Single-cell Mass Research Articles

Single cell proteomics analysis of drug response shows its potential as a drug discovery platform.

Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us a unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments.

A Sample Preparation Procedure for Isobaric Labeling-Based Single-Cell Proteomics.

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.

High-throughput single-cell mass spectrometry enables metabolic network analysis by resolving phospholipid C[double bond, length as m-dash]C isomers.

Single-cell mass spectrometry (MS) is an essential technology for sensitive and multiplexed analysis of metabolites and lipids for cell phenotyping and pathway studies. However, the structural elucidation of lipids from single cells remains a challenge, especially in the high-throughput scenario. Technically, there is a contradiction between the inadequate sample amount (i.e. a single cell, 0.5-20 pL) for replicate or multiple analysis, on the one hand, and the high metabolite coverage and multidimensional structure analysis that needs to be performed for each single cell, on the other hand. Here, we have developed a high-throughput single-cell MS platform that can perform both lipid profiling and lipid carbon-carbon double bond (C[double bond, length as m-dash]C) location isomer resolution analysis, aided by C[double bond, length as m-dash]C activation in unsaturated lipids by the Paternò-Büchi (PB) reaction and tandem MS, termed single-cell structural lipidomics analysis. The method can achieve a single-cell analysis throughput of 51 cells per minute. A total of 145 lipids were structurally characterized at the subclass level, of which the relative abundance of 17 isomeric lipids differing in the location of C[double bond, length as m-dash]C from 5 lipid precursors was determined. While cell-to-cell variations in MS1-based lipid profiling can be large, an advantage of quantifying lipid C[double bond, length as m-dash]C location isomers is the significantly improved quantitation accuracy. For example, the relative standard deviations (RSDs) of the relative amounts of PC 34:1 C[double bond, length as m-dash]C position isomers in MDA-MB-468 cells are half smaller than those measured for PC 34:1 as a whole by MS1 abundance profiling. Taken together, the developed method can be effectively used for in-depth structural lipid metabolism network analysis by high-throughput analysis of 142 MDA-MB-468 human breast cancer cells.

Ferroptosis Inducers Upregulate PD-L1 in Recurrent Triple-Negative Breast Cancer.

(1) Background: Triple-negative breast cancer (TNBC) is a distinct subgroup of breast cancer presenting a high level of recurrence, and neo-adjuvant chemotherapy is beneficial in its therapy management. Anti-PD-L1 immunotherapy improves the effect of neo-adjuvant therapy in TNBC. (2) Methods: Immune-modulation and ferroptosis-related R-packages were developed for integrative omics analyses under ferroptosis-inducer treatments: TNBC cells stimulated with ferroptosis inducers (GSE173905, GSE154425), single cell data (GSE191246) and mass spectrometry on breast cancer stem cells. Clinical association analyses were carried out with breast tumors (TCGA and METABRIC cohorts). Protein-level validation was investigated through protein atlas proteome experiments. (3) Results: Erastin/RSL3 ferroptosis inducers upregulate CD274 in TNBC cells (MDA-MB-231 and HCC38). In breast cancer, CD274 expression is associated with overall survival. Breast tumors presenting high expression of CD274 upregulated some ferroptosis drivers associated with prognosis: IDO1, IFNG and TNFAIP3. At the protein level, the induction of Cd274 and Tnfaip3 was confirmed in breast cancer stem cells under salinomycin treatment. In a 4T1 tumor treated with cyclophosphamide, the single cell expression of Cd274 was found to increase both in myeloid- and lymphoid-infiltrated cells, independently of its receptor Pdcd1. The CD274 ferroptosis-driver score computed on a breast tumor transcriptome stratified patients on their prognosis: low score was observed in the basal subgroup, with a higher level of recurrent risk scores (oncotypeDx, ggi and gene70 scores). In the METABRIC cohort, CD274, IDO1, IFNG and TNFAIP3 were found to be overexpressed in the TNBC subgroup. The CD274 ferroptosis-driver score was found to be associated with overall survival, independently of TNM classification and age diagnosis. The tumor expression of CD274, TNFAIP3, IFNG and IDO1, in a biopsy of breast ductal carcinoma, was confirmed at the protein level (4) Conclusions: Ferroptosis inducers upregulate PD-L1 in TNBC cells, known to be an effective target of immunotherapy in high-risk early TNBC patients who received neo-adjuvant therapy. Basal and TNBC tumors highly expressed CD274 and ferroptosis drivers: IFNG, TNFAIP3 and IDO1. The CD274 ferroptosis-driver score is associated with prognosis and to the risk of recurrence in breast cancer. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested for recurrent TNBC.

