Single-cell RNA-seq Studies Research Articles

Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders such as addiction and Parkinson's disease. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter (VMAT2), vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44 %), VGAT+ (37 %) and VGLUT2+ (41 %) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54 %), fewer were VGAT+ (42 %), and VGLUT2+ neurons were least abundant (16 %). Moreover, 20 % of VTA neurons and 10 % of SNc neurons expressed more than one vesicular transporter, including 45 % of VGLUT2+ neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

HDAC Inhibitors recapitulate Human Disease-Associated Microglia Signatures in vitro.

Disease-associated microglia (DAM), initially described in mouse models of neurodegenerative diseases, have been classified into two related states; starting from a TREM2-independent DAM1 state to a TREM2 dependent state termed DAM2, with each state being characterized by the expression of specific marker genes 1 . Recently, single-cell (sc)RNA-Seq studies have reported the existence of DAMs in humans 2-6 ; however, whether DAMs play beneficial or detrimental roles in the context of neurodegeneration is still under debate 7,8 . Here, we present a pharmacological approach to mimic human DAM in vitro by exposing different human microglia models to selected histone deacetylase (HDAC) inhibitors. We also provide an initial functional characterization of our model system, showing a specific increase of amyloid beta phagocytosis along with a reduction of MCP-1 secretion. Additionally, we report an increase in MITF expression, a transcription factor previously described to drive expression towards the DAM phenotype. We further identify CADM1 , LIPA and SCIN as DAM- marker genes shared across various proposed DAM signatures and in our model systems. Overall, our strategy for targeted microglial polarization bears great potential to further explore human DAM function and biology.

Tissue-specific functions of MSCs are linked to homeostatic muscle maintenance and alter with aging.

Mesenchymal stromal cells (MSCs), also known as fibro-adipogenic progenitors, play a critical role in muscle maintenance and sarcopenia development. Although analogous MSCs are present in various tissues, recent single-cell RNA-seq studies have revealed the inter-tissue heterogeneity of MSCs. However, the functional significance of MSC heterogeneity and its role in aging remain unclear. Here, we investigated the properties of MSCs and their age-related changes in seven mouse tissues through histological, cell culture, and genetic examinations. The tissue of origin had a greater impact on the MSC transcriptome than aging. By first analyzing age-related changes, we found that Kera is exclusively expressed in muscle MSCs and significantly down-regulated by aging. Kera knockout mice recapitulated some sarcopenic phenotypes including reduced muscle mass and specific force, revealing the functional importance of Kera in the maintenance of muscle youth. These results suggest that MSCs have tissue-specific supportive functions and that deterioration in these functions may trigger tissue aging.

Placozoan secretory cell types implicated in feeding, innate immunity and regulation of behavior.

Placozoa are millimeter-sized, flat, irregularly shaped ciliated animals that crawl on surfaces in warm oceans feeding on biofilms, which they digest externally. They stand out from other animals due to their simple body plans. They lack organs, body cavities, muscles and a nervous system and have only seven broadly defined morphological cell types, each with a unique distribution. Analyses of single cell transcriptomes of four species of placozoans revealed greater diversity of secretory cell types than evident from morphological studies, but the locations of many of these new cell types were unknown and it was unclear which morphological cell types they represent. Furthermore, there were contradictions between the conclusions of previous studies and the single cell RNAseq studies. To address these issues, we used mRNA probes for genes encoding secretory products expressed in different metacells in Trichoplax adhaerens to localize cells in whole mounts and in dissociated cell cultures, where their morphological features could be visualized and identified. The nature and functions of their secretory granules were further investigated with electron microscopic techniques and by imaging secretion in live animals during feeding episodes. We found that two cell types participate in disintegrating prey, one resembling a lytic cell type in mammals and another combining features of zymogen gland cells and enterocytes. We identified secretory epithelial cells expressing glycoproteins or short peptides implicated in defense. We located seven peptidergic cell types and two types of mucocytes. Our findings reveal mechanisms that placozoans use to feed and protect themselves from pathogens and clues about neuropeptidergic signaling. We compare placozoan secretory cell types with cell types in other animal phyla to gain insight about general evolutionary trends in cell type diversification, as well as pathways leading to the emergence of synapomorphies.

