiology to the level of single cells and microbiomes.

Doublet identification in single-cell sequencing data using scDblFinder.

Doublets are prevalent in single-cell sequencing data and can lead to artifactual findings. A number of strategies have therefore been proposed to detect them. Building on the strengths of existing approaches, we developed scDblFinder, a fast, flexible and accurate Bioconductor-based doublet detection method. Here we present the method, justify its design choices, demonstrate its performance on both single-cell RNA and accessibility (ATAC) sequencing data, and provide some observations on doublet formation, detection, and enrichment analysis. Even in complex datasets, scDblFinder can accurately identify most heterotypic doublets, and was already found by an independent benchmark to outcompete alternatives.

Neuron-Keratinocyte Communication in the Epidermis in Painful Diabetic Neuropathy

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration, which can progress to complete loss of cutaneous innervation and is accompanied by neuropathic pain. Uncovering the mechanisms underlying axonal degeneration in PDN remains a major challenge to finding effective and disease-modifying therapies. Sensory nerve afferents normally extend into the epidermis in close juxtaposition to keratinocytes but degenerate in diabetic skin. Our aim is to identify the changes in gene expression profiles and the interactions between dorsal root ganglion (DRG) neurons and keratinocytes to explore the mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts axonal degeneration underlying neuropathic pain in PDN. We used a mouse model of PDN where mice were fed a regular diet (RD, 11% fat) or a high-fat diet (HFD, 42% fat) for 10 weeks during which these mice develop glucose intolerance, mechanical allodynia, small fiber neuropathy. Using a single-cell RNA (scRNA-seq) sequencing approach we captured DRG and keratinocytes gene expression profiles and generated interactome maps. scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes between RD and HFD from the DRG. We were able to identify several clusters of immune cells and keratinocytes at different stages of differentiation. scRNA-seq results were validated using RNAscope on DRG and skin frozen sections. Moreover, we generated interactome maps between DRG neurons and the peripheral cells to highlight ligand-receptor interactions and we looked to identify genes that were differentially expressed in these interactions. Taken together our data highlights the importance of studying neurons in conjunction with the cells in the tissues with which they interact to identify ligand-receptor interactions that may lead to the identification of neuron signaling in a chronic pain state such as PDN. Grant support from 1R01AR77691-01. Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by small-fiber degeneration, which can progress to complete loss of cutaneous innervation and is accompanied by neuropathic pain. Uncovering the mechanisms underlying axonal degeneration in PDN remains a major challenge to finding effective and disease-modifying therapies. Sensory nerve afferents normally extend into the epidermis in close juxtaposition to keratinocytes but degenerate in diabetic skin. Our aim is to identify the changes in gene expression profiles and the interactions between dorsal root ganglion (DRG) neurons and keratinocytes to explore the mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts axonal degeneration underlying neuropathic pain in PDN. We used a mouse model of PDN where mice were fed a regular diet (RD, 11% fat) or a high-fat diet (HFD, 42% fat) for 10 weeks during which these mice develop glucose intolerance, mechanical allodynia, small fiber neuropathy. Using a single-cell RNA (scRNA-seq) sequencing approach we captured DRG and keratinocytes gene expression profiles and generated interactome maps. scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes between RD and HFD from the DRG. We were able to identify several clusters of immune cells and keratinocytes at different stages of differentiation. scRNA-seq results were validated using RNAscope on DRG and skin frozen sections. Moreover, we generated interactome maps between DRG neurons and the peripheral cells to highlight ligand-receptor interactions and we looked to identify genes that were differentially expressed in these interactions. Taken together our data highlights the importance of studying neurons in conjunction with the cells in the tissues with which they interact to identify ligand-receptor interactions that may lead to the identification of neuron signaling in a chronic pain state such as PDN. Grant support from 1R01AR77691-01.