PD-1 and TIGIT coexpressing CD8 + CD103 + tissue-resident memory cells in endometrial cancer as potential targets for immunotherapy

BackgroundImmunotherapy has shown promise in treating various cancers; however, its efficacy in endometrial cancer (EC) remains suboptimal owing to the complex dynamics of the tumour immune microenvironment. This study focuses on exploring the potential of targeting the programmed cell death protein 1 gene (PD-1) and the T cell Immunoreceptor with Ig and ITIM domains gene (TIGIT) coexpressing tissue-resident memory cells in EC. MethodsA comprehensive approach, utilizing RNA sequencing, single-cell RNA sequencing, mass cytometry, and flow cytometry, was employed to analyse the expression patterns of PD-1 and TIGIT in the EC tumor environment and to characterize the phenotypic properties of tumor-infiltrating lymphocytes (TILs), particularly tissue-resident memory (TRM) cells. Additionally, in vitro cell experiments were conducted to assess the functional impact of PD-1 and TIGIT blockade on T-cell activity. ResultsOur analysis identified a significant co-expression of PD-1 and TIGIT in TRM cells within the EC tumor microenvironment. These TRM cells displayed an exhausted phenotype with impaired cytotoxicity, enhanced proliferative capacity, and diminished cytotoxic activity. In vitro T-cell assays showed that a dual blockade of PD-1 and TIGIT more effectively restored T-cell functionality compared to single blockade, suggesting enhanced therapeutic potential. ConclusionsTRM cells co-expressing PD-1 and TIGIT represent potential targets for EC immunotherapy. Dual immune checkpoint blockade targeting PD-1 and TIGIT may offer an effective therapeutic strategy for EC, providing valuable insights for the development of immunotherapeutic approaches.

SINGLE-CELL AND SPATIAL OMICS FOR MAPPING CELLULAR SENESCENCE IN HEALTH, AGING AND DISEASE

Abstract NIH SenNet consortium aims to dissect the heterogeneity of senescent cells (SnCs) and map their impact on the microenvironment at a single cell resolution and in the spatial tissue context, which requires the implementation of an array of omics technologies to comprehensively identify, characterize, and spatially profile SnCs across tissues in humans and mice. These technologies are broadly categorized into two groups –single cell omics and spatial mapping. To achieve single cell resolution and overcome the scarcity of SnCs, high-throughput single-cell and single-nucleus transcriptomic techniques have become a mainstay tool for surveying tens of thousands of cells to identify transcriptional signatures in rare cell populations, enabling discovery of potential new SnC biomarkers. Novel single cell mass spectrometry methods are developed for unbiased discovery of proteomic signatures of SnCs. A hallmark of SnCs is the senescence-associated secretory phenotype (SASP), which requires the use of proteomics, secretomics, metabolomics and lipidomics, especially SASP-associated extracellular vesicles, for comprehensive characterization of SAPS. High resolution molecular and cellular imaging of gene expression (e.g., MERFISH) or protein markers (e.g., CODEX) is critical for the study of SnCs in the large-scale tissue context. NGS-based spatial omics sequencing is poised to bridge the gap to realize both genome scale and cellular resolution in mapping SnCs in tissue. Novel technologies such as Seq-Scope and Pixel-Seq developed within SenNet further enabled subcellular resolution. SenNet investigators also developed spatially resolved epigenome and multi-omics sequencing techniques to link transcriptional or proteomic phenotype of SnCs to epigenetic mechanism. Further integration with high-resolution imaging makes spatial omics the crucial linchpin in connecting mechanistic underpinnings and molecular signatures with morphological features and spatial distribution. All these are critical for the construction of a map of SnCs and associated niches in the native tissue environment implicated in human health, aging, and disease, which is one of the main goals of the SenNet consortium.

Single-cell immunophenotyping revealed the association of CD4+ central and CD4+ effector memory T cells linking exacerbating chronic obstructive pulmonary disease and NSCLC.

