Chromium Single Cell Research Articles

A single-cell multiomics analysis of IRF4 in mediating treatment resistance in ibrutinib-treated chronic lymphocytic leukemia (CLL).

7051 Background: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib (ibr) has revolutionized the treatment of CLL, showing efficacy in the majority of patients. Nevertheless, resistance occurs in a subset of patients, often with dismal clinical outcomes. Ibr resistance occurs through several mechanisms, including mutations affecting BTK or PLCG2, or upregulation of alternative survival pathways. We investigated at the single-cell level the molecular mechanisms that underlie ibr resistance in CLL cells and their interactions with non-malignant tumor microenvironment (TME) cell populations. Methods: We retrospectively identified 7 CLL patients with ibr resistance (as second line therapy). Samples were obtained before ibr treatment and at progression. Chromium Single Cell Multiome ATAC + Gene Expression (GEX) (10x Genomics) was applied to a mixture of CD19+CD5+ and CD19- cells to jointly analyze DNA accessibility and gene expression in the same single nuclei of CLL B and TME cells. An average of 4,800 cells (range 2,600-15,000) per sample was retained after QC filtering. Principal component analysis (PCA) was performed on GEX counts. ATAC peaks were called with MACS2, and latent semantic indexing was used for dimensionality reduction. We then computed a weighted nearest neighbour graph, identified clusters using the SLM algorithm and performed cell type assignment with established marker genes. Results: We identified 28 distinct cell clusters, representing CLL B-cells or cell types that constitute the TME, including CD4+ and CD8+ T cells, monocytes, dendritic and NK cells. CLL B-cell clusters were patient and timepoint-specific, while TME clusters were occupied by cells from multiple patients. Five patients showed multiple distinct CLL B-cell clusters pre-treatment or at progression, reflecting cellular heterogeneity within the tumor compartment. Interestingly, some clusters that were dominated by post-treatment CLL B-cells also included cells from pre-treatment samples, suggesting pre-existence of the clone giving rise to the relapse. For each patient, we performed pairwise differential expression between CLL B-cell clusters. Gene ontology analysis revealed increased activation of IRF4 and MHC-related pathways in CLL B-cells at progression compared to pre-treatment in 6 and 4 out of 7 patients, respectively. The elevated IRF4 pathway activity was underpinned by increased chromatin accessibility in IRF4 signature genes. Conclusions: Differential blockade of B-cell receptor signaling by ibr has been reported to downregulate activity of IRF4, a key transcription factor in B-cell activation. Our results suggest that CLL B-cells that are resistant to ibrutinib have re-activated IRF4 activity beyond pre-treatment levels, implying a potential role for IRF4 in ibrutinib resistance.

Interrogating early molecular events of doxorubicin-induced cardiotoxicity at single-cell level to identify new cardioprotective agents

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq Foundation Background Doxorubicin (DOX) is a highly effective chemotherapeutic agent widely used for treating various types of malignancies. Unfortunately, its clinical implication is hampered by cardiotoxic side effects which are responsible for increased mortality among cancer survivors compared to the general population. Preclinical and clinical studies have highlighted potential mechanisms of anthracycline-induced cardiotoxicity (AIC), but the core molecular basis remains largely unknown. More importantly, only a few studies have focused on the characterization of the molecular events that distinguish the early reversible phase of AIC from an advanced and irreversible state of the disease. Aim We aim to provide an unbiased and in-depth profile of early transcriptional changes induced by DOX at single cell resolution, to identify new druggable targets for the development of cardioprotective agents to prevent AIC. Methods Based on our previously established murine model of AIC, BALB/c mice were injected with saline (Vehicle) or DOX (3 weekly injections of 4 mg/kg), and hearts were collected at either 3 days (acute cardiotoxicity) or 6 weeks after the first injection (chronic cardiotoxicity). Nuclei were isolated from frozen hearts for single-nuclei transcriptomic (snRNAseq; 10x Genomics, Chromium Single Cell 3' v3.1). Seurat pipeline was mainly used for downstream bioinformatic analysis. Results Echocardiography revealed DOX-induced systolic dysfunction at 6 weeks, confirming the establishment of AIC. SnRNAseq data from 12 hearts allowed to identify 8 major cell types including cardiomyocytes (CM), endothelial and mural cells, fibroblasts, myeloid cells, B and T lymphocytes and neuronal-like cells. To unravel changes in CM, we performed unbiased subclustering which defined 6 different subpopulations, including a CM_Stressed population, characterized by enrichment of typical cardiac stress markers, such as natriuretic peptide hormone (Nppb) and myosin heavy chain 7 (Myh7). Differential abundance analysis showed enrichment of the CM-Stressed population at both 3 days and 6 weeks, suggesting that DOX drives a transcriptional change toward stress status in CM both at early and late stages of cardiotoxicity. Differential gene expression analysis revealed Golgi associated kinase 1B (Gask1B) to be significantly upregulated at both 3 days and 6 weeks in CM_Stressed. In agreement, preliminary experiments showed that Gask1b gene silencing partially rescued DOX-induced heart dysfunction in zebrafish model and hiPSC-derived CM, identifying Gask1b as a potential new player in DOX cardiotoxicity. Conclusions We generated a single-nucleus dataset of DOX-treated mouse hearts and identified transcriptional changes driven by DOX in CM at early and late stages of the disease. Furthermore, we identified Gask1b gene, whose role in cardiac pathophysiology was previously unappreciated, as a new potential marker and determinant of DOX cardiotoxicity.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes.

