Single-cell Transcriptome Profiling Research Articles

Single-Cell RNA Sequencing Reveals Dysregulated POSTN+WNT5A+ Fibroblast Subclusters in Prurigo Nodularis

Prurigo nodularis (PN) is an intensely pruritic, inflammatory skin disease with a poorly understood pathogenesis. We performed single-cell transcriptomic profiling of 28,695 lesional and nonlesional PN cells. Lesional PN has increased dysregulated fibroblasts (FBs) and myofibroblasts. FBs in lesional PN were shifted toward a cancer-associated FB-like phenotype, with POSTN+WNT5A+ cancer-associated FBs increased in PN and similarly so in squamous cell carcinoma. A multicenter cohort study revealed an increased risk of squamous cell carcinoma and cancer-associated FB-associated malignancies (breast and colorectal) in patients with PN. Systemic fibroproliferative diseases (renal sclerosis and idiopathic pulmonary fibrosis) were upregulated in patients with PN. Ligand-receptor analyses demonstrated an FB neuronal axis with FB-derived WNT5A and periostin interactions with neuronal receptors melanoma cell adhesion molecule and ITGAV. These findings identify a pathogenic and targetable POSTN+WNT5A+ FB subpopulation that may predispose cancer-associated FB-associated malignancies in patients with PN.

Abstract A104: Targeting RABL3 to disrupt KRAS trafficking and activity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers with limited treatment options. Nearly all PDAC harbors a KRAS mutation, and oncogenic KRAS signaling is a key driver. Localization of the KRAS protein to the plasma membrane is absolutely required for downstream tumorigenic activities, making trafficking of KRAS to the plasma membrane a compelling target for treatment. In a familial pancreatic cancer syndrome with a striking five cases of PDAC, we recently discovered a rare, inherited gain-of-function mutation in the gene RABL3 that impacts the kinetics of KRAS intracellular trafficking. RABL3 was found to indirectly interact with KRAS, and the familial RABL3 mutation accelerates trafficking of KRAS to the plasma membrane. Here, we present pre-clinical experiments evaluating the therapeutic potential of targeting RABL3 to disrupt KRAS trafficking and activity. To achieve inducible knockdown of RABL3, we have engineered KPCY cells (tumor cells derived from KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre; Rosa26YFP/YFP mice) to express a doxycycline-inducible shRNA targeting RABL3. In vitro, RABL3 knockdown results in robust suppression of KPCY cell proliferation. Moreover, the knockdown of RABL3 abrogated tumor growth of KPCY cells in immune-competent subcutaneous and orthotopic pancreatic cancer mouse models. Single-cell transcriptome profiling of tumors revealed immune infiltration of T cells, B cells, macrophages, and dendritic cells following RABL3 knockdown, suggesting that RABL3 knockdown creates vulnerabilities seen with suppression of KRAS activity. Validating these findings in a human context, we find a significantly elevated level of RABL3 expression across multiple human PDAC cell lines, consistent with a driver role in PDAC and a critical role in regulating KRAS trafficking. Similarly, analysis of human datasets revealed elevated RABL3 transcript levels in PDAC TCGA specimens compared to matched normal tissue as well as in PDAC patient-derived organoids compared to the GTEx cohort of normal pancreas tissues. Furthermore, there is a trend for worse patient survival with higher RABL3 expression levels. Therefore, to evaluate the potential of RABL3 knockdown to improve the potency, efficacy, and toxicity when combined with other PDAC therapeutic targets, we are currently testing the impacts of RABL3 knockdown on the therapeutic profile of newly available small-molecular KRAS inhibitors. Our findings provide pre-clinical support for targeting RABL3 to disrupt KRAS intracellular trafficking and inhibit KRAS activity for the treatment of PDAC. Citation Format: Woo-Jeong Jeong, Katherine J. Aney, Ethan Chen, Annan Yang, Giselle A. Uribe, Andrew Aguirre, Sahar Nissim. Targeting RABL3 to disrupt KRAS trafficking and activity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A104.