Tobacco smoking generates airway inflammation in chronic obstructive pulmonary disease (COPD), and its involvement in the development of lung cancer is still among the leading causes of early death. Therefore, we aimed to have a better understanding of the disbalance in immunoregulation in chronic inflammatory conditions in smoker subjects with stable COPD (stCOPD), exacerbating COPD (exCOPD), or non-small cell lung cancer (NSCLC). Smoker controls without chronic illness were recruited as controls. Through extensive mapping of single cells, surface receptor quantification was achieved by single-cell mass cytometry (CyTOF) with 29 antibodies. The CyTOF characterized 14 main immune subsets such as CD4+, CD8+, CD4+/CD8+, CD4-/CD8-, and γ/δ T cells and other subsets such as CD4+ or CD8+ NKT cells, NK cells, B cells, plasmablasts, monocytes, CD11cdim, mDCs, and pDCs. The CD4+ central memory (CM) T cells (CD4+/CD45RA-/CD45RO+/CD197+) and CD4+ effector memory (EM) T cells (CD4+/CD45RA-/CD45RO+/CD197-) were FACS-sorted for RNA-Seq analysis. Plasma samples were assayed by Luminex MAGPIX® for the quantitative measurement of 17 soluble immuno-oncology mediators (BTLA, CD28, CD80, CD27, CD40, CD86, CTLA-4, GITR, GITRL, HVEM, ICOS, LAG-3, PD-1, PD-L1, PD-L2, TIM-3, TLR-2) in the four studied groups. Our focus was on T-cell-dependent differences in COPD and NSCLC, where peripheral CD4+ central memory and CD4+ effector memory cells showed a significant reduction in exCOPD and CD4+ CM showed elevation in NSCLC. The transcriptome analysis delineated a perfect correlation of differentially expressed genes between exacerbating COPD and NSCLC-derived peripheral CD4+ CM or CD4+ EM cells. The measurement of 17 immuno-oncology soluble mediators revealed a disease-associated phenotype in the peripheral blood of stCOPD, exCOPD, and NSCLC patients. The applied single-cell mass cytometry, the whole transcriptome profiling of peripheral CD4+ memory cells, and the quantification of 17 plasma mediators provided complex data that may contribute to the understanding of the disbalance in immune homeostasis generated or sustained by tobacco smoking in COPD and NSCLC.

Quantification of Nitric Oxide in Single Cells Using the Single-Probe Mass Spectrometry Technique.

Nitric oxide (NO) is a small molecule that plays important roles in biological systems and human diseases. The abundance of intracellular NO is tightly related to numerous biological processes. Due to cell heterogeneity, the intracellular NO amounts significantly vary from cell to cell, and therefore, any meaningful studies need to be conducted at the single-cell level. However, measuring NO in single cells is very challenging, primarily due to the extremely small size of single cells and reactive nature of NO. In the current studies, the quantitative reaction between NO and amlodipine, a compound containing the Hantzsch ester group, was performed in live cells. The product dehydro amlodipine was then detected by the Single-probe single-cell mass spectrometry technique to quantify NO in single cells. The experimental results indicated heterogeneous distributions of intracellular NO amounts in single cells with the existence of subpopulations.

Immune profiling and tracking of two-dimensional transition metal dichalcogenides in cells and tissues