CGAS-STING Mediated Inflammation In Diabetic Wound Macrophages Is Regulated By Histone Demethylase JMJD3

Abstract Recent work has shown that the cGAS-STING inflammatory pathway is elevated in conditions that are marked by chronic inflammation, such as obesity and diabetes. Using single cell RNA (scRNA) sequencing from human wounds, we show that the Sting gene is increased in diabetic wound macrophages. Histone demethylase JMJD3 specifically demethylates lysine 27 on histone 3 (H3K27me3) - a repressive mark for gene transcription. Using diabetic mice, we note on ChIP analysis, a decrease in the H3K27me3 repressive mark at the STING promoter in diabetic wound macrophages relative to littermate controls, with subsequently enhanced STING protein levels by western blot. Additionally, scRNA from wound macrophages obtained from a novel murine strain deficient in Jmjd3 in myeloid-cell lineages (Jmjd3f/fLyz2Cre+), revealed significantly decreased Sting gene levels. Further, ChIP analysis of H3K27me3 levels at the STING promoter from Jmjd3f/fLyz2Cre+ wound macrophages reveal increased H3K27me3 levels compared to littermate controls, with a concomitant decrease in STING protein as determined by western blot. Mechanistically, we use mRNA, protein and ChIP studies to reveal that in diabetic wound macrophages, IL6 is responsible for increased JMJD3 leading to increased JAK1,3/STAT3 activity and increased STING production. Wound healing curves from diabetic mice locally treated with macrophage-specific nanoparticles against JMJD3 showed significantly improved healing, and decreased STING protein production in these wounds. Taken together, this data highlights a unique epigenetic regulator of STING production in diabetic wound macrophages and provides a viable cell-specific, nanotherapeutic approach to non-healing diabetic wounds. Supported by NIH (F32DK126471), Vascular and Endovascular Surgical Society Resident Research Award

Activation of Keratinocyte Gq-linked G-Protein Coupled Receptors Remodels Dorsal Root Ganglion Neurons Single-Cell Transcriptomic Profile

Keratinocytes are closely juxtaposed to cutaneous nerve terminals, enabling communication between keratinocytes and cutaneous nerves. We investigated potential mechanisms that mediate this communication. We genetically expressed stimulatory DREADDs (hM3Dq) into K14 basal keratinocytes (K14) in mice as a tool for mimicking the activation of Gq-linked G protein-coupled receptors (GPCRs) in K14 expressing cells. We have shown that activation of these DREADDs reduced innervation of the epidermis indicating that activation of K-14 Gq-linked GPCRs can regulate nerve fiber degeneration in the epidermis. We used bulk and single-cell RNA (scRNA-seq) sequencing from the skin and Dorsal Root Ganglion (DRG) taken from mice expressing hM3Dq into K14 keratinocytes comparing control mice receiving saline and mice receiving CNO to activate K14 keratinocytes expressing hM3Dq. Bulk RNA sequencing of the epidermis revealed changes in a set of genes associated with neurite outgrowth, including class III semaphorins, potentially impacting cutaneous nerve degeneration. Using RNAScope we found dramatic changes in the expression of semaphorins and their receptors in distinct DRG neurons subtypes. Using single-cell RNA-seq from lumbar DRG we profiled the transcriptome of 10,000 individual DRG cells and found that hM3Dq mediated stimulation of keratinocytes induced changes in DRG single-cell gene expression profile resulting in the identification of a novel DRG neuronal subtype among the nonpeptidergic nociceptors (NP1.2). Our findings indicate that activation of basal keratinocytes Gq-linked GPCRs can result in profound changes in DRG neuronal transcriptomic profile resulting in a phenotypical switch (plasticity) of DRG neurons. Furthermore, our results suggest possible therapeutic effects of activating or blocking specific keratinocyte Gq-linked GPCRs for promoting axon regeneration in small fiber neuropathy. Grant support from 1R01AR77691-01.

Human KIR+ CD8+ T cells target pathogenic T cells in Celiac disease and are active in autoimmune diseases and COVID-19