Ribogenesis boosts controlled by HEATR1-MYC interplay promote transition into brain tumour growth

Cell commitment to tumourigenesis and the onset of uncontrolled growth are critical determinants in cancer development but the early events directing tumour initiating cell (TIC) fate remain unclear. We reveal a single-cell transcriptome profile of brain TICs transitioning into tumour growth using the brain tumour (brat) neural stem cell-based Drosophila model. Prominent changes in metabolic and proteostasis-associated processes including ribogenesis are identified. Increased ribogenesis is a known cell adaptation in established tumours. Here we propose that brain TICs boost ribogenesis prior to tumour growth. In brat-deficient TICs, we show that this dramatic change is mediated by upregulated HEAT-Repeat Containing 1 (HEATR1) to promote ribosomal RNA generation, TIC enlargement and onset of overgrowth. High HEATR1 expression correlates with poor glioma patient survival and patient-derived glioblastoma stem cells rely on HEATR1 for enhanced ribogenesis and tumourigenic potential. Finally, we show that HEATR1 binds the master growth regulator MYC, promotes its nucleolar localisation and appears required for MYC-driven ribogenesis, suggesting a mechanism co-opted in ribogenesis reprogramming during early brain TIC development.

Synovium and infrapatellar fat pad share common mesenchymal progenitors and undergo coordinated changes in osteoarthritis.

Osteoarthritis (OA) affects multiple tissues in the knee joint, including the synovium and intra-articular adipose tissue (IAAT) that are attached to each other. However, whether these two tissues share the same progenitor cells and hence function as a single unit in joint homeostasis and diseases is largely unknown. Single-cell transcriptomic profiling of synovium and infrapatellar fat pad (IFP), the largest IAAT, from control and OA mice revealed five mesenchymal clusters and predicted mesenchymal progenitor cells (MPCs) as the common progenitors for other cells: synovial lining fibroblasts (SLFs), myofibroblasts (MFs), and preadipocytes 1 and 2. Histologic examination of joints in reporter mice having Dpp4-CreER and Prg4-CreER that label MPCs and SLFs, respectively, demonstrated that Dpp4+ MPCs reside in the synovial sublining layer and give rise to Prg4+ SLFs and Perilipin+ adipocytes during growth and OA progression. After OA injury, both MPCs and SLFs gave rise to MFs, which remained in the thickened synovium at later stages of OA. In culture, Dpp4+ MPCs possessed mesenchymal progenitor properties, such as proliferation and multilineage differentiation. In contrast, Prg4+ SLFs did not contribute to adipocytes in IFP and Prg4+ cells barely grew in vitro. Taken together, we demonstrate that the synovium and joint fat pad are one integrated functional tissue sharing common mesenchymal progenitors and undergoing coordinated changes during OA progression.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with “secretory”, “contractile” and “vascular” transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.

Up-regulated SLC25A39 promotes cell growth and metastasis via regulating ROS production in colorectal cancer.

Background: The mitochondrial transporter SLC25A39 has been implicated in the import of mitochondrial glutathione (mGSH) from the cytoplasm, crucial for mitigating oxidative stress and preserving mitochondrial function. Despite the well-established involvement of mitochondria in cancer, the functional impact of SLC25A39 on CRC progression remains elusive. Methods: The mRNA and protein expressions were detected by PCR, immunohistochemistry, and Western blot, respectively. Cell activity, cell proliferation, colony formation, and apoptosis were measured by CCK8 assay, EdU incorporation assay, plated colony formation assay, and flow cytometry, respectively. Cell migration was detected by wound healing and transwell chamber assay. The tumor microenvironment (TME), immune checkpoint molecules, and drug sensitivity of CRC patients were investigated using R language, GraphPad Prism 8 and online databases. Results: Here, we report a significant upregulation of SLC25A39 expression in CRC. Functional assays revealed that overexpression of SLC25A39 promoted CRC cell proliferation and migration while inhibiting apoptosis. Conversely, SLC25A39 knockdown suppressed cell growth and migration while enhancing apoptosis in vitro. Additionally, reduced SLC25A39 expression attenuated tumor growth in xenograft models. Mechanistically, elevated SLC25A39 levels correlated with reduced reactive oxygen species (ROS) accumulation in CRC. Furthermore, bioinformatic analyses unveiled the high SLC25A39 levels was associated with decreased expression of immune checkpoints and reduced responsiveness to immunotherapy. Single-cell transcriptomic profiling identified diverse cellular expression patterns of SLC25A39 and related immune regulators. Lastly, drug sensitivity analysis indicated potential therapeutic avenues targeting SLC25A39 in CRC. Conclusion Our findings underscore the pivotal role of SLC25A39 in CRC progression and suggest its candidacy as a therapeutic target in CRC management.