Long-term Tolerance to Islet Transplantation via Targeted Reduction of beta cell-specific T cells.

Activated beta-cell reactive CD4 + and CD8 + T effector cells undergo a profound DNA-damage response which is targetable by small molecule inhibitors of the p53 and cell cycle pathways that lead to apoptosis. The use of a combination of MDM2 and WEE1 inhibitors, which termed "p53 potentiation with checkpoint abrogation" (PPCA), conferred significant therapeutic efficacy in treating mouse models of new onset T1D. Specific targeting of these T effector cells by PPCA results in a loss of inflammatory T cell subsets, notably proliferation CD4 + Th0 and Th1 subsets and CD8 + T effector memory cells, as determined by single cell RNA-seq studies with the preservation of T regulatory cells. When autologous islet grafts are given to established diabetic NOD mice, a single course of PPCA results in long-term islet graft acceptance, restoration of normoglycemia and loss of beta cell specific CD4 + and CD8 + T cells. PPCA shows promise as a potential means of estimating islet graft tolerance in T1D recipients of islet graft transplantation.

SCIntRuler: Guiding the integration of multiple single-cell RNA-seq datasets with a novel statistical metric.

The growing number of single-cell RNA-seq (scRNA-seq) studies highlights the potential benefits of integrating multiple datasets, such as augmenting sample sizes and enhancing analytical robustness. Inherent diversity and batch discrepancies within samples or across studies continue to pose significant challenges for computational analyses. Questions persist in practice, lacking definitive answers: Should we use a specific integration method or opt for simply merging the datasets during joint analysis? Among all the existing data integration methods, which one is more suitable in specific scenarios? To fill the gap, we introduce SCIntRuler, a novel statistical metric for guiding the integration of multiple scRNA-seq datasets. SCIntRuler helps researchers make informed decisions regarding the necessity of data integration and the selection of an appropriate integration method. Our simulations and real data applications demonstrate that SCIntRuler streamlines decision-making processes and facilitates the analysis of diverse scRNA-seq datasets under varying contexts, thereby alleviating the complexities associated with the integration of heterogeneous scRNA-seq datasets. The implementation of our method is available on CRAN as an open-source R package with a user- friendly manual available: https://cloud.r-project.org/web/packages/SCIntRuler/index.html.

Proportion and distribution of neurotransmitter-defined cell types in the ventral tegmental area and substantia nigra pars compacta.

Most studies on the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) have focused on dopamine neurons and their role in processes such as motivation, learning, movement, and associated disorders such as addiction. However there has been increasing attention on other VTA and SNc cell types that release GABA, glutamate, or a combination of neurotransmitters. Yet the relative distributions and proportions of neurotransmitter-defined cell types across VTA and SNc has remained unclear. Here, we used fluorescent in situ hybridization in male and female mice to label VTA and SNc neurons that expressed mRNA encoding the canonical vesicular transporters for dopamine, GABA, or glutamate: vesicular monoamine transporter (VMAT2), vesicular GABA transporter (VGAT), and vesicular glutamate transporter (VGLUT2). Within VTA, we found that no one type was particularly more abundant, instead we observed similar numbers of VMAT2+ (44%), VGAT+ (37%) and VGLUT2+ (41%) neurons. In SNc we found that a slight majority of neurons expressed VMAT2 (54%), fewer were VGAT+ (42%), and VGLUT2+ neurons were least abundant (16%). Moreover, 20% of VTA neurons and 10% of SNc neurons expressed more than one vesicular transporter, including 45% of VGLUT2+ neurons. We also assessed within VTA and SNc subregions and found remarkable heterogeneity in cell-type composition. And by quantifying density across both anterior-posterior and medial-lateral axes we generated heatmaps to visualize the distribution of each cell type. Our data complement recent single-cell RNAseq studies and support a more diverse landscape of neurotransmitter-defined cell types in VTA and SNc than is typically appreciated.

Ploidy inference from single-cell data: application to human and mouse cell atlases.