Abstract Previous reports show a small subset of CD8+ T cells expressing Ly49 proteins in mice can suppress autoimmunity in a model of demyelinating disease. Here we find a markedly increased frequency of CD8+ T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various human autoimmune diseases. Increased KIR+ CD8+ T cells in the gut also correlate with disease activity in Celiac disease (CeD) patients. Moreover, KIR+ CD8+ T cells can efficiently eliminate pathogenic gliadin-specific CD4+ T cells from CeD patients’ leukocytes in vitro. Together with gene expression data, this shows that these cells are the likely equivalent of the mouse Ly49+ CD8+ T cells. Furthermore, we observe elevated levels of KIR+ CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Single-cell RNA and parallelized TCR sequencing reveals that expanded KIR+ CD8+ T cells from these different diseases and healthy subjects display shared phenotypes and similar T cell receptor sequences. Selective ablation of the murine counterpart in virus-infected mice leads to exacerbated autoimmunity developed after infection. These results characterize a regulatory CD8+ T cell subset in humans which we hypothesize functions to control pathogenic cells in autoimmune and infectious diseases, with important implications for the cellular dynamics and possible therapeutic approaches to suppress unwanted autoimmunity. Supported by National Institutes of Health U19-AI057229 Howard Hughes Medical Institute Stanford Diabetes Research Center (P30DK116074)

Doublet identification in single-cell sequencing data using scDblFinder

Doublets are prevalent in single-cell sequencing data and can lead to artifactual findings. A number of strategies have therefore been proposed to detect them. Building on the strengths of existing approaches, we developed scDblFinder, a fast, flexible and accurate Bioconductor-based doublet detection method. Here we present the method, justify its design choices, demonstrate its performance on both single-cell RNA and accessibility (ATAC) sequencing data, and provide some observations on doublet formation, detection, and enrichment analysis. Even in complex datasets, scDblFinder can accurately identify most heterotypic doublets, and was already found by an independent benchmark to outcompete alternatives.

Identification of two immune-related risk score signatures through integrated analysis of multi-omics data in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Immunotherapy has become a major treatment for advanced HCC, but the therapeutic effects remain unsatisfactory. In this study, we constructed an immune cell risk score (ICS) and an immune cell-related gene risk score (ICRGS) for the prognosis prediction of HCC through integrated analysis of bulk and single-cell RNA (scRNA) sequencing data. These two risk score signatures both showed good predictive values in the training and validation cohorts. The potential interactions among these prognostic immune cell types were elucidated by cell–cell communication analysis. The results of enrichment analysis and gene set enrichment analysis (GSEA) of the prognostic genes showed that metabolic-related processes were involved in the immune response of HCC. Furthermore, the results of correlation analyses further confirmed the hub genes that were strongly correlated with immune cells. Finally, potential therapeutic drugs targeting these hub genes were screened by CellMiner based on NCI-60 cell line set. Taken together, two useful models for the prognosis prediction of HCC patients were constructed in this study. The functional differences between the two groups of HCC patients separated by ICS or ICRGS provide fundamental knowledge for finding synergistic therapeutic targets for HCC immunotherapy.

Multidimensional single-cell analysis of human peripheral blood reveals characteristic features of the immune system landscape in aging and frailty.

Frailty is an intermediate status of the human aging process, associated with decompensated homeostasis and death. The immune phenotype of frailty and its underlying cellular and molecular processes remain poorly understood. We profiled 114,467 immune cells from cord blood, young adults and healthy and frail old adults using single-cell RNA and TCR sequencing. Here we show an age-dependent accumulation of transcriptome heterogeneity and variability in immune cells. Characteristic transcription factors were identified in given cell types of specific age groups. Trajectory analysis revealed cells from non-frail and frail old adults often fall into distinct paths. Numerous TCR clonotypes were shared among T-cell subtypes in old adults, indicating differential pluripotency and resilience capabilities of aged T cells. A frailty-specific monocyte subset was identified with exclusively high expression of long noncoding RNAs NEAT1 and MALAT1. Our study discovers human frailty-specific immune cell characteristics based on the comprehensive dimensions in the immune landscape of aging and frailty.

Evolutionarily conservative and non-conservative regulatory networks during primate interneuron development revealed by single-cell RNA and ATAC sequencing

The differences in size and function between primate and rodent brains, and the association of disturbed excitatory/inhibitory balance with many neurodevelopmental disorders highlight the importance to study primate ganglionic eminences (GEs) development. Here we used single-cell RNA and ATAC sequencing to characterize the emergence of cell diversity in monkey and human GEs where most striatal and cortical interneurons are generated. We identified regional and temporal diversity among progenitor cells which give rise to a variety of interneurons. These cells are specified within the primate GEs by well conserved gene regulatory networks, similar to those identified in mice. However, we detected, in human, several novel regulatory pathways or factors involved in the specification and migration of interneurons. Importantly, comparison of progenitors between our human and published mouse GE datasets led to the discovery and confirmation of outer radial glial cells in GEs in human cortex. Our findings reveal both evolutionarily conservative and nonconservative regulatory networks in primate GEs, which may contribute to their larger brain sizes and more complex neural networks compared with mouse.