The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Abstract PR003: T cell receptor repertoire and diversity are prognostic markers in bladder cancer

Abstract T cells represent essential effector cells in the intrinsic antitumor immune response. Cancer-specific T cells can recognize cancer neoantigens through their T cell receptors (TCRs). The expansion of these clones is believed to be an early response to malignancy. We hypothesized that the T cell landscape offers insights into immune competency. We aimed to characterize the TCR repertoire in patients with bladder cancer and explore correlations with disease outcomes. We analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer (MIBC) treated with chemotherapy followed by radical cystectomy. Peripheral and tumor TCR repertoires were produced using ultradeep amplicon-based sequencing of the TCR beta chain. T cell fractions were inferred from whole exome sequencing data of blood DNA using TcellExTRECT. Latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. The T cell subtype compositions in tumor and blood were investigated in four patients with bladder cancer using the Chromium Single Cell Immune profiling kit from 10X Genomics. Low pre-treatment peripheral TCR diversity was associated with worse overall survival (HR = 2.3, p = 0.024, n = 119) in patients with MIBC, particularly when combined with a low fraction of circulating T cells (HR= 4.7, p = 0.00049, n = 67). We validated these findings in two independent cohorts of patients with MIBC (p = 0.01, n = 107; p = 0.042, n = 79). The low-diversity TCR repertoires were characterized by large expanded T cell clones that persisted over time. Longitudinal analysis indicated a potential adverse impact of treatment, evidenced by a reduction in TCR diversity and circulating T cells over time in patients with initially high-diversity repertoires. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections and cytomegalovirus DNA detection was strongly associated with low TCR diversity (p = 5.3 × 10−5). We observed a notable disparity between tumor and peripheral blood TCR repertoires. Single-cell sequencing revealed that regulatory T cells were prevalent in the tumor, whereas a combination of cytotoxic and naïve T cells dominated the blood. More explicitly, expanded clones in the blood were commonly annotated as terminally differentiated effector memory T cells expressing high levels of cytotoxic and exhausted genes. Our results suggest that high peripheral TCR diversity and high T cell fraction are markers of general immune competence, reflecting the ability to eradicate cancer and other diseases. In contrast, the expanded persistent clones predominating the low peripheral TCR diversity repertoires are likely exhausted and less likely to exhibit tumor specificity or effective disease-fighting capabilities. Our findings underline the crucial role of the immune system in determining disease outcomes and highlight the potential for improving immune health as a promising approach for future treatment and prevention. Citation Format: Nanna Kristjánsdóttir, Asbjørn Kjær, Iver Nordentoft, Randi I Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Trine Strandgaard, Deema Radif, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen B Jensen, Nicolai J. Birkbak, Lars Dyrskjøt. T cell receptor repertoire and diversity are prognostic markers in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR003.

Abstract PO1-25-06: Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells

Abstract Background In recent years, the advancement of adoptive cell transfer-based immunotherapies has significantly contributed to the evolution of cancer treatment. The crucial step in achieving successful treatment lies in the identification of T cells that exhibit reactivity towards tumor-specific antigens. However, the heterogeneous composition of tumor antigen-specific T cells poses a challenge as there are no standardized biomarkers available for their identification. To overcome this limitation, we investigated alternative approaches, employing single cell RNA and VDJ sequencing to identify antigen-specific T cells. Methods First, peripheral blood mononuclear cells (PBMCs) obtained from healthy human donors were stained with carboxyfluorescein succinimidyl ester (CFSE) to facilitate the identification of proliferating antigen-specific T cells. Then the PBMCs were exposed to 1 ug/ml of CMV pp65 peptide. After a 2-day incubation period with the CMV peptide, interleukin-2 (IL-2) was administered to stimulate T cell expansion. At various time points (0, 1, 2, 3, 5, and 7 days) following the CMV peptide treatment, CD8+ T cells were isolated through fluorescence-activated cell sorting (FACS) analysis. On day 7, the CD8+ T cells were further segregated into CFSE- and CFSE+ cell populations. The single-cell transcriptomes and T cell receptor (TCR) repertoire of the CD8+ T cells were subsequently examined using the Chromium Single Cell Reagent Kit. Results The TCR repertoire analysis of CD8+ CFSE- T cells revealed the dominance of 10 major clones, accounting for 93.9% of the total cells. These clones were confirmed to be CMV-specific through various in vitro assays, including NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assay. In contrast, CD8+ CFSE+ T cells did not exhibit significant overlap with the major TCR clones found in CFSE- CD8+ T cells, comprising only 0.02% of the total cells. Gene set enrichment analysis demonstrated that the upregulated genes in CFSE- CD8+ T cells, when compared to CFSE+ T cells, were related to cell cycle proliferation and T cell cytotoxicity. Furthermore, these genes exhibited a matching upregulation pattern with CFSE- T cells from another batch of the same donor, as well as from two other healthy donors. In addition, there was a moderate increase in the expression of the upregulated genes in CFSE- CD8+ T cells at day 3 compared to day 0-2. In this end, we selected a set of nine genes that were consistently upregulated in CFSE- CD8+ T to serve as markers for antigen-specific T cells, employing a logistic regression model. This gene set showed high sensitivity (97.7%) and specificity (95.6%) in identifying T cell clones that respond to CMV antigen at day 3. Conclusions We have discovered potential markers for antigen-specific CD8+ T cells. These results can offer the promising opportunity for earlier isolation of T cells that exhibit reactivity towards tumor-specific antigens. This breakthrough has significant implications, as it can potentially contribute to cost savings in adoptive cell transfer-based immunotherapies. Citation Format: Byung-Kwan Jeong, Young-Ae Kim, Jungwook Park, Baknoon Ham, Jiheyong Kim, Chae Lyul Lim, Hee Jin Lee. Integrated analysis of single-cell RNA and VDJ-sequencing data to identify T cell marker expressed on antigen-specific T cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-06.

Linking Altered Nephrogenesis with Hypertension using Single Nuclei RNA Sequencing of Fetal Kidney at E15.5, E16.5 and E17.5 in a Novel Nephron Deficient Rat Model

