Flow Cytometry-based Analysis Research Articles

Essential Fluidics for a Flow Cytometer.

Flow cytometry is an inherently fluidic process that flows particles on a one-by-one basis through a sensing region to discretely measure their optical and physical properties. It can be used to analyze particles ranging in size from nanoparticles to whole organisms (e.g., zebrafish). It has particular value for blood analysis, and thus most instruments are fluidically optimized for particles that are comparable in size to a typical blood cell. The principles of fluid dynamics allow for particles of such size to be precisely positioned in flow as they pass through sensing regions that are tens of microns in length at linear velocities of meters per second. Such fluidic systems enable discrete analysis of cell-sized particles at rates approaching 100 kHz. For larger particles, the principles of fluidics greatly reduce the achievable rates, but such high rates of data acquisition for cell-sized particles allow rapid collection of information on many thousands to millions of cells and provides for research and clinical measurements of both rare and common cell populations with a high degree of statistical confidence. Additionally, flow cytometers can accurately count particles via the use of volumetric sample delivery and can be coupled with high-throughput sampling technologies to greatly increase the rate at which independent samples can be delivered to the system. Due to the combination of high analysis rates, sensitive multiparameter measurements, high-throughput sampling, and accurate counting, flow cytometry analysis is the gold standard for many critical applications in clinical, research, pharmaceutical, and environmental areas. Beyond the power of flow cytometry as an analytical technique, the fluidic pathway can be coupled with a sorting mechanism to collect particles based on desired properties. We present an overview of fluidic systems that enable flow cytometry-based analysis and sorting. We introduce historical approaches, explanations of commonly implemented fluidics, and brief discussions of potential future fluidics where appropriate. © 2024 Wiley Periodicals LLC.

Proteomics reveals dynamic metabolic changes in human hematopoietic stem progenitor cells from fetal to adulthood

BackgroundHematopoietic stem progenitor cells (HSPCs) undergo phenotypical and functional changes during their emergence and development. Although the molecular programs governing the development of human hematopoietic stem cells (HSCs) have been investigated broadly, the relationships between dynamic metabolic alterations and their functions remain poorly characterized.MethodsIn this study, we comprehensively described the proteomics of HSPCs in the human fetal liver (FL), umbilical cord blood (UCB), and adult bone marrow (aBM). The metabolic state of human HSPCs was assessed via a Seahorse assay, RT‒PCR, and flow cytometry-based metabolic-related analysis. To investigate whether perturbing glutathione metabolism affects reactive oxygen species (ROS) production, the metabolic state, and the expansion of human HSPCs, HSPCs were treated with buthionine sulfoximine (BSO), an inhibitor of glutathione synthetase, and N-acetyl-L-cysteine (NAC).ResultsWe investigated the metabolomic landscape of human HSPCs from the fetal, perinatal, and adult developmental stages by in-depth quantitative proteomics and predicted a metabolic switch from the oxidative state to the glycolytic state during human HSPC development. Seahorse assays, mitochondrial activity, ROS level, glucose uptake, and protein synthesis rate analysis supported our findings. In addition, immune-related pathways and antigen presentation were upregulated in UCB or aBM HSPCs, indicating their functional maturation upon development. Glutathione-related metabolic perturbations resulted in distinct responses in human HSPCs and progenitors. Furthermore, the molecular and immunophenotypic differences between human HSPCs at different developmental stages were revealed at the protein level for the first time.ConclusionThe metabolic landscape of human HSPCs at three developmental stages (FL, UCB, and aBM), combined with proteomics and functional validations, substantially extends our understanding of HSC metabolic regulation. These findings provide valuable resources for understanding human HSC function and development during fetal and adult life.

Alteration of β-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as β-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of β-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface β-glucan while low pH triggers an increase in β-glucan. There is no inverse pattern between exposure levels of β-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in β-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in β-glucan triggers an increase in the virulence of C. auris. This study demonstrates that β-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

Activated CD4+ T Cell Proportion in the Peripheral Blood Correlates with the Duration of Cytokine Release Syndrome and Predicts Clinical Outcome after Chimeric Antigen Receptor T Cell Therapy.