Tumor cell senescence-induced macrophage CD73 expression is a critical metabolic immune checkpoint in the aging tumor microenvironment.

Background: The role of senescent cells in the tumor microenvironment (TME) is usually bilateral, and diverse therapeutic approaches, such as radiotherapy and chemotherapy, can induce cellular senescence. Cellular interactions are widespread in the TME, and tumor cells reprogram immune cells metabolically by producing metabolites. However, how senescent cells remodel the metabolism of TME remains unclear. This study aimed to explore precise targets to enhance senescent cells-induced anti-tumor immunity from a metabolic perspective. Methods: The in vivo senescence model was induced by 8 Gy×3 radiotherapy or cisplatin chemotherapy, and the in vitro model was induced by 10 Gy-irradiation or cisplatin treatment. Metabonomic analysis and ELISA assay on tumor interstitial fluid were performed for metabolites screening. Marker expression and immune cell infiltration in the TME were analyzed by flow cytometry. Cell co-culture system and senescence-conditioned medium were used for crosstalk validation in vitro. RNA sequencing and rescue experiments were conducted for mechanism excavation. Immunofluorescence staining and single-cell transcriptome profiling analysis were performed for clinical validation. Results: We innovatively reveal the metabolic landscape of the senescent TME, characterized with the elevation of adenosine. It is attributed to the senescent tumor cell-induced CD73 upregulation of tumor-associated macrophages (TAMs). CD73 expression in TAMs is evoked by SASP-related pro-inflammatory cytokines, especially IL-6, and regulated by JAK/STAT3 pathway. Consistently, a positive correlation between tumor cells senescence and TAMs CD73 expression is identified in lung cancer clinical specimens and databases. Lastly, blocking CD73 in a senescent background suppresses tumors and activates CD8+ T cell-mediated antitumor immunity. Conclusions: TAMs expressed CD73 contributes significantly to the adenosine accumulation in the senescent TME, suggesting targeting CD73 is a novel synergistic anti-tumor strategy in the aging microenvironment.

Single-cell profiling of the pig cecum at various developmental stages.

The gastrointestinal tract is essential for food digestion, nutrient absorption, waste elimination, and microbial defense. Single-cell transcriptome profiling of the intestinal tract has greatly enriched our understanding of cellular diversity, functional heterogeneity, and their importance in intestinal tract development and disease. Although such profiling has been extensively conducted in humans and mice, the single-cell gene expression landscape of the pig cecum remains unexplored. Here, single-cell RNA sequencing was performed on 45 572 cells obtained from seven cecal samples in pigs at four different developmental stages (days (D) 30, 42, 150, and 730). Analysis revealed 12 major cell types and 38 subtypes, as well as their distinctive genes, transcription factors, and regulons, many of which were conserved in humans. An increase in the relative proportions of CD8 + T and Granzyme A (low expression) natural killer T cells (GZMA low NKT) cells and a decrease in the relative proportions of epithelial stem cells, Tregs, RHEX + T cells, and plasmacytoid dendritic cells (pDCs) were noted across the developmental stages. Moreover, the post-weaning period exhibited an up-regulation in mitochondrial genes, COX2 and ND2, as well as genes involved in immune activation in multiple cell types. Cell-cell crosstalk analysis indicated that IBP6 + fibroblasts were the main signal senders at D30, whereas IBP6 - fibroblasts assumed this role at the other stages. NKT cells established interactions with epithelial cells and IBP6 + fibroblasts in the D730 cecum through mediation of GZMA-F2RL1/F2RL2 pairs. This study provides valuable insights into cellular heterogeneity and function in the pig cecum at different development stages.