This study sought to elucidate molecular mechanisms linking altered nephrogenesis in the HSRA rat, a novel model of nephron deficiency, to the development of hypertension and chronic kidney disease (CKD) later in life. The HSRA model gives rise to offspring born with a single kidney (-S) 50-75% of the time. The single kidneys from HSRA-S animals exhibit reduced nephron endowment compared to either kidney from two-kidney offspring (-C). We hypothesize that early molecular factors during development contribute to the observed reduction in nephron number in -S animals. Timed breeding and embryonic kidney isolation from -S and -C animals were performed at gestational days (GD) 15.5, 16.5, or 17.5. Single nuclei RNA sequencing (snRNA-seq) was conducted using n=3 -S kidneys and n=3 -C kidneys for each timepoint. The library was prepared using a 10x Chromium Single Cell 3’ kit, followed by sequencing on an Illumina NextSeq2000. The average number of cells captured per sample was 16,997, yielding an average of 25,335 reads/cell, with a median of 1,512 genes/cell identified. Bioinformatic analysis using Cell Ranger and Seurat (v4) revealed various renal progenitor cell types including metanephric mesenchymal (MM) and ureteric bud cells, alongside tubular, stromal, and glomerular endothelial progenitors. Podocytes emerged by GD17.5, originating from the MM cell population, as evidenced by their extension from this cluster. Global differential expression analysis revealed minimal transcriptional differences between -S and -C fetal kidneys at GD 16.5 and 17.5. However, a down-regulation of ribosomal genes was observed on GD15.5 in the -S group, suggesting premature cellular differentiation contributing to the diminished nephron formation. Markedly, cell type-specific gene expression differences were observed at GD 16.5 and 17.5, highlighting the superior resolution that snRNAseq provides compared to bulk RNA sequencing methods. We are currently performing trajectory analysis using Monocle3 to investigate cellular fate pathways and uncover genes involved during nephron progenitor cell differentiation. These preliminary findings shed light on the cellular and molecular landscape during critical developmental stages in the kidney of the HSRA rat that could link developmental factors affecting nephron number to hypertension and CKD pathologies later in life. This research was generously supported by the Graduate School of Health Sciences at UMMC. MRG is supported by NIH R01HL137673. The UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS, including Molecular Center of Health and Disease-COBRE (P20GM144041-Garrett), Mississippi INBRE (P20GM103476-Flynt), and Cardiorenal and Metabolic Diseases-COBRE (P20GM104357-Hall). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract LB309: Influence of pathological pericytes on PDAC tumor progression and tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in men and women in the U.S., with a 5-year survival rate of 12 %. This poor prognosis is partly due to the aggressive nature of the disease, lack of early detection methods, its complex and dense tumor microenvironment (TME), and lack of effective treatment options. Within the PDAC TME, morphologically aberrant leaky vessels are responsible for hypoxia and impaired immune response, which likely reduces the efficacy of cancer therapies. We and others showed that pericyte coverage significantly correlates with vascular integrity/function and intratumoral hypoxia. Therefore, understanding perivascular heterogeneity and the unique contribution of each phenotype to the TME evolution is a critical step toward personalized treatment options for PDAC. Consequently, we hypothesize that correcting such undesirable phenotype via vascular normalization will alleviate the harsh TME and enhance the treatment efficacy. Our study revealed that tumor-associated pericytes across all PDAC tumor tissues exhibited ectopic αSMA expression up to 10 times higher than normal pericytes. This aberrant pericyte phenotype correlated with vascular leakiness, hypoxia, and vessel leakiness. Our attempt to elucidate the underlying mechanism of pericyte phenotype switching using an in-vitro culture system shows that pancreatic cancer cell-derived extracellular vesicles are a potent inducer of αSMA expression in pericytes present mechanical abnormalities and immune-suppressive features. To further determine the pathological signature of tumor pericytes and identify potential target molecules toward vascular normalization, we performed single-cell RNA sequencing (scRNA-seq). Pericytes were isolated from WT pancreas and KPC tumors at 15-20 weeks to obtain desired viable single-cell population suitable for 10x library preparation and Illumina sequencing.We isolated and barcode at least 5000 cells using the 10X genomics system. Sequencing libraries were prepared using the Chromium Single Cell 3ʹ reagent kit v3. They were sequenced until a sequencing depth of 100,000 reads per cell was achieved, and the data was analyzed using Cell Ranger and various other analytical tools (e.g., Seuret, Conos, ScMerge, singleR). We generated custom scripts from the single-cell data to answer the relevant questions. Sequences were demultiplexed based on barcodes and assigned unique molecular identifiers (UMIs). We visualized clusters using graphing methods, such as t-SNE plots and UMAP clustering. This approach helped identify groups of cells with unique and differential transcriptional landscapes and group them into cellular subtypes based on their gene expression profiles. In conclusion, our study indicates that tumor-associated pericytes undergo phenotype switching under the influence of pancreatic cancer cells, and these aberrant pericytes might contribute to non-optimal vascular integrity and function. Single-cell RNA sequencing helped understand the molecular signature of the pericyte subpopulation, providing a powerful tool to develop novel targeting strategies for vascular normalization. Future work includes exploring the vascular normalization approach by suppressing pericyte phenotype switching to enhance vascular function and chemo and immunotherapeutic efficacy. Citation Format: Vikneshwari Natarajan, Sangdeuk Ha, Jiha Kim. Influence of pathological pericytes on PDAC tumor progression and tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB309.

Abstract 1273: Tumor progression and tumor microenvironment of pancreas cancer ascites revealed by scRNA-seq and spatial transcriptomics