Ploidy is relevant to numerous biological phenomena, including development, metabolism, and tissue regeneration. Single-cell RNA-seq and other omics studies are revolutionizing our understanding of biology, yet they have largely overlooked ploidy. This is likely due to the additional assay step required for ploidy measurement. Here, we developed a statistical method to infer ploidy from single-cell ATAC-seq data, addressing this gap. When applied to data from human and mouse cell atlases, our method enabled systematic detection of polyploidy across diverse cell types. This method allows for the integration of ploidy analysis into single-cell studies. Additionally, this method can be adapted to detect the proliferating stage in the cell cycle and copy number variations in cancer cells. The software is implemented as the scPloidy package of the R software and is freely available from CRAN.

Abstract 6614: Integrating single-cell RNA-seq and large genome-wide association study data to identify colorectal cancer cell-of-origin populations

Abstract Background: Single-cell RNA sequencing (scRNA-seq) enables the precise identification of distinct cell populations and the measurement of gene expression within each population. Recently, integrating scRNA-seq and genome-wide association study (GWAS) data has shown promise in identifying cell-of-origin populations in human diseases. Colorectal cancer (CRC) progresses through a phenotypic continuum from normal colon to adenoma, and ultimately CRC. However, the specific disease cell-of-origin populations underlying each stage of CRC development at a single-cell resolution have not been well studied. Additionally, while transcriptome-wide association studies (TWAS) have identified more than 200 putative risk genes in CRC, the precise disease cell-of-origin populations associated with these genes remain elusive. Materials and methods: We analyzed scRNA-seq datasets from 31 normal colon, 8 colon serrated polyps, and 15 conventional adenoma tissues of individuals participated the Colorectal Molecular Atlas Project (COLON MAP). We next conducted an integrative analysis of the aforementioned scRNA-seq data, with extensive epigenomics data and summary statistics from a large CRC GWAS conducted among European ancestry populations (78,473 CRC cases and 107,143 controls) using single-cell disease relevance score (scDRS), to investigate cell-of-origin populations underlying the CRC development. We conducted correlation analysis between gene expression and individual cell risk scores derived from scDRS. Additionally, we performed differential expression analysis comparing disease cell-of-origin populations with other cell populations at each stage. Results: We identified multiple cell-of-origin populations associated with CRC, including absorptive cells (ABS) (P < 1 × 10-4) and goblet cells (GOB) (P < 7 × 10-3) in the normal colon, ABS (P < 2 × 10-3), enteroendocrine cells (EE) (P < 0.04), and serrated-specific cells (SSC) (P < 1 × 10-4) in serrated polyps, and ABS (P < 9 × 10-3) and SSC (P < 6 × 10-4) in adenomas. Among the previously reported 205 risk genes from TWAS, a majority show high correlations of their expressions with the risk scores of the single cells (median value R2 = 0.07, 0.14, 0.137 at normal colon, serrated polyps, adenoma, respectively). Moreover, 57 of these TWAS-reported genes (27.8% of 205 genes) exhibited evidence of differential expression in disease cell-of-origin populations compared to other cell populations at a nominal p-value < 0.05 in at least one stage (Binomial test, P < 0.01 for all). Conclusion: This study reveals the cell-of-origin populations that influence the development of CRC, and provides evidence of the role of risk genes in these populations. These findings are crucial for identifying causal genes and understanding the cellular mechanisms that drive the biology and etiology of CRC. Citation Format: Chao Li, Zhishan Chen, Qing Li, Jungyoon Choi, Quanhu Sheng, Wanqing Wen, Xiang Shu, Xiao-ou Shu, Jirong Long, Qiuyin Cai, Bhuminder Singh, Martha J. Shrubsole, Ken Lau, Wei Zheng, Xingyi Guo. Integrating single-cell RNA-seq and large genome-wide association study data to identify colorectal cancer cell-of-origin populations [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 6614.

Abstract 3708: Characterization of EpCAM-positive cells for specific detection of disseminated cancer cells in bone marrow of early breast cancer patients