MQuad enables clonal substructure discovery using single cell mitochondrial variants

Mitochondrial mutations are increasingly recognised as informative endogenous genetic markers that can be used to reconstruct cellular clonal structure using single-cell RNA or DNA sequencing data. However, identifying informative mtDNA variants in noisy and sparse single-cell sequencing data is still challenging with few computation methods available. Here we present an open source computational tool MQuad that accurately calls clonally informative mtDNA variants in a population of single cells, and an analysis suite for complete clonality inference, based on single cell RNA, DNA or ATAC sequencing data. Through a variety of simulated and experimental single cell sequencing data, we showed that MQuad can identify mitochondrial variants with both high sensitivity and specificity, outperforming existing methods by a large extent. Furthermore, we demonstrate its wide applicability in different single cell sequencing protocols, particularly in complementing single-nucleotide and copy-number variations to extract finer clonal resolution.

Vitamin C Deficiency Causes Cell Type-Specific Epigenetic Reprogramming and Acute Tubular Necrosis in a Mouse Model.

Vitamin C deficiency is found in patients with variable kidney diseases. However, the role of vitamin C as an epigenetic regulator in renal homeostasis and pathogenesis remains largely unknown. We showed that vitamin C deficiency leads to acute tubular necrosis (ATN) using a vitamin C-deficient mouse model (Gulo knock-out). DNA/RNA epigenetic modifications and injured S3 proximal tubule cells were identified in the vitamin C-deficient kidneys using whole-genome bisulfite sequencing, methylated RNA immunoprecipitation sequencing, and single-cell RNA sequencing. Integrated evidence suggested that epigenetic modifications affected the proximal tubule cells and fenestrated endothelial cells, leading to tubule injury and hypoxia through transcriptional regulation. Strikingly, loss of DNA hydroxymethylation and DNA hypermethylation in vitamin C-deficient kidneys preceded the histologic sign of tubule necrosis, indicating the causality of vitamin C-induced epigenetic modification in ATN. Consistently, prophylactic supplementation of an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, promoted DNA demethylation and prevented the progression of cisplatin-induced ATN. Vitamin C played a critical role in renal homeostasis and pathogenesis in a mouse model, suggesting vitamin supplementation may be an approach to lower the risk of kidney injury.

Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients

Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe coronavirus disease 2019 (COVID-19) and an increased risk of death from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T cells made resistant to a common immunosuppressant, tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated tacrolimus-resistant SARS-CoV-2-specific T cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single-cell level, including flow cytometry, single-cell RNA (scRNA) Cellular Indexing of Transcriptomes and Epitopes (CITE), and T cell receptor (TCR) sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with tacrolimus, to prevent an overshooting immune response manifested as bystander T cell activation in the setting of severe COVID-19 immunopathology, and tacrolimus-resistant SARS-CoV-2-specific T cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology while providing viral clearance in severe non-transplant COVID-19 cases.

In Situ Single-Molecule Imaging of MicroRNAs in Switchable Migrating Cells under Biomimetic Confinement.

Spatial imaging of RNAs in single cells is extremely charming for deciphering of regulatory mechanisms in multiple migration modes during tumor metastasis. Herein, enzyme-free-mediated cascade amplified nanoprobes were designed for in situ single-molecule imaging of dual-microRNAs (miRNAs) in switchable migrating cells. Differential expression and localization of dual-miRNAs were clearly exhibited in multiple cell lines attributed to enhanced sensitivity via the cascade signal amplification strategy. Significantly, in situ three-dimensional (3D) imaging of dual-miRNAs in transition of cell migration phenotypes was successfully reconstructed in both non-confined and confined microenvironments in vitro, of which differential spatial distribution was observed in a single cell. This is very promising for exploring key roles of spatial RNA distribution in migrating cells at the single-molecule level, which will advance revealing the molecular mechanism and physical principle in 3D cell migration in vivo.