Abstract Peritoneal metastasis of pancreas cancer is one of the ultimate modes of spread in the cancer. Little is known about how cancer cells spread into abdominal cavity and what is different from hematogenous metastasis. It is also not well characterized how tumor microenvironment prevent cancer cells from spreading in abdominal cavity. We performed single-cell RNA-seq analysis and special transcriptomic analysis to clarify the progression and environment of the cancer. We collected and processed ascites samples of 20 PDAC samples from 19 patients. Sixteen ascites samples were obtained from abdominal paracentesis and two cases in the postmortem period. For one patient ascites was collected both pre and postmortem. All but one patient received chemotherapy prior to ascites collection. Routine clinical cytology evaluation of ascites samples from 17 patients indicated it was malignant in 11 patients and nonmalignant in six patients. All ascites samples were purified by gradient centrifuge using Ficoll-Paque Plus to remove erythrocytes and necrotic debris. For the platform, we adopted the 10x Genomics Chromium Single Cell Gene Expression platform for single-cell RNA-seq library preparation. Spatial transcriptomics was performed for eight FFPE samples from the two autopsy patients with paired cancer ascites using 10x Visium. The CytAssist was used to transfer transcripts of pre-sectioned and pre-stained tissues with 11 × 11 mm Visium Capture Area slides. The sample locations were metastatic tumors to bowel wall, rib bone, mesentery, iliac nerve, diaphragm, peritoneum, pelvis, and primary pancreas tumor. A total of 248,263 cells were analyzed in single-cell RNA-seq. Cell population in pancreas cancer ascites consisted of macrophages, lymphocytes, dendritic cells, plasma cells, plasmacytoid dendritic cells, mesothelial cells, and cancer cells. Cancer cells were detected in twelve samples whereas no cancer cells were detected in eight samples. Gene ontology and gene set enrichment analyses showed macrophages in ascites with cancer cells involved neutrophil activation while those in ascites without cancer cells retained antigen presentation capacity and inducing interferon gamma pathway. Mesothelial cells in ascites with cancer cells showed more apoptosis and lymphocyte proliferation while those in ascites without cancers cells triggered fibrosis and mitosis. These data suggest that macrophage and mesothelial cells could be categorized into anti-tumor cells and tumor associated cells. Ongoing investigations are exploring ligand-receptor relationships. We are going to compare the expression profile of tumor cells in ascites fluid and in tissue from various organs by integrating single cell RNA-seq data and special transcriptomics data. Citation Format: Shigeaki Umeda, Elias-Ramzey Karnoub, Fiyinfolu Balogun, Alejandro Jiménez-Sánchez, Christine A. Iacobuzio-Donahue. Tumor progression and tumor microenvironment of pancreas cancer ascites revealed by scRNA-seq and spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1273.

Abstract 7331: Establishing a single-cell eQTL dataset of lung tissues from Asian never-smokers to identify cell-type specific lung cancer susceptibility genes

Abstract Lung cancer is one of the most prevalent and deadly forms of cancer worldwide. Whilst smoking is the main determinant, genetic factors also play a crucial role as genome-wide association studies (GWAS) have identified >50 loci associated with lung cancer risk. Yet, for most of these loci, it is still unknown how they contribute to lung cancer risk. Expression quantitative trait loci (eQTL) studies have been powerful in linking GWAS variants to potential target genes, providing genetic mechanisms underlying common diseases such as cancers. However, the current eQTL resources lack ancestral diversity and are primarily based on bulk tissues. Emerging single-cell eQTL (sc-eQTL) approaches can detect context-specific gene regulation but are mainly of blood samples or cultured cells and still representing European populations. This limits our abilities to test GWAS variants in cancer-relevant cell types as well as in diverse populations. To generate a resource to characterize lung cancer GWAS loci, we are building a lung sc-eQTL dataset of Asian population while addressing common challenges of tissue sc-eQTL. Namely, processing fresh tissue in a population scale is logistically challenging and costly, and epithelial cells (including cell types of lung cancer origin) are vulnerable to the dissociation and freezing/thawing process. To address these issues, we incorporated sample multiplexing and cell type balancing. We collected fresh tumor-distant normal lung tissues from 131 never-smoking Korean women and dissociated them before cryopreservation. We then performed single-cell RNA sequencing (scRNA-seq) using 10x Chromium Single Cell 3’ v3.1 chemistry with multiplexing of ~6 samples/batch. To enrich for epithelial cells, we utilized flow cytometry with surface markers of four major lung cell types (epithelial: EpCAM+/CD45-, immune: EpCAM-/CD45+, endothelial and stromal: EpCAM-/CD45-) before 10X library preparation. Concurrently, we performed DNA genotyping and imputation using matched blood samples. Following scRNA-seq (~36,000 reads/cell) we performed a genotype-based sample demultiplexing using Demuxlet. By integrating Demuxlet and Scrublet, we identified ~89% of the detected cells as singlets. After applying QC to filter empty droplets and low-quality cells, we obtained 428,619 cells or 3,272 cells/patient. Cell annotation guided by Azimuth label transfer using the Human Lung Cell Atlas, identified 28 cell types including 7 of epithelial origins. We will further perform eQTL analyses for individual cell types using pseudo-bulk and LIMIX (linear mixed model) or SAIGE-qtl (poisson regression) methods followed by aggregation across the cell types. By incorporating lung cancer GWAS data, we will identify cell-type specific susceptibility genes. Our dataset will provide a unique resource for lung cancer research. Citation Format: Thong Luong, Erping Long, Jinhu Yin, Bolun Li, Ju Hye Shin, Elelta Sisay, Alexander Kane, Alyxandra Golden, Yoon Soo Chang, Nicholas Banovich, Nathaniel Rothman, Jinyoung Byun, Qing Lan, Christopher Amos, Jianxin Shi, Jin Gu Lee, Eun Young Kim, Jiyeon Choi. Establishing a single-cell eQTL dataset of lung tissues from Asian never-smokers to identify cell-type specific lung cancer susceptibility genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7331.

Molecular characterization of metastatic progression in patient derived xenograft models of malignant uveal melanoma

Aims/Purpose: It has been observed that large UM lesions with significant infiltration of macrophages and CD8+ T cells exhibit specific genetic profiles that increase the risk of liver metastasis. Additionally, recent discoveries of DNA methylation changes in Class 2 tumours undergoing metastasis suggest a potential role of epigenetic alterations in UM progression.Methods: To identify factors involved in UM metastasis, we implanted 25 viable UM tumours (consisting of 11 Class 1 tumours with a good prognosis and 14 Class 2 tumours with a poor prognosis) subcutaneously into immunodeficient NSG mice. From these, we successfully established ten patient‐derived xenografts (PDXs) using Class 2 tumour tissues. Two metastatic PDXs were derived from liver (UM50) and peritoneal (UM58) metastases spontaneously developed on mice.Results: Comprehensive characterization of both primary tumours and PDX tissues was performed using bulk transcriptomics and methylomics techniques. Among the enriched metabolic pathways identified in the liver metastasis‐derived PDX tissue of sample UM50 compared to the PDX tissue from the primary tumour, four were related to epigenetic regulation, including the activation of rRNA expression by ERCC6 and EHMT2, methylation of histones and DNA by Polycomb Repressive Complex 2 (PRC2), regulation of rRNA expression by NAD‐dependent Deacetylase Sirtuin‐1, and the DNA methylation pathway.Conclusions: These findings highlight the potential importance of epigenetic deregulation in UM progression and provide a foundation for future investigations. Further analysis using the Chromium Single Cell ATAC platform to examine chromatin accessibility at the single‐cell level in primary and metastatic PDX tissues of UM50 and UM58 that are ongoing can contribute to unravelling the precise role of epigenetic alterations in UM metastasis. Continued research in this area may lead to the development of novel therapeutic strategies targeting epigenetic mechanisms and improving outcomes for patients with metastatic UM.