Abstract Obviously, breast cancer cells that disseminated from the primary tumor before surgical resection are responsible for metastasis. However, detection of disseminated cancer cells (DCC) in breast cancer patients during minimal residual disease is still difficult, impeding the study of early systemic cancer and the extraction of clinically relevant diagnostic or therapeutic information. Others and we have shown previously that EpCAM is a suitable DCC detection marker, however not entirely specific in bone marrow. To improve DCC detection, we here compared different methods to enrich DCC exploiting information from our ongoing single cell RNA-seq study. Based on our ongoing scRNA-seq study (see poster of Elia Raya), we generated a qPCR assay to classify candidate DCC based on an expression signature of four genes. This assay categorizes plasma cell-like EpCAM+ cells from healthy donors and candidate DCC from breast cancer patients with high fidelity. With this assay at hand, we compared two enrichment strategies: depletion of CD11b, CD319 and Glycophorin A positive cells from density gradient enriched mononuclear cells (MNC) vs. positive selection of EpCAM+ cells and subsequent addition of markers that separate out DCC among EpCAM+ cells. For this we applied 10x genomics profiling of EpCAM+ cells from bone marrow. We depleted and screened the bone marrow of 221 patients suffering from non-metastatic early breast cancer for EpCAM-positive cells. From 87 patient samples we could isolate EpCAM-positive cells. We performed single cell RNA-sequencing on 64 EpCAM-positive cells from 54 patients of which 84% were classified as cancer cells and 16% of bone marrow origin. Applying the selected gene signature, 76% of isolated cells were classified as DCC, 16% were undefined and 8% as non-cancer bone marrow cell (NCC). Upon positive selection of EpCAM+ cells implementing additional markers, we isolated 53 cells from 14 patients. Of these, 68% showed a DCC or DCC-like-signature, 15 % a NCC and 17% an undefined profile. To get a better understanding of these EpCAM-positive confounder populations we currently perform a scRNA-seq profiling using 10X on the enriched EpCAM+ cells. Our data shows that EpCAM is a promising marker to identify DCCs in the bone marrow of early cancer patients, but it also indicates that we find several groups of confounder cells. To differentiate between these cell types during staining and in our qPCR assay we are currently testing additional markers. Citation Format: Jonas Roth, Elia Raya, Sarah Hücker, Huiqin Koerkel-Qu, Stephan Seitz, Christoph Klein. Characterization of EpCAM-positive cells for specific detection of disseminated cancer cells in bone marrow of early breast cancer patients [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 3708.

Immune Response Associated Gene Signatures in Aortic Dissection Compared to Aortic Aneurysm.

Thoracic aortic dissections (TAD) are life-threatening events mostly requiring immediate surgical treatment. Although dissections mainly occur independently of thoracic aortic aneurysms (TAA), both share a high comorbidity. There are several indications for an involvement of the immune system in the development of TAD, just as in TAA. Nevertheless, specific disease-relevant genes, biomolecular processes, and immune-specific phenotypes remain unknown. RNA from isolated aortic smooth muscle cells from TAD (n = 4), TAA (n = 3), and control patients were analyzed using microarray-based technologies. Additionally, three publicly available bulk RNA-seq studies of TAD (n = 23) and controls (n = 17) and one single-cell RNA-seq study of TAA (n = 8) and controls (n = 3) were analyzed. Differentially expressed genes were identified and used to identify affected pathways in TAD. Five selected genes were validated by quantitative real-time polymerase chain reaction (PCR). We identified 37 genes that were significantly dysregulated in at least three TAD studies-24 of them were not shown to be associated with TAD, yet. Gene ontology analysis showed that immune response was significantly affected. Five of the genes (CCL2, RNASE2, HAVCR2, CXCL8, and IL6R) were revealed as core genes that affect immune response in TAD. We compared the gene expression of those genes to TAA and found that CXCL8, IL6R, and potentially also CCL2 were upregulated in TAD. The identified immune-related genes showed TAD-specificity, independent of possible pre-existing comorbidities like TAA. So, these genes represent potential biomarkers and therapeutic targets linked to the immune response in acute TAD. Additionally, we identified a set of differentially expressed genes that represents a resource for further studies.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Human Pluripotent Stem Cells Derived Endothelial Cells Repair Choroidal Ischemia.