Two-dimensional (2D) transition metal dichalcogenides (TMDs), such as molybdenum disulfide (MoS2) and tungsten disulfide (WS2), have attracted considerable interest in biomedicine due to their unique combination of physicochemical properties. The effect of nanomaterials on immune cells and their biodistribution are critical aspects of their clinical translation. However, understanding the interactions of these emerging 2D nanomaterials with the complex pool of peripheral blood mononuclear cells (PBMCs) at the single-cell level and their in situ localization in the main organs still needs to be discovered, preventing their translation in medical settings. Here, we report in-depth immune profiling of water-based and defect-free 2D formulations of MoS2 and WS2 through the simultaneous label-free tracking of their immune cell interactions, both ex vivo in human PBMCs and in vivo in mice by high-dimensional analytical approaches, as well as their biodistribution. For comparison, we studied graphene, the hitherto most explored 2D material for biomedical applications. First, we assessed the impact at the protein and gene level by multiplex protein arrays and RNA sequencing, demonstrating a very modest effect of MoS2 and WS2 on immune cell functionality compared to graphene. Then, a single-cell view of the effects of MoS2 and WS2 on 16 primary human immune cell types in terms of viability and functionality was obtained by single-cell mass cytometry by time-of-flight (CyTOF). We explored over 30 markers looking at multiple cell parameters. Finally, we present evidence that MoS2 and WS2 are visible, without the need for labeling, at the single-cell and tissue level by CyTOF, imaging mass cytometry, and multiplexed ion beam imaging by time-of-flight (MIBI-TOF). In particular, MoS2 and WS2 could be detected in the molybdenum (95Mo) and tungsten (180–186W) channels, respectively, which are not used for commercial mass cytometry tags, allowing for the simultaneous interrogation of a wide variety of biological parameters ex vivo and in vivo following intravenous administration of the TMDs. Indeed, we demonstrated the accumulation of TMDs in the main organs by MIBI-TOF and they could also be identified in specific immune cell subsets by CyTOF. Among the two TMDs studied, WS2 exhibited the highest brightness and signal intensity in all the cell subpopulations and tissues analyzed. In conclusion, we identified TMDs as immune-compatible nanoplatforms, traceable at the single-cell and tissue (sub-organ) levels, thus opening up new perspectives for their exploration in biomedicine.

Recent developments in ionization techniques for single-cell mass spectrometry.

The variation among individual cells plays a significant role in many biological functions. Single-cell analysis is advantageous for gaining insight into intricate biochemical mechanisms rarely accessible when studying tissues as a whole. However, measurement on a unicellular scale is still challenging due to unicellular complex composition, minute substance quantities, and considerable differences in compound concentrations. Mass spectrometry has recently gained extensive attention in unicellular analytical fields due to its exceptional sensitivity, throughput, and compound identification abilities. At present, single-cell mass spectrometry primarily concentrates on the enhancement of ionization methods. The principal ionization approaches encompass nanoelectrospray ionization (nano-ESI), laser desorption ionization (LDI), secondary ion mass spectrometry (SIMS), and inductively coupled plasma (ICP). This article summarizes the most recent advancements in ionization techniques and explores their potential directions within the field of single-cell mass spectrometry.

SUMOylation inhibitors activate anti-tumor immunity by reshaping the immune microenvironment in a preclinical model of hepatocellular carcinoma.

High levels of heterogeneity and immunosuppression characterize the HCC immune microenvironment (TME). Unfortunately, the majority of hepatocellular carcinoma (HCC) patients do not benefit from immune checkpoint inhibitors (ICIs) therapy. New small molecule therapies for the treatment of HCC are the goal of our research. SUMOylation inhibitors (TAK-981 and ML-792) were evaluated for the treatment of preclinical mouse HCC models (including subcutaneous and orthotopic HCC models). We profile immune cell subsets from tumor samples after SUMOylation inhibitors treatment using single-cell RNA sequencing (scRNA-seq), mass cytometry (CyTOF), flow cytometry, and multiple immunofluorescences (mIF). We discover that SUMOylation is higher in HCC patient samples compared to normal liver tissue. TAK-981 and ML-792 decrease SUMOylation at nanomolar levels in HCC cells and also successfully reduced the tumor burden. Analysis combining scRNA-seq and CyTOF demonstrate that treatment with SUMOylation inhibitors reduces the exhausted CD8+T (Tex) cells while enhancing the cytotoxic NK cells, M1 macrophages and cytotoxic T lymphocytes (CTL) in preclinical mouse HCC model. Furthermore, SUMOylation inhibitors have the potential to activate innate immune signals from CD8+T, NK and macrophages while promoting TNFα and IL-17 secretion. Most notably, SUMOylation inhibitors can directly alter the TME by adjusting the abundance of intestinal microbiota, thereby restoring anti-tumor immunity in HCC models. This preclinical study suggests that SUMO signaling inhibitors may be beneficial for the treatment of HCC.

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance.

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry.