Cell Specific Early Dysfunctional Gene Clusters in the APPNL-G-F (APPKI) Alzheimer's Model

Alzheimer's Disease (AD) occurrence is expected to triple by the year 2060. Due to a lack of the understanding of causative mechanisms we are devoid of effective therapies. We have explored the hippocampus and cortex of the 2-month old APPNL-G-F (APPKI) using 10× Genomics Chromium Single Cell Multiome Assay for Transposase Accessible Chromatin (ATAC) + Gene Expression (GEX) to define at the transcriptome level, cell specific alterations in immune, lipid , metabolic and neurodevelopment related gene clusters. Additionally, we have assessed DNA-methylation to uncover related CpG islands and gene promoters.

Single‐nuclei transcriptomic identifies type‐specific neuronal cell vulnerability and non‐neuronal molecular changes in Amnestic and Logopenic Variant Primary Progressive Aphasia Alzheimer’s disease

AbstractBackgroundIndividuals with neuropathological criteria for Alzheimer’s disease (AD) may manifest with atypical clinical syndromes. Past work showed that the neurobiological basis for these differences is related to specific neuronal vulnerabilities for tau pathology. For instance, amnestic cases have a higher burden of neurofibrillary changes in CA1. In contrast, logopenic variant primary progressive aphasia (lvPPA) cases have a higher tau burden in the superior temporal gyrus (STG). Single‐cell technology enables investigations on the molecular basis of differential neuronal vulnerability in AD. Therefore, we investigated factors underlying selective vulnerability using brain samples from individuals with pure AD with different clinical manifestations.MethodsnRNA Sequencing using the Chromium Single Cell 3′ (10X Genomics, USA) on nuclei cells extracted from the CA1 sector and posterior STG of postmortem brain tissue of 24 individuals either meeting pathological criteria for AD (A3B3C3; 12 amnestic and eight lvPPA) and healthy controls (A≤1B≤1C≤1; n = 4) (Table 1, Fig. 1A/B). Bioinformatics analyses were conducted using Cell Ranger and R software. Comparisons between cell subpopulations were conducted with Wald statistical test, and p‐values < .05 were considered significant.ResultAfter quality control, we recovered 98,180 nuclei with a mean of 2,130 genes per nuclei. Upon cross‐sample alignment and t‐stochastic neighborhood embedding clustering (Fig. 1C), we found 21 excitatory neuronal subpopulations (Exc‐sub) in CA1 and 26 in STG, and 22 and 27 inhibitory neuronal subpopulations (Inh‐sub) in CA1 and STG, respectively; 16 astrocytes subpopulations in both areas and 20 microglia subpopulations in CA1 and 17 in STG (Fig. 2). One STG Exc‐sub, expressing CUX2 and LAMP5 genes showed vulnerability in lvPPA patients. Also, one STG Inh‐sub, expressing the ADARB2 gene, showed vulnerability for all AD patients. Furthermore, two astrocytes subpopulations (one in both areas and one in STG) and three STG microglia subpopulations showed proportional composition changes comparing AD patients and HC.ConclusionOur preliminary study identified a vulnerable population of excitatory neurons related to lvPPA. We are conducting validation studies using quantitative pathology to confirm these results. Furthermore, analysis of a higher number of cases is ongoing and will continue to inform on factors associated with neuronal vulnerability.

Characterization of Hematopoietic Stem and Progenitor Cells of PNH Patients Using Single-Cell RNA Sequencing Analysis

The etiology of paroxysmal nocturnal hemoglobinuria (PNH) is somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs), and affected PNH-type cells lack cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs). In PNH, GPI-AP deficient cells clonally expand and occupy a large proportion of mature cells. Several hypotheses have been proposed to explain clonal expansion of PNH-type HSPCs, including reduced sensitivity to apoptosis and escape from immune-mediated cytotoxicity. However, definitive mechanisms remain unclear. To further understand PNH pathophysiology at high multi-dimensional resolution, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from PNH patients. Fresh BM samples were obtained from eight PNH patients (male, n = 2, female, n = 6; median age, 51 [range, 21-67] years), who had large PNH-type granulocyte populations and stable clone sizes (median, 45 [range, 16-99] %), and five age- and sex-matched healthy donors (HDs). PNH-type and wild-type (WT) lineage-CD34+HSPCs were enriched from bone marrow mononuclear cells (BMMNCs) by flow cytometric sorting. Sorted cells were processed through the Chromium Controller (10x genomics, Pleasanton, CA, USA). scRNA-Seq libraries were constructed using the Chromium Single Cell 3' Reagent Kits (10x genomics) according to the manufacturer's instructions. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA, USA). We obtained 81,470 high-quality cells from PNH patients and 56,545 cells from HDs for further analysis. Cells were clustered with Seurat and visualized in UMAP. Cell clusters were assigned to 6 HSPC subsets based on expression of canonical marker genes (Figure 1A). To investigate compositional changes in BMMNCs between PNH patients and HDs, we performed differential abundance analysis with Milo and identified a prominent decrease in hematopoietic stem cells (HSCs) and lymphoid progenitors, as compared to HDs (Figure 1B). We then evaluated differential abundance between PNH-type and WT HSPCs in PNH patients, identifying no significant differences in each HSPC subsets (Figure 1B). These data were consistent with flow cytometric profiling of BMMNCs, similar proportions of PNH-type cells in each HSPC subsets in individual patients despite with profound decrease in PNH-type lymphocytes (Figure 1C). We next sought to investigate skewed gene sets in PNH-type HSPCs using gene set enrichment analysis. Comparison between PNH-type and WT HSPCs identified upregulation of inflammatory signaling pathways and downregulation of the cell-cycling pathways in PNH-type HSPCs, as compared to WT HSPCs (Figure 1D). We made comparisons within individual subsets. In HSCs, genes involved in IFN-γ response and oxidative phosphorylation were upregulated in PNH-type HSCs, while cell-cycling genes (ie. E2F targets and G2M checkpoint) were not dysregulated, as compared to WT HSCs. In summary,no significant skewing in lineage differentiation of PNH-type HSPCs was observed, suggesting PNH-type lymphocytes were negatively selected during the maturation process in lymphoid tissues. Upregulation of the inflammatory and immune response pathways in PNH-type HSPCs indicated a proinflammatory phenotype of PNH-type HSPCs. Upregulation of cell cycle-related pathways was not observed in PNH-type HSCs compared to WT HSCs, consistent with stable PNH clone sizes in our cohort of PNH patients.