Choroidal atrophy is a common fundus pathological change closely related to the development of age-related macular degeneration (AMD), retinitis pigmentosa, and pathological myopia. Studies suggest that choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first cells lost in choroidal atrophy. It is found that endothelial cells derived from human pluripotent stem cells (hPSC-ECs) through the MESP1+ mesodermal progenitor stage express CECs-specific markers and can integrate into choriocapillaris. Single-cell RNA-seq (scRNA-seq) studies show that hPSC-ECs upregulate angiogenesis and immune-modulatory and neural protective genes after interacting with ex vivo ischemic choroid. In a rat model of choroidal ischemia (CI), transplantation of hPSC-ECs into the suprachoroidal space increases choroid thickness and vasculature density. Close-up examination shows that engrafted hPSC-ECs integrate with all layers of rat choroidal vessels and last 90 days. Remarkably, EC transplantation improves the visual function of CI rats. The work demonstrates that hPSC-ECs can be used to repair choroidal ischemia in the animal model, which may lead to a new therapy to alleviate choroidal atrophy implicated in dry AMD, pathological myopia, and other ocular diseases.

Imaging Mass Cytometry Reveals a Highly Heterogenous Cell Makeup of the Follicular Lymphoma Tumour Microenvironment

The highly variable clinical course and response to treatment of follicular lymphoma (FL) is strongly influenced by the tumor microenvironment (TME). While both pro- and anti-tumor effects depend on cell-cell interactions within the TME, these mechanisms remain incompletely understood. Bulk (Dave et al. NEJM 2004) and single-cell RNA-seq studies have identified signatures associated with prognosis, e.g., ‘T cell depleted’ microenvironments that are linked with inferior clinical outcomes (Han et al. Blood Cancer Discov. 2022). While such research has shed light on the TME, single-cell studies often lack spatial context and conventional immunohistochemistry is limited by a low number of parameters. Imaging mass cytometry (IMC) allows multiplexed analysis of up to 40 markers in tissue simultaneously, offering a unique opportunity to study the spatial features of single cells within the TME, informing both disease biology and predictors of clinical outcome. To our knowledge, IMC has more commonly been applied to solid malignancies. FL presents unique challenges in this remit, such as its morphologic variability and phenotyping cells within highly structured, dense lymph-node tissue composed of inherent tight-knit microenvironments. To analyse FL tissue by IMC, we first optimized a 31-plex panel to identify key immune and non-immune lineages including tumor cells, T and NK cells, macrophages, fibroblasts, dendritic, endothelial and reticular cells. Additionally, we wished to identify functional states such as memory, effector or regulatory phenotypes in addition to other parameters including activation, exhaustion and proliferation. Formalin-fixed paraffin embedded (FFPE) biopsies from 100 untreated FL patients enrolled on the Gallium trial (NCT01332968) of frontline chemo-immunotherapy, were subjected to IMC. In total, 381 highly multiplexed images were acquired; 13 were excluded due to suboptimal image quality. Following image pre-processing and deep-learning based cell segmentation, we analysed >3.5 million cells across 368 high-dimensional images (mean=9659 cells, SD=4600). To effectively phenotype large numbers of cells, we constructed a workflow incorporating ‘ASTIR’ (Geuenich et al. Cell Syst. 2021) - a publicly available marker-based algorithm - and cell clustering to identify population subsets. We identified 95 cell populations including Lymphoma subsets (variable CD10, CD11c, CD80 expression), CD4+ and CD8+ T cell subsets (memory, naïve, exhausted etc) and a range of non-lymphoid cells including myeloid and stromal cells. Of particular interest, we found a CD4+ CD3+ PD1+ ICOS+ population which are likely T follicular helper cells (Tfh). On average, Tfh-like cells constitute 1.9% of cells per image (SD=3.1%), although we a found considerable range in their presence. Given previous associations with Tfh cells and FL prognosis we are investigating this variability in further detail. In addition, we have identified ~30 samples enriched for CD57+ T cells, a phenotype suggestive of functional senescence. Amongst substantial heterogeneity of identified cell phenotypes (Fig A), we have identified variable sample sets characterised by enrichment or depletion of i) Lymphoma vs. T-cells (Fig B) and ii) Macrophage vs. T-cell subsets, consistent with previous single cell studies. Here, we report the use of IMC to examine the TME of untreated FL, observing marked heterogeneity of cell phenotypes across FL. Future work seeks to examine prognostic associations between the frequency of cell subsets and their spatial interactions, with this clinically annotated dataset derived from the front-line GALLIUM study. This work will provide a detailed characterization of how the spatial context of FL TME influences disease behaviour, response to treatment and survival. Given the high-throughput and high-plex nature of this study, ultimately, we aim to distil this multitude of information into condensed biomarker(s), to provide potential insights into enhancing diagnostic and/or prognostic capabilities. Therefore, we aim to derive meaningful clinical correlates associated to outcome that can be ultimately translated into practice.