Global Characterization of Peripheral B Cells in Parkinson's Disease by Single-Cell RNA and BCR Sequencing.

Immune system plays important roles in the pathogenesis of Parkinson’s disease (PD). However, the role of B cells in this complex disease are still not fully understood. B cells produce antibodies but can also regulate immune responses. In order to decode the relative contribution of peripheral B cell subtypes to the etiology of PD, we performed single cell RNA and BCR sequencing for 10,466 B cells from 8 PD patients and 6 age-matched healthy controls. We observed significant increased memory B cells and significant decreased naïve B cells in PD patients compared to healthy controls. Notably, we also discovered increased IgG and IgA isotypes and more frequent class switch recombination events in PD patients. Moreover, we identified preferential V and J gene segments of B cell receptors in PD patients as the evidence of convergent selection in PD. Finally, we found a marked clonal expanded memory B cell population in PD patients, up-regulating both MHC II genes (HLA-DRB5, HLA-DQA2 and HLA-DPB1) and transcription factor activator protein 1 (AP-1), suggesting that the antigen presentation capacity of B cells was enhanced and B cells were activated in PD patients. Overall, this study conducted a comprehensive analysis of peripheral B cell characteristics of PD patients, which provided novel insights into the humoral immune response in the pathogenesis of PD.

Abstract PD6-03: Spatio-molecular dissection of the breast cancer metastatic microenvironment

Abstract Metastatic breast cancer (MBC) remains incurable due to inevitable development of therapeutic resistance. Although tumor cell intrinsic mechanisms of resistance in MBC are beginning to be elucidated by bulk sequencing studies, the roles of the tumor microenvironment and intratumor heterogeneity in therapeutic resistance remain underexplored due to both technological barriers and limited availability of samples. To comprehensively capture these characteristics we have adapted a research biopsy protocol to collect tissue for an array of single-cell and spatio-molecular assays whose performance we have optimized for MBC, including single-cell and single-nucleus RNA sequencing, Slide-Seq, Multiplexed Error-Robust FISH (MERFISH), Expansion Sequencing (ExSEQ), Co-detection by Indexing (CODEX) and Multiplexed Ion Beam Imaging (MIBI). To date, we have successfully performed single-cell or single-nucleus RNAseq in 67 MBC biopsies and generated detailed accompanying clinical annotations for each. These samples provide a representation of the clinicopathological diversity of MBC including different breast cancer subtypes (44 HR+/HER2-, 3 HR-/HER2+, 3 HR+/HER2+, 16 TNBC, 1 unknown), common anatomic sites of metastasis (37 liver, 9 axilla, 7 breast, 5 bone, 3 chest wall, 3 neck, 1 brain, 1 lung, 1 skin), metastatic presentations (53 recurrent, 14 de novo) and histologic subtypes in the breast (45 IDC, 7 ILC, 6 mixed, 3 DCIS, 1 mucinous, 5 unknown/NA). Following optimization, both single-cell and single-nucleus RNA seq perform well in these MBC biopsies recovering all expected cell types including the malignant, stromal (e.g. fibroblasts, endothelial cells), myeloid (e.g. monocytes, macrophages) and lymphoid compartments (e.g. T cells, B cells, NK cells) as well as relevant oncogenic programs (e.g. cell cycle programs in all compartments; EMT-like and ER signaling programs in the malignant compartment, immune checkpoint programs in the lymphoid compartment; and fibroblast activation and vascular homeostasis programs in the stromal compartment). In addition to differences between the two techniques, these data demonstrate substantial intratumor heterogeneity in cell type composition. For example in liver biopsies the average number of cells per sample compartment by single nucleus RNA-seq was 6745 malignant (56%, SD 4216), 4637 stromal (41%, SD 3727), 1196 lymphoid (8%, SD 1617) and 874 myeloid (6%, SD 852); in breast biopsies the average number of cells per compartment by single nucleus RNA-seq was 6421 malignant (70%, SD 3497), 1628 stromal (24%, SD 117), 333 lymphoid (4%, SD 170) and 213 myeloid (3%, SD 117). Additionally, we find both inter- and intra-tumor heterogeneity in expression patterns and programs including, for example, expression of ER, PR and HER2 within clinical receptor subtypes (log normalized counts for ER expression in tumor cells by single cell RNA-seq: HR+/HER2- 0.921 (SD 0.714); HR+/HER2+ 0.768 (SD 0.624); HR-/HER2+ 0.018 (SD 0.122); and HR-/HER2- 0.005 (SD 0.066). For a subset of 13 biopsies we are also completing the spatiomolecular characterization methods on serial sections of a single adjacent biopsy. This unique experimental setup was designed to enable efficient comparison and integration of these assays. In spite of differences between experimental techniques and readouts, cell typing can be approached by annotation transfer from matching single cell or single nucleus RNAseq data, enabling exploratory analyses including evaluation of spatial phenotypes and cell type colocalization. Overall, these single cell and spatial data afford a comprehensive atlas including cell types, cell states/programs, cell interactions and spatial organization in MBC lesions. Future analyses will include serial biopsies over time and integration of clinicopathologic data including therapeutic response and resistance. Citation Format: Daniel L Abravanel, Johanna Klughammer, Timothy Blosser, Yury Goltsev, Sizun Jiang, Yunjao Bai, Evan Murray, Shahar Alon, Yi Cui, Daniel R Goodwin, Anubhav Sinha, Ofir Cohen, Michal Slyper, Orr Ashenberg, Danielle Dionne, Judit Jané-Valbuena, Caroline BM Porter, Asa Segerstolpe, Julia Waldman, Sébastien Vigneau, Karla Helvie, Allison Frangieh, Laura DelloStritto, Miraj Patel, Jingyi We, Kathleen Pfaff, Nicole Cullen, Ana Lako, Madison Turner, Isaac Wakiro, Sara Napolitano, Abhay Kanodia, Rebecca Ortiz, Colin MacKichan, Stephanie Inga, Judy Chen, Aaron R Thorner, Asaf Rotem, Scott Rodig, Fei Chen, Edward S Boyden, Garry P Nolan, Xiaowei Zhuang, Orit Rozenblatt-Rosen, Bruce E Johnson, Aviv Regev, Nikhil Wagle. Spatio-molecular dissection of the breast cancer metastatic microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-03.