Role of Microenvironment in Influencing the Activity of CD19 CAR T Cells in B-Cell Precursor Acute Lymphoblastic Leukemia (B-ALL)

Background: As a therapy based on immune cells, CAR T cells treatment elicits an acute response involving both innate and adaptive immunity which is followed by a process of resolution. A major interest is to identify, within the tumor microenvironment (TME), factors that influence the activity and potency of anti-CD19 CAR T cells. The B-ALL TME provides factors that protect leukemia and, conversely, suppress effector T cells. However, studies on the interaction between the bone marrow (BM) TME and CAR T cells are still limited. We hypothesized that the immunological niche of the BM reacts to CAR T-cell-mediated inflammation by modulating the immune response through the activation of inhibitory pathways and molecules. Methods: We conducted an academic phase I/II study (NCT03389035) with donor-derived anti-CD19 CAR T cells generated with Sleeping Beauty transposon (CARCIK-CD19) in B-ALL patients relapsed after alloHSCT. Hematology laboratory data, CAR T,and CD3+ cell kinetics were analyzed in 43 pediatric and adult B-ALL patients who underwent commercial and investigational anti-CD19 CAR T cell treatment. The scRNA-seq libraries of BM samples were generated using Chromium Single Cell 5′ Reagent Kit of the 10x Genomics (v3.1). Validation was performed in 20 pre-/post- CAR-T matched BM samples by a 30-color flow cytometry panel. Results: Following the peak of expansion, CAR T-cell counts decreased in the third and four-week post-infusion. A similar dynamic of expansion and contraction was observed in CD3+ T cells, total white blood cells (WBC), and monocytes, indicating that a widespread resolution of CAR T-driven inflammation occurs during the first month after infusion. Interestingly, the intensity of the response seems to differ depending on the cell product used. To elucidate the mediators involved in the resolution of CAR T-cell-mediated inflammation, the single-cell transcriptome of patients' BM cells at early time points post-CAR T-cell infusion (1-2 months) was compared to pre-treatment samples at the moment of disease relapse and the infusion products. Eighteen sequencing libraries were analyzed, 6 libraries per patient, for a total of 71407 high-quality cells at an average of 65000 reads per cell. Unbiased clustering of BM cells and infusion products was performed by UMAP embedding. Notably, extensive integration of the dataset coming from the individual patients was observed.The more representative clusters were classified into infusion product, CD4 and CD8 endogenous population, B cells, myeloid cells, pDC, NK, and NK-T cells. We observed profound changes in the composition of BM after CAR T cells infusion compared to pre-treatment samples. Complete disappearance of the B cells associated cluster was observed after treatment. CAR T-cell treatment generated a remodeling of the BM microenvironment, with an increase in the myeloid cells (specifically monocytes), NK, NK-T, and exhausted CD8+ T cell populations. After CAR T-cell infusion, myeloid cells displayed a higher resemblance to myeloid-derived suppressor cells (MDSCs). GeneSet Enrichment Analysis (GSEA) showed significant enrichment in pathways associated with immunosuppression both in the myeloid compartment and in endogenous T cells and in CAR T cells. Of note, the genes involved are also strictly correlated to the generation of terminally exhausted T cells and the emergence of MDSCs. Spectral flow cytometry-based experiments validated our observation of an increase of monocytes, MDSC-like cells, NK, NK-T, and CD8 terminal cells. Modeling intercellular communication using NicheNet suggested the induction of immunosuppressive genes in myeloid cells by both CAR T cells and endogenous T cells. Using the same tool with all niches as the sender and T cells as the receiver, we observed that myeloid cells induced significant signaling in both CAR T cells and endogenous T cells. Conclusions: Through sc-RNAseq and spectral flow cytometry, we have characterized changes in the BM TME after CAR T cell therapy. Specifically, CAR T cells-mediated myeloid activation is associated with pathways of immune dysregulation that may dampen CAR T cell expansion and antagonize the effects of the therapy. Furthermore, these data support the hypothesis that transcriptomic interrogation can infer pathways relevant to the communication between CAR T cells and the TME.