Hematopoietic Stem and Progenitor Cells Acquire an Inflammatory Memory of the Acute Myeloid Leukemia Niche

Recent studies suggest that acute myeloid leukemia (AML) generates inflammation in the bone marrow (BM) niche to an extent that compromises the function of healthy hematopoietic stem and progenitor cells (HSPC) and impacts patient prognosis. AML xenograft studies have identified several pro-inflammatory mediators (IL-6, IL-1b) that are secreted into the BM niche by leukemic blasts. Prior work by our laboratory also showed contributions involving other AML-secreted factors, in regulating HSPCs in the leukemic niche. HSPC are cellular components of the innate immune system, but the full extent of their inflammatory adaptation by AML is unknown. To overcome some of the experimental constraints inherent in the xenograft approach, we first established non-conditioned congenic grafts of C1498 murine AML cells in C57B/6 recipients. Assessment of the BM revealed that, even at low AML burden, these animals contained a range of elevated pro-inflammatory factors. Single cell RNA-Seq studies of healthy HSPCs, obtained concurrently at sacrifice confirmed an inflammatory transcriptional signature in HSC and MPP populations, with GSEA enrichment for interferon-alpha and -gamma signaling pathways. These observations led us to hypothesize that AML-experienced (inflammation-primed) healthy HSPCs may acquire a durable long-term inflammatory memory that shapes their response to subsequent stimulation during relapse or infection. To test this hypothesis, we adoptively transferred FACS-sorted healthy AML-experienced HSPCs from C1498-engrafted mice (HSPC AML) into secondary recipients. HSPCs transplanted from PBS-injected recipients (HSPC PBS) serve as controls. To assess whether HSPC AML would respond to immune challenge more aggressively compared to HSPC PBS, we exposed these animals with a single-injection of lipopolysaccharide (LPS), followed by tissue harvest and analysis after 24 hours. Bulk RNA-Seq analysis of HSPC AML from the LPS-challenged recipients showed amplified GSEA signatures for tumor necrosis factor- and NF-kB signaling pathways compared to HSPC PBS. In addition, LPS-challenged HSPC AML showed amplified expression by several krüppel-like factors (KLF) genes. Analysis for transposase-accessible chromatin loci (ATAC-Seq), concurrently obtained from LPS-challenged HSPC AML echoed the enrichment of KLF motifs as well. Collectively, the C1498 model suggests that initial AML exposure programs HSPC AML for an amplified inflammatory and differentiation program. To further ascertain disease relevance and reproducibility we turned to a doxycycline-inducible (i)MLL-AF9 AML model. Given the low leukemic burden in the BM of C1498-grafted animals, we wanted to test the hypothesis that secreted factors accounted, at least in part, for the inflammatory memory. Indeed, injection of AML secreted factors from iMLL-AF9 models revealed a strong inflammatory signature in bulk RNA-Seq studies. To determine if innate immune priming in HSPC can be directly mediated by AML crosstalk, we exposed ex vivo-expanded HSPCs to purified secreted factors (SF AML) from (i)MLL-AF9 AML blasts, followed by a secondary inflammatory challenge with LPS seven days later. Gene expression analysis of SF AML-experienced LPS-challenged HSPCs confirmed myeloid-skewed progenitor differentiation and increased expression of inflammatory targets. Together, our data show for the first time that phenotypically normal, AML-experienced HSPCs carry a durable inflammatory legacy whereby primary exposure to AML secreted factors alters their response to secondary stimuli. Our experiments phenocopy observations of trained immunity in other experimental systems, and suggest the acquisition of an innate immune memory in the AML niche. Immune priming may impact the role of these HSPC in the context of inflammation associated with aging, or clones with predisposing molecular lesions such as TET2 or DNMT3A.