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Epigenetic Scarring Leads to Irreversible NK Cell Dysfunction in Myeloid Malignancies

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to the same spectrum of myeloid malignancies and have extremely poor outcomes in the relapsed/refractory setting. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, patients with MDS and AML often relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have been mostly unsuccessful in patients with MDS and AML. This could be due to the immunosuppressive tumor microenvironment in the bone marrow niche or to intrinsic dysfunction in the immune effector cells of these patients. Natural killer (NK) cells are innate lymphocytes that play an important role in cancer immune surveillance and have distinct advantages over T cells as candidates for immunotherapy. Myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity. Little is known about the mechanisms of NK cell immune evasion developed by myeloid blasts. Here, we show that NK cells from patients with myeloid malignancies display a global dysfunction with severely impaired secretory function and killing capacity. Single cell RNAseq and mass cytometry experiments revealed that these NK cells from MDS and AML patients display an exhausted phenotype at the transcriptomic and proteomic levels. We also show that these NK cells have an altered metabolism compared with age matched healthy control NK cells. We show that this dysfunction is mediated by a crosstalk between myeloid blasts and NK cells and cell-cell contact dependent release of transforming growth factor beta (TGF-β). This crosstalk leads to a profound epigenetic reprogramming of NK cells driven by transcription factors known to mediate exhaustion and immune suppression. Myeloid blast-induced NK dysfunction and epigenetic state of exhaustion can be prevented by pharmacologically inhibiting the TGF-β pathway or knockout of TGFBR2 in NK cells. However, our data reveal that once this dysfunction occurs it is irreversible owing to epigenetic scarring driven by the transcription factor BATF. In fact, we show that TGF-β induces the expression of BATF in NK cells, which in turn mediates a gene regulatory program leading to NK cell dysfunction by driving the expression of exhaustion and inhibitory genes (e.g. HAVCR2, ENTPD1, CTLA4, TGFBR2). Collectively, our findings reveal a novel mechanism of NK cell immune evasion manifested by stable epigenetic rewiring and inactivation of NK cells by myeloid blasts. Our data support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies rather than therapies aiming at reversing or rescuing the function of autologous NK cells.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Prior Knowledge Integration Improves Relapse Prediction and Identifies Relapse Associated Mechanisms in Childhood B Cell Acute Lymphoblastic Leukemia

Introduction Despite decades of research and advancement in frontline treatments of B cell acute lymphoblastic leukemia (B-ALL), relapse remains the major barrier to survive this diagnosis in children and young adults. Although the majority of children will respond to initial treatment, 50% of children who relapse will die. Hence, there is a critical unmet need to prevent relapse in these patients. Previously, using proteomic features and predictive modeling we reported that patients who will eventually suffer relapse have cells predictive of their future relapse detectable at the time of diagnosis. These cells sit at the pro-B to pre-B transition in B-cell development and have elevated expression of signaling proteins: SYK, rpS6, CREB and 4EBP1. Although, our model outperformed standard risk models such as NCI criteria and MRD relapse risk, our model lacks higher accuracy and precision. Here, we utilize novel immunological Elastic-Net (iEN) model for relapse prediction which incorporates per-feature prior knowledge into the predictive model and improves the predictive power of the model. Methods and Results We analyzed 160 diagnosis bone marrow samples of children with newly diagnosed B-ALL treated on the cooperative group protocols. These samples comprised multiple cytogenetic subtypes and were annotated for clinical outcome (MRD status, relapse status, time to relapse) and patient attributes. Cells in these samples were analyzed using single-cell mass cytometry and expression of markers defining B cell developmental stages(CD34, CD38, CD127, CD24, TdT, CD179a, CD179b, IgHi, IgHs, CD19, CD20), signaling molecules (p4EBP1, pSTAT5, pPLC-γ2, pAKT, pSYK, pRPS6, pERK1-ERK2, pCREB) and transcription factors(TFs) (IKAROS, PAX5) were detected in the basal condition as well as after short-term ex vivo stimulation. Following data acquisition and preprocessing, cells were developmentally classified to the closest healthy B-cell population using a single-cell developmental classifier. Following cell-type classification, 1) frequency of cells with each of signaling molecules and TFs in the unstimulated state and in response to each of 3 stimulations, 2) age and WBC counts for each patient were extracted. These data were split in 90% training and 10% validation cohort. Prior knowledge for signaling molecules and TFs in response to each ex vivo stimulation condition is included after evaluation by a panel of expert cancer biologists such that molecules more consistent with known relapse biology have score of 1 and less consistent molecules have <1. This prior knowledge and training phase data were utilized to construct the iEN. The performance of model was determined using ten-fold cross-validation. Once constructed the model was applied to a validation cohort. The iEN predicts relapse status with AUROC 0.85 ± 0.06 and outperforms our published model as well as standard risk models. Piecewise regression analysis of iEN revealed that pro-BI and pro-BII cells highly expressing pSYK, pCREB, pRPS6, p4EBP1 and pAKT have the highest coefficient magnitudes and are associated with relapse, consistent with our prior results. These molecules act within the network downstream of the pre-B cell receptor, suggesting relapse associated cells to have activated preBCR signaling. The transcriptomic profile of these cells revealed enrichment of PI3K-mTOR pathway genes, consistent with activated signaling profiles predicted by iEN. However, these cells were also enriched for expression of genes associated with metabolic pathways: oxidative phosphorylation, glycolysis, and fatty acid metabolism. Using a CyTOF panel focused on metabolic proteins, we confirmed co-expression of preBCR associated molecules and metabolic proteins (GLUT1, Enolase 1 and lactate dehydrogenase A) in pro-BI (TdT+/CD24-/CD179a+/CD179b+) and pro-BII (TdT+/CD24+/IgMi-/IgMs-) cells from relapse patients. Together this data suggests a relationship between active preBCR signaling with cellular metabolism and relapse. Conclusions Supervised predictive models integrating specific domain knowledge may improve prediction of relapse and identification of relapse associated mechanisms in B-ALL. Further analysis and integration of clinical data are underway and will be presented.