Bone Marrow Interstitial Light-Chain Amyloid and Its Microenvironment

Amyloid deposition and neighboring tissue responses remain poorly understood. Patient series have shown that with Congo red staining of marrow biopsies about 20% of AL patients have interstitial deposits, 40% have vascular and 40% have no amyloid seen and that marrow amyloid is rare in patients with myeloma. (Ann Hematol. 2011;90(1):101-6; Am J Clin Pathol. 2003;120(4):610-6) The constituents of the deposits have been summarized for 65 AL biopsies, a third with extensive interstitial deposits; apolipoproteins E, A-IV and J are prominently present in the examples offered as well as in an extensive summary of the amyloid proteome.(Amyloid. 2022;29(3):156-64; Crit Rev Biochem Mol Biol. 2021;56(5):526-42) With the goal of achieving a better understanding of the impact AL amyloid deposits might have on the marrow microenvironment we undertook a systematic assessment, within the limitations of specimen availability and quality, of the CD138-depleted marrow mononuclear cells (MNC) and marrow and blood plasma from AL patients with (+MA) and without (-MA) interstitial marrow amyloid. Data on the 49 consenting AL patients are archived in the Harvard Dataverse (https://doi.org/10.7910/DVN/TDFMAI). We compared +MA and -MA CD138-depleted MNC from AL patients by gene expression profiling (GEP) on the Illumina platform; reads were mapped to the UCSC hg19 human genome with Tophat 2/Bowtie 2. Normalization and differential expression analyses were performed with Cuffdiff.(Methods Mol Biol. 2016;1374:339-61) We also compared a different set of +MA and -MA CD138-depleted MNC by single-cell RNA-sequencing (scRNA seq) employing the 10X Genomics Chromium Single Cell 3′ method in our core facility and processing the samples for evaluation through CellRanger, Seurat v. 4.0.3 and the Azimuth marrow reference-based method at the 31 cell type resolution. In addition, we compared peripheral blood and marrow plasma from these patients by immunoprecipitation and immunoblot with respect to apolipoproteins and free light chains (FLC) in complex with the erythroid protein TMCC2 and by ELISA for marrow apolipoprotein E and J levels. GEP showed no differential expression of genes for proteolytic enzymes or apolipoproteins between the groups but +MA cases had significantly up-regulated erythroid genes involved in oxygen transport, including transmembrane and coiled-coil domain family 2 ( TMCC2). In +MA marrows at the single-cell level, CD14+ monocytes were increased by 22%, granulocyte-monocyte progenitors decreased by 66%, and erythroid-megakaryocyte progenitors and early and late erythroid progenitors increased up to five-fold. Gene enrichment studies showed that in +MA marrows pathways for TNFα signaling via NFκ-B were significantly active (Figure 1); immune activation and monocyte hypoxia were also significantly enriched. As shown in Figure 2, apolipoprotein J is strongly associated with FLC in blood and E is not, while in +MA marrow plasma apolipoproteins J and E are strongly present in association with FLC. By ELISA, there is also significantly more apolipoprotein E and J in +MA marrow plasma. Our data suggest that erythroid-related activity is enhanced in marrows with interstitial amyloid and that a red blood cell related protein, TMCC2, is associated with apolipoproteins and FLC, possibly providing a loose cargo of chaperones that may abet fibril formation by FLC with predisposed secondary structures whose fragmentation sites are made optimally accessible by the loose complex; sites in the FR4 and 5' constant region of AL FLC may be highly vulnerable if exposed in such fashion to perivisceral proteases associated with matrix remodeling. The marrow microenvironment is clearly impacted by the presence of interstitial amyloid. In HSC from +MA marrows there is increased signaling in pathways that affect lineage commitments such as TNF-α/NF-κB and PI3 kinase/AKT/mTOR. In monocytes from +MA specimens there is evidence of modulation of NF-kB. Both alterations are possibly due to apolipoprotein E, although there is no evidence that monocyte transcription of APO E in +MA CD138-depleted marrow MNC is increased. In conclusion, marrows with interstitial amyloid provide opportunities to study amyloid's impact on cellular behavior, regenerative activity and chaperone involvement in amyloid deposition.

Determinants of Response to Anti CD-19 CAR-T Cells for Diffuse Large B-Cell Lymphoma in Pre-Treatment Peripheral Blood Mononuclear Cells Using Single-Cell RNA-Seq

Background: Chimeric antigen receptor T (CAR-T) cell therapy has become the standard of care in patients with relapsed/refractory diffuse large cell lymphoma (R/R DLBCL), providing durable remission in 40% of patients. Lack of response is likely to be attributed to inter- and intra-patient variability, including PBMC heterogeneity and apheresis material composition, CAR-T infusion product, in-vivo expansion and activity of CAR-T cells and the development of tumor-intrinsic mechanisms. Current methods for evaluating CAR-T cell activation and potency have limited predictive value for clinical outcomes. To address this challenge, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) obtained from patients with R/R LBCL patients at the time of apheresis for the manufacturing of anti-CD19 CAR-T cell therapy. Methods: Between 10/2020 and 03/2022, 31 patients who underwent lymphopheresis were enrolled. Samples were obtained - 1) At day of lymphopheresis from peripheral blood; 2) On the day of infusion of CAR-T from the product; and 3) On day +7 after infusion from peripheral blood. All patients had day +30 PET-CT. In addition, PBMCs samples from healthy donors were used as control to analyze baseline characteristics compared to the DLBCL patients. PBMCs from patients of healthy donors were purified from frozen blood samples by density gradient separation, stained using human CD45 antibody and loaded onto a 10x Chromium system. libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit. The data was processed and analyzed using the Seurat R package. The study was approved by the local Ethic committee. Results: Patient characteristics: 31 RR DLBCL patients (42% females; median age 72, range 47-81 years), all treated with CAR-T cells therapy as third line of therapy, participated in the study. All patients underwent lymphopheresis, followed by CAR-T production. Disease status at lymphodepletion was complete remission (CR, n=4, 13%), partial remission (n=6, 19%), and progressive disease (n=21, 68%) and patients received either tisagenlecleucel (n=20, 65%) or axicabtagene ciloleucel (n=11, 35%) . PET scan performed on day 30 post treatment revealed that 68% and 32% obtained CR, and PD, respectively. scRNA analysis revealed differences in PBMC composition between healthy donors and LBCL patients (Figure 1A), with lower levels of naïve and memory T cells and elevated levels of activated monocytes in LBCL patients, suggesting a shift towards an inflamed and pre-dysfunctional immune state. Categorization of LBCL patients into responders (PR/CR on day +30 PET-CT) and non-responders (PD on day +30 PET-CT) revealed differences in immune cell subsets between responders and non-responders (Figure 2A), with enriched inflammation-related pathways in non-responders, In contrast, responders exhibited a distinct healthy-like immune profiles, particularly high levels of naive T cells, similar to that obtained in their age matched healthy controls. Of note, non-responders showed alterations in immune cell subsets, including increased levels of T regulatory cells and effector NK cells. Furthermore, non-responders demonstrated a prominent inflammatory immune profile, characterized by gene pathways related to inflammation and activation, such as type I and III IFN-G, and TNFb response (Figure 1B). Notably, an IFN-G score was significantly higher in non-responder's monocytes subpopulations, compared to responders. Conclusions: These findings indicate that the success of CAR-T therapy could be determined by the molecular characteristics and subpopulation distribution of each patient's PBMC. The diverse immune profiles of DLBCL patients might impact the effectiveness of their immune cells in generating successful CAR-T products or providing a conducive environment for CAR T cells to combat cancer. Furthermore, these findings emphasize the potential of scRNA-seq in evaluating PBMC from CAR-T patients prior to treatment, allowing the potential calculation of an “immune potency score” associated with the probability of a positive outcome from CAR-T treatment of DLBCL patients. This score could potentially be used by oncologists in decision-making processes to determine the appropriate candidates for CAR-T therapy.