A Large-Scale Meta-Analysis Reveals Positive Feedback between Macrophages and T Cells That Sensitizes Tumors to Immunotherapy.

Identification of robust immunogenomic connections, particularly macrophage T-cell interactions, in a large-scale pan-cancer meta-analysis and development of a predictive model for immunotherapy response that outperformed existing models could facilitate clinical decision-making.

Single-cell sequencing identifies inflammation-promoting fibroblast-neutrophil interaction in peri-implantitis.

To reveal the cellular composition and molecular environment of the periodontal and peri-implant inflammatory infiltrates through a single-cell sequencing technique, which may explain the pathological difference between these two diseases. A special focus was placed on the phenotypes and potential roles of neutrophils and fibroblasts in peri-implant/periodontal tissue immunity. High-throughput single-cell transcriptomic profiling of peri-implant tissues from patients with peri-implantitis as well as periodontal tissues from patients with periodontitis and healthy donors was performed. Immunofluorescence analysis was carried out to further validate the identified cell subtypes and their involvement in peri-implantitis and periodontitis. Based on our single-cell resolution analysis, a quantified proportional increase of neutrophil (Neu) subtypes was shown in peri-implantitis. Among these, a predominance of Neutro_CXCR2 was revealed. We also found the involvement of inflammation-promoting fibroblasts as well as a predominance of CXCL8+ fibroblast-CXCR2+ neutrophil interaction in peri-implantitis. Our study indicated that the predominance of CXCL8+ fibroblast-CXCR2+ neutrophil interaction might underline the enhanced host response in peri-implantitis compared with periodontitis. This information offers a molecular basis by which fibroblast and neutrophil subtypes might be diagnostically and therapeutically targeted in peri-implantitis.

Single-cell transcriptome profiling of primary tumors and paired organoids of pancreatobiliary cancer

Single-cell RNA-seq (scRNA-seq) and cancer organoid model have shown promise in investigating tumor microenvironment heterogeneity and facilitating chemotherapeutic drug testing to inform treatment selection. It is still unknown whether the scRNA-seq results based on organoid can faithfully reflect the heterogeneity of primary pancreatobiliary cancer. To reveal the similarities and differences between primary tumors and their matched organoids at transcriptome level, we conducted scRNA-seq for paired primary tumors and organoids from one cholangiocarcinoma (CCA) and two pancreatic ductal adenocarcinoma (PDAC) patients. We identified inter-patient and intra-tumor heterogeneity and found that the organoids retained copy number variation (CNV) patterns of primary tumors. There was no significant difference in cancer stem cell (CSC) properties between the primary tumors and the organoids, whereas organoid from one PDAC case had increased mesenchymal-score and decreased epithelial-score compared with the primary tumors. All organoids showed a transition tendency from the classical subtype to the basal-like subtype in the transcriptional level. Organoids and primary tumors differed in metabolic and unfolded protein response (UPR) signatures. In addition, we revealed the heterogeneity of cancer associated fibroblasts (CAFs) and T cells, and explored the developmental trajectory of T cells. Our findings facilitate further understanding of organoid model and confirm its application prospects in pancreatobiliary cancer.

Dictionary of immune responses to cytokines at single-cell resolution

Cytokines mediate cell–cell communication in the immune system and represent important therapeutic targets1–3. A myriad of studies have highlighted their central role in immune function4–13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine–cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1β induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell–cell communication networks in any immune response.

Immune and oxidative stress disorder in ovulation-dysfunction women revealed by single-cell transcriptome.