Single-Cell Genomics and Proteomics Reveals Venetoclax-Resistant Monocytic Differentiation of TP53 LOH Clones in TP53 Mutant AML

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by accumulation of abnormal hematopoietic stem and progenitor cells. TP53 mutations, present in 8 - 30 % of AML, are associated with high risk of relapse and poor prognosis. The mechanisms underlying their persistence are not well understood. Here, we attempt to describe the clonal architecture and immunophenotypes of TP53 mutant ( TP53-mut) clones during therapy. Leveraging advancements in single-cell sequencing and machine learning models, this study integrated single-cell mass cytometry (CyTOF) and single-cell DNA + protein sequencing (seq) profiling of ten paired pre- and post-treatment samples from five TP53-mut AML patients. Methods: Patients 1 and 3 received treatment with CD47AB (Magrolimab), 5'-Azacitidine (Aza), and Venetoclax (Ven); Patient 2 received IMGN632 (CD123-targeting ADC), Aza and Ven. Patient 4 received p97 Inhibitor CB-5339; Patient 5 received CD47 inhibitor (ALX148), Aza, Ven. Consequently, Ven/Aza served as the therapeutic backbone for most patients. For single cell DNA+protein analysis, we sequenced a total of 44,550 cells from these 10 samples. Samples with at least 70% viability were stained with the Total-Seq™-D Human Heme Oncology Cocktail, containing 45 oligo-conjugated antibodies for surface markers relevant to AML. For mutation analysis, we utilized a validated AML custom panel (Morita et al. 2020) covering recurrent mutations in 37 genes. For CyTOF, we multiplexed samples and stained with 50 antibodies characterizing lineage, apoptosis, and signaling pathways. We successfully constructed a machine learning model for integrating both data types using our gradient boosting algorithm established pipelines and were able to predict DNA mutation status in CyTOF data with over 95% accuracy. Results: Analysis of the clonal architecture revealed the presence of TP53 mutations not only in AML blasts but also in lymphocyte subpopulations in all TP53-mut clones from the patients in this study. For example, in patient 1, as a representative case, TP53 mutations were observed in 16.5% of CD4+ T cells, 35.9% of CD8+ T cells, and 19.2% of NK cells. Clonal evolution analysis showed sensitivity of TP53-mut clones with TP53 Loss of heterozygosity ( TP53 mut + LOH) to Ven/Aza and monocytic differentiation in resistant cells, potentially a manifestation of their stem-like nature. Patient 1 displayed three distinct subclones: clone 1 harbored an ETV6 mutation (p.V322S), clone 2, derived from clone 1, acquired additional IDH1 (p.R132L) and TP53 (p.R241G) mutations, clone 3, originating from clone 2 and comprising the highest proportion, exhibited loss of the other TP53 allele (LOH), and decreased from 96.2% to 55.2% post-treatment (Figure 1A). Meanwhile, clone 1 and clone 2 increased from 1.9% to 27.3% and 1.9% to 17.4% respectively. Dissecting the phenotypic changes, clone 3 showed significant pre- and post-treatment shifts in cellular composition. Pre-treatment, it consisted predominantly of 89.7% CD34+ blasts and only 0.2% CD16+ monocytic cells. Post-treatment, CD34+ blasts decreased to 9.3%, while CD16+ monocytic cells increased to 67.8%. CyTOF analysis corroborated our findings (Figure 1B) with trajectory analysis revealing that AML cells had a stemness profile (CD34+CD38- and C-kit+) located at the beginning of the trajectory. Conversely, post-treatment AML cells displayed a loss of stemness and expressed myeloid differentiation markers, including CD33, CD11b, CD14, and CD68. We concluded that Ven-based treatments induce monocytic differentiation in resistant AML cells. CyTOF analysis of the proteomic profiles of resistant cells revealed an increase in Ki67, BCL-XL, and 4EBP1 and a decrease in CD34 and C-KIT, suggesting a potential resistance mechanism involving the upregulation of anti-apoptotic molecules and mTOR pathway. Notably, CD36, a fatty acid transporter, exhibited increased expression in monocytic cells, suggesting an altered metabolism in the survival monocytic cells. Conclusion: This study establishes a genotype-phenotype connection through single-cell proteogenomic profiling of TP53-mutated AML, describing the clonal evolution and immunophenotypic dynamics during treatment while proposing a potential mechanism of resistance.