Multi-Omics Characterization of Tumor Microenvironment Heterogeneity and Immunotherapy Resistance Through Cell States-Based Subtyping in Bladder Cancer.

Due to the strong heterogeneity of bladder cancer (BC), there is often substantial variation in the prognosis and efficiency of immunotherapy among BC patients. For the precision treatment and assessment of prognosis, the subtyping of BC plays a critical role. Despite various subtyping methods proposed previously, most of them are based on a limited number of molecules, and none of them is developed on the basis of cell states. In this study, we construct a single-cell atlas by integrating single cell RNA-seq, RNA microarray, and bulk RNA-seq data to identify the absolute proportion of 22 different cell states in BC, including immune and nonimmune cell states derived from tumor tissues. To explore the heterogeneity of BC, BC was identified into four different subtypes in multiple cohorts using an improved consensus clustering algorithm based on cell states. Among the four subtypes, C1 had median prognosis and best overall response rate (ORR), which characterized an immunosuppressive tumor microenvironment. C2 was enriched in epithelial-mesenchymal transition/invasion, angiogenesis, immunosuppression, and immune exhaustion. Surely, C2 performed the worst in prognosis and ORR. C3 with worse ORR than C2 was enriched in angiogenesis and almost nonimmune exhaustion. Displaying an immune effective environment, C4 performed the best in prognosis and ORR. We found that patients with just an immunosuppressive environment are suitable for immunotherapy, but patients with an immunosuppressive environment accompanied by immune exhaustion or angiogenesis may resist immunotherapy. Furthermore, we conducted exploration into the heterogeneity of the transcriptome, mutational profiles, and somatic copy-number alterations in four subtypes, which could explain the significant differences related to cell states in prognosis and ORR. We also found that PD-1 in immune and tumor cells could both influence ORR in BC. The level of TGFβ in a cell state can be opposite to the overall level in the tissues, and the level in a specific cell state could predict ORR more accurately. Thus, our work furthers the understanding of heterogeneity and immunotherapy resistance in BC, which is expected to assist clinical practice and serve as a supplement to the current subtyping method from a novel perspective of cell states.