Ovulation dysfunction is now a widespread cause of infertility around the world. Although the impact of immune cells in human reproduction has been widely investigated, systematic understanding of the changes of the immune atlas under female ovulation remain less understood. Here, we generated single cell transcriptomic profiles of 80,689 PBMCs in three representative statuses of ovulation dysfunction, i.e., polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and menopause (MENO), and identified totally 7 major cell types and 25 subsets of cells. Our study revealed distinct cluster distributions of immune cells among individuals of ovulation disorders and health. In patients with ovulation dysfunction, we observed a significant reduction in populations of naïve CD8 T cells and effector memory CD4 T cells, whereas circulating NK cells and regulatory NK cells increased. Our results highlight the significant contribution of cDC-mediated signaling pathways to the overall inflammatory response within ovulation disorders. Furthermore, our data demonstrated a significant upregulation of oxidative stress in patients with ovulation disorder. Overall, our study gave a deeper insight into the mechanism of PCOS, POI, and menopause, which may contribute to the better diagnosis and treatments of these ovulatory disorder.

Abstract A136: Inferring therapeutic vulnerability within tumors through integration of pan-cancer cell line and single-cell transcriptomic profiles

Abstract Single-cell sequencing techniques have greatly advanced our current understanding of intratumoral heterogeneity through identifying tumor subpopulations with distinct biologies and therapeutic responses. However, translating biological differences into treatment strategies is challenging, as we still lack tools to facilitate efficient drug discovery that tackles heterogeneous tumors. One key step in development of such approaches centers around accurate prediction of drug response at the single-cell level to offer therapeutic options to specific cell subpopulations. Here, we present a transparent computational framework (nicknamed scIDUC) to predict therapeutic efficacies on an individual-cell basis by integrating single-cell transcriptomic profiles with large, data-rich pan-cancer cell line screening datasets. Our method detects shared expression patterns between the two data sources and utilizes such information to project cellular drug response. This method achieves high accuracy, with predicted sensitivities easily able to separate cells into their true cellular drug resistance status as measured by effect size (Cohen's d > 1.0); this holds when using single-cell RNA-seq from both cell line and in vivo models. More importantly, we examine our method’s utility with three distinct prospective tests, and in each our predicted results are accurate and mirrored biological expectations. In the first two tests, we investigated predicting drugs for cell subpopulations that are resistant to standard-of-care (SOC) therapies due to intrinsic resistance or effects of tumor microenvironments. In both, our results showed high consistency with results from the original studies. In the third test, we generated SOC therapy resistant cell lines, used scIDUC to identify drugs predicted effective on the resistant line, and validated the predictions with in vitro experiments. Together, scIDUC quickly and directly translates scRNA-seq data into meaningful cellular drug response for individual cells, displaying the potential to be used by researchers as a first-line tool for nuanced and heterogeneity-aware drug discovery. Citation Format: Weijie Zhang, Danielle Maeser, Adam Lee, Yingbo Huang, Robert F Gruener, Sampreeti Jena, Anand G Patel, R. Stephanie Huang. Inferring therapeutic vulnerability within tumors through integration of pan-cancer cell line and single-cell transcriptomic profiles [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A136.

Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers

Circulating Tcells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor Tcells. By single-cell transcriptomic profiling of 36 neoantigen-specific Tcell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ Tcells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific Tcells revealed that NeoTCRPBL Tcells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor Tcells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

East Asian-specific and cross-ancestry genome-wide meta-analyses provide mechanistic insights into peptic ulcer disease

Peptic ulcer disease (PUD) refers to acid-induced injury of the digestive tract, occurring mainly in the stomach (gastric ulcer (GU)) or duodenum (duodenal ulcer (DU)). In the present study, we conducted a large-scale, cross-ancestry meta-analysis of PUD combining genome-wide association studies with Japanese and European studies (52,032 cases and 905,344 controls), and discovered 25 new loci highly concordant across ancestries. An examination of GU and DU genetic architecture demonstrated that GUs shared the same risk loci as DUs, although with smaller genetic effect sizes and higher polygenicity than DUs, indicating higher heterogeneity of GUs. Helicobacter pylori (HP)-stratified analysis found an HP-related host genetic locus. Integrative analyses using bulk and single-cell transcriptome profiles highlighted the genetic factors of PUD being enriched in the highly expressed genes in stomach tissues, especially in somatostatin-producing D cells. Our results provide genetic evidence that gastrointestinal cell differentiations and hormone regulations are critical in PUD etiology.