CCRG-02. DEVELOPMENTALLY PARTITIONED PROLIFERATIVE COMPARTMENTS IN GLIOBLASTOMA

Abstract It is now well established from single-cell RNA-seq studies that glioblastoma (GBM) tumors are complex, maligned neuro-developmental ecosystems harboring diverse tumor cell types. Neoplastic cells can resemble astrocytes, neural progenitors, oligodendrocyte progenitor cells, mesenchymal cells, and radial glial cells that contribute to tumor growth and homeostasis in specific ways. However, GBM single-cell data sets have failed to produce general models for transitions in and out of specific developmental and proliferative states in tumors. One reason is that human GBM tumors do not neatly resolve into developmental hierarchies. Here we focused on modeling GBM tumor cellular heterogeneity by defining "proliferative compartments" in single-cell transcriptomic data derived from primary tumors and early passage tumorsphere cultures. Previously, we observed that each tumor cell entering S-phase has a unique developmental signature that can be leveraged to define broader partitioned proliferative compartments (PPCs) with distinct developmental gene expression and genomic alteration patterns. Thus, we extracted the S-phase cells from a tumor, defined the proliferative compartments in that tumor using de novo clustering, determined the marker genes for each compartment, and defined the broader PPCs across tumors by de novo clustering based on similarity in marker genes. From a cohort of six tumors we observed eight broader PPCs and found that tumors can contain as many as five PPCs or as few as two. Because tumor growth and recurrence both require cell proliferation, we propose that patient-specific PPCs represent the engines of GBM progression.

T-Cell Signaling Mediates the Epigenetic Priming of Germinal Center B-Cell Plasticity and Stem Functionality

Chemo-immunotherapy can prolong life expectancy of patients with Diffuse Large B-cell Lymphomas (DLBCL). However, the emergence of donor-derived lymphoma cases after stem cell transplantation and the relapse of a third of DLBCL patients, strongly suggests the existence of persisting lymphoma repopulating cells (LRC) with stem-like functionality. This apparently controversial concept arises from the fact that DLBCLs originate from mature rather than progenitor cells. Identifying the biological and epigenetic features of these elusive LRC, and how they arise from normal germinal center (GC) B cells, is therefore critical and remains underexplored. Although fully mature, GC B cells naturally manifest remarkable phenotypic plasticity as compared to other cell types. We hypothesized that this plasticity reflects stem-like programming and functionality. Along these lines, we found that GC B cells are uniquely enriched for pluripotent stem cell transcriptional signatures, and increased chromatin accessibility at stem cell enhancers. We reasoned that the capacity to undergo induced Pluripotent Stem Cells (iPSC) reprogramming could serve as a surrogate assay for stem-like plasticity. Therefore, we investigated the potential of GC B cells to form iPSCs using a doxycycline-dependent mouse strain that enables the inducible expression of Yamanaka transcription factors (TF) in any given cell. Strikingly, GC B cells isolated from our mice manifested a 10-fold (p<0.0001) increase in iPSC reprogramming as compared to other mature B cells. GC B cells are a heterogenous mix of functionally distinct subpopulations. Using single cell RNA-seq and iPSC assays in sorted cells, we noted that the rare population (~3%) of GC B cells selected by T-cell help manifested the stem-like transcriptional profile and iPSC reprogramming phenotype. Moreover, blocking T-cell help during the GC reaction in vivo using CD40 blockade abrogated iPSC forming capacity. Lymphoma-associated EZH2 Y641F mutations impair GC B-cell interactions with T FH cells due to aberrant repression of immune synapse genes. Accordingly, EZH2 Y641F GC B cells manifested loss of the iPSC phenotype. T-cell help induces MYC in GC B cells. However, induction of MYC in mature naïve B cells did not confer them with iPSC potential indicating that other mechanisms must be at play. Lymphoma-associated Btg1 Q36Hmutations confer fitness in GC B cells by enhancing their response to T-cell help. Reciprocal to EZH2 Y641F, Btg1 Q36Hmutant GC B cells manifested further significant increase in iPSC formation. Performing multi-ome (simultaneous single-cell RNAseq and ATACseq) studies in GC B cells revealed massive gain in chromatin accessibility of bona fide stem cell super-enhancers in GC B cells that received T-cell help. There was also a reduced accessibility at genes that maintain B-cell lineage (e.g. Pax5) and GC phenotypes (e.g. Foxo1). Finally, lymphoma-associated Histone 1 mutations induce upregulation of stem cell programs in GCs and could enhance iPSC reprogramming in fibroblasts. We therefore hypothesized that H1 deficiency would overcome the GC dependency on T-cells for their plasticity phenotype. Indeed, increased iPSC formation occurred across all GC subpopulations in H1 C/E-/- (HIST1C/HIST1E knockout) mice. We speculate that restricting plasticity to B cells under selection by T cells, limits the potential for GC B cells to acquire high levels of plasticity, hence reducing the potential for these cells to initiate lymphomas. Consistent with this notion, we found that DLBCL patients enriched for these stem cell signatures manifested inferior clinical outcomes.