Abstract 14843: Unveiling the Phenotype of Smooth Muscle Foam Cells in Human Atherosclerotic Lesions: Insights From Bulk and Single Cell RNA Sequencing

Background and Aims: Our previous studies of human coronary atherosclerosis and ApoE-/- mice indicated that at least 50% and ~70% of foam cells are of smooth muscle cell (SMC) origin, respectively. We also found that SMCs generate a distinct class of foam cells, due to several gene expression and regulatory differences compared to macrophage foam cells. In the current investigation we utilized transcriptomic approaches to identify and characterize the phenotype of SMC foam cells of human coronary plaques and in SMCs treated with aggregated LDL (ag-LDL). Methods: Sections of fresh human coronary artery were subjected to gentle digestion and the isolated cells used for single-cell RNA Sequencing (scRNA-Seq) (n=3) using 10X Genomics Chromium Single Cell 3’ Reagent Kits v3 Technology. Human vascular SMCs were treated with 100 μg/mL ag-LDL for 24 h (n=3) and stained with the fluorescent lipid probe BODIPY493/503. BODIPY high SMCs were collected and subjected to bulk RNA sequencing along with non-treated SMCs as control. Results: Single cell RNA sequencing of cells isolated from human coronary arteries followed by unsupervised Seurat-based clustering (R version 4.2.0) identified 15 distinct clusters including 8 SMC clusters with varying degrees of differentiation and macrophage foam and non-foam cells. The top 70 upregulated genes in ag-LDL treated SMCs were used to identify clusters corresponding to SMC foam cells in the scRNA-seq dataset. We found that these genes are mostly enriched in 2 clusters of less differentiated SMCs. SMC and macrophage foam cell clusters display distinct characteristics. SMC foam cell clusters exhibit upregulation of complement and coagulation cascades, as well as ECM-receptor interaction genes. In contrast, the macrophage foam cell cluster shows enrichment of proinflammatory genes, genes involved in the response to unfolded protein, and both pro- and anti-apoptotic genes. Conclusion: Our studies provide novel tools to investigate the nature of SMC foam cells in human atherosclerosis and may provide unique markers for SMC foam cells. Gaining a comprehensive insight of the significance and characteristics of SMC foam cells is crucial in elucidating their ultimate role and potential as a therapeutic target in atherosclerosis.

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (<10%; micro-MK low) and high rate of micro-MK (>40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.

Single-Cell RNA Sequencing Analysis of the Early Postnatal Mouse Lens Epithelium.

The lens epithelium maintains the overall health of the organ. We used single-cell RNA sequencing (scRNA-seq) technology to assess transcriptional heterogeneity between cells in the postnatal day 2 (P2) epithelium and identify distinct epithelial cell subtypes. Analysis of these data was used to better understand lens growth, differentiation, and homeostasis on P2. scRNA-seq on P2 mouse lenses was performed using the 10x Genomics Chromium Single Cell 3' Kit (v3.1) and short-read Illumina sequencing. Sequence alignment and preprocessing of data were conducted using 10x Genomics Cell Ranger software. Seurat was employed for preprocessing, quality control, dimensionality reduction, and cell clustering, and Monocle was utilized for trajectory analysis to understand the developmental progression of the lens cells. CellChat and GO analyses were used to explore cell-cell communication networks and signaling interactions. Lens epithelial cells (LECs) were divided into seven subclusters, classified by specific gene markers. The expression of crystallin, cell-cycle, and metabolic genes was not uniform, indicating distinct functional roles of LECs. Trajectory analysis predicted a bifurcation of differentiating and cycling cells from an Igfbp5+ progenitor pool. We also identified heterogeneity in signaling molecules and pathways, suggesting that cycling and progenitor subclusters have prominent roles in coordinating crosstalk. scRNA-seq corroborated many known markers of epithelial differentiation and proliferation while providing further insight into the pathways and genes directing these processes. Interestingly, we demonstrated that the developing epithelium can be divided into distinct subpopulations. These clusters reflect the transcriptionally diverse roles of the epithelium in proliferation, signaling, and maintenance.

High-Throughput Antibody Discovery Using Barcode Enabled Antigen Mapping (BEAM

Abstract Multi-analyte single cell technologies have potential to greatly accelerate antibody discovery by identifying antigen-specific B cells. Barcode Enabled Antigen Mapping (BEAM) provides a high-throughput, multimodal analysis of antigen-bound B cells (BEAM-Ab) using 10x Genomics Chromium Single Cell Immune Profiling v2 Solution and novel computational approaches to discover B-cell receptor (BCR) sequences for further functional characterization. To demonstrate the antigen sensitivity of BEAM-Ab, we spiked in 5% Hen Egg Lysozyme (HEL) and 5% gp120 transgenic B cells in 90% wild type non-transgenic splenocytes. The cells were screened using a panel of barcoded antigens and then CD19+PE+ (antigen+) B cells were isolated using flow cytometry. Single cell gene expression, BCR, and antigen barcode analysis indicated clonotype specificity to the respective antigens in the sorted cells. Furthermore, we demonstrated BEAM-Ab specificity by analyzing a convalescent COVID patient sample against an antigen panel containing five different COVID antigens and a negative control. Analysis of this COVID sample suggests the clonal expansion of B cells recognizing the ectodomain of the wild type SARS-CoV2 spike protein, receptor binding domain of spike protein, and the ectodomain of the D614G mutant spike protein. We did not observe any clonal expansion to Omicron-specific antigen or the non-specific negative control in our dataset. Our data demonstrates that BEAM-Ab is an effective antibody discovery tool. BEAM-Ab unlocks the ability to rapidly screen large numbers of samples with an accurate single cell resolution and antigen specificity, with the entire workflow generating the candidate sequences just one week after sample processing.