Characterizing Inferred Functional and Phenotypical States of Newly Diagnosed Acute Myeloid Leukemia from Single Cell Transcriptomic Profiling

Background: Acute myeloid leukemia (AML) is a heterogeneous disease, marked by leukemia stem cells (LSCs), which significantly contribute to treatment resistance and dismal prognosis. While appreciable efforts have been made to study the diversity of functional and phenotypic states of LSCs, integrating these states from inferred single cell profiling has not been explored. Moreover, the spatial distribution and variability of hierarchical leukemic states within diverse cytogenetic groups, and their potential connection to transcriptional profiles, remain understudied, highlighting a gap in our understanding of this complex disease. Methods: To investigate the heterogeneity across different cytogenetics,we performed single cell RNA profiling (scRNA) of bone marrow (BM) mononulear cells from 20 newly diagnosed adult AML patients with diploid (n=7), del5/5q (n=5), del7/7q (n=5) and double deletion (del5/5q & del7/7q; n=3). A total of 111,130 cells passed quality assessment, of which 56,168 (50.5%) cells were identified as AML cells by integrating flow cytometry, immunohistochemistry, copy number variation, and FISH with the scRNA expression profiles. Functional states of AML cells were predicted by a recently proposed hierarchy of AML LSCs representing distinct maturation states (Zeng et al Nat Med 2022). Phenotypical states were annotated by Symphony using a healthy BM and validated in newly sequenced healthy BM samples. We also applied deconvolution scRNA analysis with spatial GeoMx profiling to identify spatial location of primitive versus differentiated AML cells. Results: Differential gene expression and gene set enrichment analysis (GSEA) revealed transcriptional and biological diversity across cytogenetic groups. Del7/7q cells showed high expression of human leukocyte antigen (HLA) genes and CD74, consistent with enrichment for a inflammatory phenotype revealed by GSEA. Del5/5q group demonstrated enrichment in proliferative pathways and heat shock proteins. Some diploid cells showed high expression of monocytic markers, while the others were highly expressing GMP markers, indicating the co-existence of different subclones. Differentiation states of leukemic cells are reflected in the hierarchy of cellular compositions. There is a diverse spectrum of cell types within each cytogenetic group and each patient. However, AML cells from diploid patients were enriched for GMP-like and monocyte-like cells, while phenotypically HSC-like cells showed an incremental trend in the sequence of diploid, del5/5q, del7/7q, and double deletion (Figure 1A). The inferred functional LSC states in our data also showed diverse distribution across cytogenetic groups, with nondiploid cells harboring more LSC. We also evaluated the biological differences of cytogenetic groups within each phenotypical and functional subset. Our data revealed consistent upregulation of proliferation in del5/5q and inflammation in del7/7q with double deletion preserving both features. However, diploid cells were downregulated in almost all. These data suggest cytogenetics as a major driver of ITH even for phenotypically or functionally similar AML cells. By correlating phenotypical axis with LSC states, we found a shared transcriptional program in LSC-like states across phenotypical axis except for Mono-like and DC-like cells, demonstrating the broad distribution of inferred functional LSC spanning the phenotypical axis from primitive to differentiated cells (Figure 1B). This was also observed in other LSC signatures, suggesting that AML cells with LSC capacity exist across the phenotypic of HSC-like cells to more phenotypically differentiated cells. We then performed spatial deconvolution using GeoMx of CD34 + or CD68 + myeloid areas of interest using the phenotypical signature. GeoMx revealed that primitive cells were more likely to be proximal to bone, while more differentiated states were enriched in distal region from the bone, indicating a spatial migration at time of differentiation. Conclusions: ScRNA profiling of newly diagnosed AML revealed the existence of heterogeneous LSC functional states underlying varying degrees of differentiation. We identified cytogenetics as a major source of ITH. Also, primitive cells were likely to localize to bone regions. These findings provide new insights in developing specific therapeutic strategies to improve clinical outcomes.