Integrative single-cell meta-analysis reveals disease-relevant vascular cell states and markers in human atherosclerosis

Coronary artery disease (CAD) is characterized by atherosclerotic plaque formation in the arterial wall. CAD progression involves complex interactions and phenotypic plasticity among vascular and immune cell lineages. Single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures, but human cell phenotypes remain controversial. Here, we perform an integrated meta-analysis of 22 scRNA-seq libraries to generate a comprehensive map of human atherosclerosis with 118,578 cells. Besides characterizing granular cell-type diversity and communication, we leverage this atlas to provide insights into smooth muscle cell (SMC) modulation. We integrate genome-wide association study data and uncover a critical role for modulated SMC phenotypes in CAD, myocardial infarction, and coronary calcification. Finally, we identify fibromyocyte/fibrochondrogenic SMC markers (LTBP1 and CRTAC1) as proxies of atherosclerosis progression and validate these through omics and spatial imaging analyses. Altogether, we create a unified atlas of human atherosclerosis informing cell state-specific mechanistic and translational studies of cardiovascular diseases.

Scellpam: an R package/C++ library to perform parallel partitioning around medoids on scRNAseq data sets

Background Partitioning around medoids (PAM) is one of the most widely used and successful clustering method in many fields. One of its key advantages is that it only requires a distance or a dissimilarity between the individuals, and the fact that cluster centers are actual points in the data set means they can be taken as reliable representatives of their classes. However, its wider application is hampered by the large amount of memory needed to store the distance matrix (quadratic on the number of individuals) and also by the high computational cost of computing such distance matrix and, less importantly, by the cost of the clustering algorithm itself.Results Therefore, new software has been provided that addresses these issues. This software, provided under GPL license and usable as either an R package or a C++ library, calculates in parallel the distance matrix for different distances/dissimilarities (L_1, L_2, Pearson, cosine and weighted Euclidean) and also implements a parallel fast version of PAM (FASTPAM1) using any data type to reduce memory usage. Moreover, the parallel implementation uses all the cores available in modern computers which greatly reduces the execution time. Besides its general application, the software is especially useful for processing data of single cell experiments. It has been tested in problems including clustering of single cell experiments with up to 289,000 cells with the expression of about 29,000 genes per cell.Conclusions Comparisons with other current packages in terms of execution time have been made. The method greatly outperforms the available R packages for distance matrix calculation and also improves the packages that implement the PAM itself. The software is available as an R package at https://CRAN.R-project.org/package=scellpam and as C++ libraries at https://github.com/JdMDE/jmatlib and https://github.com/JdMDE/ppamlib The package is useful for single cell RNA-seq studies but it is also applicable in other contexts where clustering of large data sets is required.

Immune checkpoint blockade induces distinct alterations in the microenvironments of primary and metastatic brain tumors.

In comparison with responses in recurrent glioblastoma (rGBM), the intracranial response of brain metastases (BrM) to immune checkpoint blockade (ICB) is less well studied. Here, we present an integrated single-cell RNA-Seq (scRNA-Seq) study of 19 ICB-naive and 9 ICB-treated BrM samples from our own and published data sets. We compared them with our previously published scRNA-Seq data from rGBM and found that ICB led to more prominent T cell infiltration into BrM than rGBM. These BrM-infiltrating T cells exhibited a tumor-specific phenotype and displayed greater activated/exhausted features. We also used multiplex immunofluorescence and spatial transcriptomics to reveal that ICB reduced a distinct CD206+ macrophage population in the perivascular space, which may modulate T cell entry into BrM. Furthermore, we identified a subset of progenitor exhausted T cells that correlated with longer overall survival in BrM patients. Our study provides a comprehensive immune cellular landscape of ICB's effect on metastatic brain tumors and offers insights into potential strategies for improving ICB efficacy for brain tumor patients.