IL-1β-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling

The source and roles of fibroblasts and T-cells during maladaptive remodeling and myocardial fibrosis in the setting of pulmonary arterial hypertension (PAH) have been long debated. We demonstrate, using single-cell mass cytometry, a subpopulation of endogenous human cardiac fibroblasts expressing increased levels of CD4, a helper T-cell marker, in addition to myofibroblast markers distributed in human fibrotic RV tissue, interstitial and perivascular lesions in SUGEN/Hypoxia (SuHx) rats, and fibroblasts labeled with pdgfrα CreERt2/+ in R26R-tdTomato mice. Recombinant IL-1β increases IL-1R, CCR2 receptor expression, modifies the secretome, and differentiates cardiac fibroblasts to form CD68-positive cell clusters. IL-1β also activates stemness markers, such as NANOG and SOX2, and genes involved in dedifferentiation, lymphoid cell function and metabolic reprogramming. IL-1β induction of lineage traced primary mouse cardiac fibroblasts causes these cells to lose their fibroblast identity and acquire an immune phenotype. Our results identify IL-1β induced immune-competency in human cardiac fibroblasts and suggest that fibroblast secretome modulation may constitute a therapeutic approach to PAH and other diseases typified by inflammation and fibrotic remodeling.

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection.

The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, accumulates in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTβ signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders, including neuropsychiatric lupus and multiple sclerosis.

High-dimensional immune profiling using mass cytometry reveals IL-17A-producing γδ T cells as biomarkers in patients with T-cell-activated idiopathic severe aplastic anemia

Severe aplastic anemia (SAA) is a bone marrow failure syndrome characterized by activated T cells. Features of T-cell activation in the pathophysiology of SAA remain unknown. To understand T cell activation states, we investigated the atlas of peripheral immune cells and the secreted cytokine network with single cell mass cytometry analysis. We found decreased γδ T-cell frequencies in all patients with SAA, together with a significantly increased proportion of interleukin (IL)-17A-producing cell subsets. Cytokine network analysis of immune cells showed significant positive relationship between IL and 17A production from immune cells and disease severity of severe aplastic anemia. On separating SAA into two distinct subgroups based on T-cell activation stage, the proportion of γδ T cells tended to decrease in the T-cell-activated SAA group compared with non-T-cell-activated group. And the proportion of IL-17A-producing γδ T cells (γδT17) within γδ T cells was newly found to be significantly higher in the T-cell-activated SAA group, implying that IL-17A production by γδ T cells was associated with T-cell activation. Overall, our study revealed a role of γδT17 cells in mediating autoreactive T-cell activation in SAA and provided a novel diagnostic indicator for monitoring autoreactive T-cell activation status during the progression of aplastic anemia in the clinic.