Defining the Drivers of Idelalisib-Related Early Autoimmune Toxicity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) patients with disease resistant to BTK and BCL-2 inhibitors can potentially be treated with PI3K inhibitors to overcome resistance and improve outcomes. PI3K inhibitors such as idelalisib and duvelisib have shown promising efficacy in CLL patients, but severe toxicities remain a significant hurdle to their implementation in the clinic (PMID: 24615777). By investigating the molecular mechanisms that lead to severe early autoimmune toxicity with idelalisib, we hope to predict or prevent toxicity, thus expanding the use of this efficacious PI3K inhibitor class of drugs. To this end, the Brown lab showed that idelalisib-induced hepatitis is autoimmune. CyTOF analysis in CLL patients with early severe toxicity in a front-line idelalisib clinical trial cohort showed a significant decrease in CD4+ T regulatory cells (Treg) with a relative shift in favor of T effectors in peripheral blood. In addition, the Brown lab showed that the Th17 pathway is more active at baseline, with a further increase in patients with toxicity (PMID: 27247136, 36732326). Since the CYTOF data is restricted to fewer mostly T-cell markers in our studies, an unbiased single cell RNA sequencing (scRNA-seq) approach is warranted to explore the full effects of idelalisib on immune cells and CLL cells in patients with and without autoimmune toxicity. The question remains what molecular mechanisms drive the idelalisib-induced decrease in the Treg population, its effect on natural killer (NK) and CD8 T-cell function, and the activation of the Th17 pathway in CLL patients with early toxicity. We therefore carried out the first systematic effort to understand the mechanisms of idelalisib toxicity in molecular detail using combined scRNA and scTCR-seq. In this study, we profile T-cell enriched PBMCs of matched pre-treatment and on-idelalisib treatment samples from CLL patients with early severe toxicity and without any toxicity (n=4 CLL patients from each group, a total of 16 samples) using 10X single cell transcriptomic and TCR profiling. Raw sequencing data files were processed using the Cell Ranger suite (version 7.1.0) from 10x Genomics. Sample identification based on SNP information was done using the Popscle suite. Seurat package (version 4.9.9.9) was used for the single cell analysis. After filtering cells with fewer than 600 features, less than 1000 UMIs, mitochondrial content higher than 15%, 51,487 cells were left for the downstream analysis. To explore transcriptional heterogeneity and perform initial cell clustering, principal component analysis and nonlinear dimensional reduction using UMAP were applied using 20 dimensions. Leiden algorithm was used for cluster identification. Cell types were identified using Azimuth with the PBMC reference dataset and confirmed with expression plots. The initial observations of the cell proportions show that baseline samples from patients with early idelalisib toxicity have fewer Tregs, NK, and CD4+ cytotoxic T (CTL) cells compared to patients without toxicity. In addition, we observed a decline in Tregs, gamma-delta (γδ) T, and CD4+ CTL cells and a concurrent increase in proliferative CD4+ and CD8+ T-cells and proliferative NK on idelalisib treatment in the early toxicity group. On the contrary, CD4+ CTL and CD4+ T effector memory (TEM) cells are enriched with idelalisib therapy in patients without toxicities. At the same time, more CD4+ T-Cell Memory (TCM), γδ T, CD8+ naive T, CD8+ TCM, and Mucosal-associated invariant T (MAIT) cells are present in pretreatment samples from patients with toxicity compared to patients without toxicity. Furthermore, proliferative T-cells are depleted with idelalisib therapy in patients without toxicity whereas they are enriched in patients with early toxicity. To further characterize idelalisib-induced depletion of Tregs, we reclustered them to identify distinct subgroups and found particular clusters of Treg were depleted in patients with early toxicity. In the non-toxicity group, more diversity of Tregs was identified with depletion of some clusters but also an increase in a cluster. At present, we are integrating the scRNA & scTCR-seq data to characterize the cellular diversity and heterogeneous phenotypic states of the T-cell populations and updated results unraveling molecular mechanisms that drive early severe toxicity in CLL patients with idelalisib treatment will be presented at the meeting.

High Dimensional Single-Cell Profiling Identifies Immune and Metabolic Heterogeneity in Immune Thrombocytopenia

Introduction Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by excessive platelet destruction and decreased platelet production. Ongoing efforts to characterize the pathophysiology of ITP have yielded certain insights, but the details of the immune and metabolic characteristics of the disease remain elusive. Tacrolimus has been demonstrated to be a promising therapeutic option for ITP patients in our previous report ( 63rd ASH, 2021), but the mechanisms are incompletely understood. Methods A total of 15 patients diagnosed with ITP and 8 healthy control (HC) individuals were enrolled between March and July 2023 in this study. Peripheral blood from ITP patients was collected at two time points: when admitted to the cohort without previous tacrolimus treatment and when receiving tacrolimus treatment for at least 28 days. We employed mass cytometry (cytometry by time-of-flight, CyTOF) with a panel of 27 phenotypic, 7 metabolic, and 8 functional markers to explore the immune landscape, metabolic characteristics, and phosphorylated signaling states at single-cell resolution. Additionally, a 10x Genomics platform was applied to perform single-cell RNA sequencing (scRNA-seq). Primary findings from our transcriptomic and proteomic analyses were further confirmed and extended by flow cytometry and RT-PCR. Results Using t-distributed stochastic neighbor embedding, a CyTOF panel of 42 markers partitioned the 32 clusters into 7 subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells, monocytes/macrophages, dendritic cells, and γδ T cells). Quantification of immune cell distribution across cell clusters showed a relatively higher level of CD8+ T cells and lower CD4+:CD8+ T-cell ratios in the patients with ITP than in the HC individuals. Additionally, the proportion of NK cells was significantly lower in patients than in control individuals. With further investigation of CD4+ and CD8+ T cells, we observed a decrease of naïve T cells and Treg cells and skewed accumulation of Th1 cells and Tc1 cells in patients with ITP relative to HC individuals. We further investigated the metabolic and functional status of T cells with CyTOF. One-carbon metabolism (MTHFD2 and SHMT2) was found to be elevated in Th1 cells from ITP patients compared with those from HC individuals. Based on CyTOF analysis as well as flow cytometry validation, a subgroup of Th1 cells with elevated ERK1/2 phosphorylation and MTHFD2 expression was speculated to be associated with the pathogenesis of ITP. A longitudinal study demonstrated that the dysregulated immune subset exhibited HC-like expression after treatment with tacrolimus. To gain further insights into the molecular characteristics of immune cell subsets and metabolic status that are differentially expanded in ITP patients, we performed scRNA-seq analysis on peripheral blood mononuclear cell (PBMC) isolated from 5 ITP patients pre- or posttreatment with tacrolimus and 3 HC individuals. Similar to the CyTOF data, a relative increase in Th1 cells and memory T cells and a reduction in naïve T cells and Treg cells were found in the ITP patient group compared with the HC group. Among the 9 subclustered T-lineage cell types, the CD4+ CXCR3+ IFNγ+ Th1 cells with functional pathways such as TCR pathway activation, IL-17 signaling pathway, and MAPK signaling pathway draw our attention. Next, we examined the metabolic status of immune cells of ITP patients to elucidate the underlying mechanisms of dysfunction of immune cells. Our analysis suggested that aberrant metabolism including glucose metabolism, nucleotide metabolism, and one-carbon metabolism caused by Th1 cells were involved in the dysregulated immune response in ITP. Moreover, ITP patients had elevated levels of metabolic checkpoints (mTOR, HIF-1α, and c-Myc) and low expression of immune checkpoint regulators (CTLA4, TIM3, and LAG3) in Th1 cells. Patients who recovered from tacrolimus treatment had a lower proportion of Th1 cells and lower expression levels of metabolic enzymes than nonresponsive patients. Conclusion Using integrated single-cell proteomic and transcriptomic profiling, this study depicts the broad immune and metabolic landscape of PBMC from ITP patients. In particular, our data unveil that inflammatory Th1 cells play a critical role in the pathophysiology of ITP and suggest a novel therapeutic opportunity of tacrolimus towards reprogramming immune metabolism.