Objective Chimeric antigen receptor (CAR) T cell therapy is an emerging and effective therapy for relapsed or refractory diffuse large B cell lymphoma (R/R DLBCL). The characteristic toxicities of CAR T cell therapy include cytokine release syndrome (CRS) and prolonged cytopenia. We investigated the factors associated with these complications after CAR T cell therapy by analyzing lymphocyte subsets following CAR T cell infusion. Methods We retrospectively analyzed peripheral blood samples on days 7, 14, and 28 after tisagenlecleucel (tisa-cel) infusion by flow cytometry at our institution between June 2020 and September 2022. Patients Thirty-five patients with R/R DLBCL who received tisa-cel therapy were included. Results A flow cytometry-based analysis of blood samples from these patients revealed that the proportion of CD4+CD25+CD127+ T cells (hereafter referred to as "activated CD4+ T cells" ) among the total CD4+ T cells on day 7 after tisa-cel infusion correlated with the duration of CRS (r=0.79, p<0.01). In addition, a prognostic analysis of the overall survival (OS) using time-dependent receiver operating characteristic curves indicated a significantly more favorable OS and progression-free survival of patients with a proportion of activated CD4+ T cells among the total CD4+ T cells <0.73 (p=0.01, and p<0.01, respectively). Conclusion These results suggest that the proportion of activated CD4+ T cells on day 7 after tisa-cel infusion correlates with the CRS duration and predicts clinical outcomes after CAR T cell therapy. Further studies with a larger number of patients are required to validate these observations.

Flow cytometry-based quantitative analysis of cellular protein expression in apoptosis subpopulations: A protocol

Flow cytometry techniques utilizing dual staining with annexin V and propidium iodide (PI) provide a robust method for quantitatively analyzing apoptosis induction. Annexin V binds phosphatidylserine exposed on the outer leaflet of the plasma membrane during early apoptosis, while PI permeates late apoptotic/necrotic cells. Simultaneous staining allows differentiation of viable, early apoptotic, and late apoptotic/necrotic populations. This approach can be enhanced by using fluorochrome-conjugated antibodies to stain specific proteins, enabling the simultaneous tracking of protein expression changes in defined cell subpopulations during apoptosis. This multiparametric approach provides key insights into signaling regulation and the mechanisms underlying the apoptotic response to cytotoxic treatments. Here we present a protocol that combines annexin V-FITC/PI staining with APC-conjugated antibody labeling in MDA-MB-231 breast cancer cells treated with doxorubicin. This protocol enables both the quantitative assessment of apoptosis induction and the tracking of decreased CD44 expression from viable to apoptotic cells. This protocol also provides guidelines for appropriate filter selection, compensation controls, gating strategies, and troubleshooting. This robust protocol holds significant potential for elucidating signaling networks involved in apoptosis and therapeutic resistance across various cellular models.

Flow cytometry-based peripheral blood analysis as an easily friendly tool for prognostic monitoring of acute ischemic stroke: a multicenter study.

Acute ischemic stroke (AIS) is a leading cause of mortality, severe neurological and long-term disability world-wide. Blood-based indicators may provide valuable information on identified prognostic factors. However, currently, there is still a lack of peripheral blood indicators for the prognosis of AIS. We aimed to identify the most promising prognostic indicators and establish prognostic models for AIS. 484 subjects enrolled from four centers were analyzed immunophenotypic indicators of peripheral blood by flow cytometry. Least absolute shrinkage and selection operator (LASSO) regression was applied to minimize the potential collinearity and over-fitting of variables measured from the same subject and over-fitting of variables. Univariate and multivariable Cox survival analysis of differences between and within cohorts was performed by log-rank test. The areas under the receiving operating characteristic (ROC) curves were used to evaluate the selection accuracy of immunophenotypic indicators in identifying AIS subjects with survival risk. The prognostic model was constructed using a multivariate Cox model, consisting of 402 subjects as a training cohort and 82 subjects as a testing cohort. In the prospective study, 7 immunophenotypic indicators of distinct significance were screened out of 72 peripheral blood immunophenotypic indicators by LASSO. In multivariate cox regression, CTL (%) [HR: 1.18, 95% CI: 1.03-1.33], monocytes/μl [HR: 1.13, 95% CI: 1.05-1.21], non-classical monocytes/μl [HR: 1.09, 95% CI: 1.02-1.16] and CD56high NK cells/μl [HR: 1.13, 95% CI: 1.05-1.21] were detected to decrease the survival probability of AIS, while Tregs/μl [HR:0.97, 95% CI: 0.95-0.99, p=0.004], BM/μl [HR:0.90, 95% CI: 0.85-0.95, p=0.023] and CD16+NK cells/μl [HR:0.93, 95% CI: 0.88-0.98, p=0.034] may have the protective effect. As for indicators' discriminative ability, the AUC for CD56highNK cells/μl attained the highest of 0.912. In stratification analysis, the survival probability for AIS subjects with a higher level of Tregs/μl, BM/μl, CD16+NK cells/μl, or lower levels of CD56highNK cells/μl, CTL (%), non-classical monocytes/μl, Monocytes/μl were more likely to survive after AIS. The multivariate Cox model showed an area under the curve (AUC) of 0.805, 0.781 and 0.819 and 0.961, 0.924 and 0.982 in the training and testing cohort, respectively. Our study identified 7 immunophenotypic indicators in peripheral blood may have great clinical significance in monitoring the prognosis of AIS and provide a convenient and valuable predictive model for AIS.

Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G1-to-S/G2 transition. Treatment with the small molecule ISRIB to bypass the effects of PERK-dependent phosphorylation of the translation initiation factor eIF2α had no such effect, suggesting that cell cycle timing depends on PERK's kinase activity but is independent of eIF2α phosphorylation. Using complementary light and electron microscopy and flow cytometry-based analyses, we also demonstrate that the ER enlarges before mitosis. Together, our results suggest coordination between UPR signaling and the cell cycle to maintain ER physiology during cell division.

Abstract 4215: Single-cell proteomic characterization of the tumor microenvironment in humanized NOG-EXL patient-derived xenograft (PDX) models

Abstract Introduction: CD34+ hematopoietic stem cell (HSC) derived humanized mice engineered to express human cytokine transgenes are promising models for the evaluation of novel immunotherapeutic treatments. While flow cytometry-based analyses have confirmed the presence of many human immune cell populations within humanized mice bearing patient-derived tumor xenografts (PDX), comprehensive high-dimensional immunophenotyping of the tumor microenvironment (TME) of these models remains limited. Beyond this, the relative influence of PDX vs. donor CD34+ HSC populations on TME composition is poorly understood. Here, we characterize the quantity and diversity of immune cell subpopulations in the TME, bone marrow, and spleen of humanized NOG-EXL (huNOG-EXL) mice using Cytometry by Time-Of-Flight (CyTOF) data with &amp;gt; 60 features. Methods: We performed CyTOF analysis on 250 dissociated samples from primary tumors (n = 15; n = 6 head and neck cancer, n = 6 ovarian cancer, n = 3 renal cell carcinoma), as well as xenograft (n = 59), spleen (n = 88), and bone marrow (n = 88) samples from huNOG-EXL mice (Taconic). Each sample was profiled with two partially overlapping antibody panels; an innate immunity-focused panel (40 features), and an adaptive immunity-focused panel (40 features). Results: CyTOF data were collected from over 10 million live immune cells for each of the adaptive and innate antibody panels. Unbiased clustering of single-cell marker expression revealed clusters corresponding to all major lymphoid and myeloid cell types. Therapeutically relevant cell states were identified including exhausted CD8+ T cells marked by PD-1 and Tim-3 expression, and PD-L1+ macrophages. Hierarchical clustering of bone marrow and spleen samples suggested that systemically, immune composition was not driven by tumor-intrinsic factors alone with many samples clustering on the basis of CD34+ stem cell donor ID. Conversely, clustering PDX samples revealed strong consistency in xenografts originating from the same primary tumor sample regardless of the chosen stem cell donor. The immune composition of primary tumor samples showed greater similarity with matched PDX samples versus unmatched PDX samples, further supporting that tumor-intrinsic factors drive immune composition in xenografts. Conclusions: We provide a high-resolution characterization of the immune phenotypes present in huNOG-EXL PDX models across three cancer types. These data captured substantial heterogeneity in the immune composition of humanized xenograft samples, and we demonstrate that immune composition in humanized xenografts is strongly driven by tumor-intrinsic features. Overall, this resource data supports the use of these models for immuno-oncology studies with future work focused on functionally validating specific immune cell populations of interest. Citation Format: Daniel Stueckmann, Jalna Meens, Sally Zhang, Shirley Hui, Sujana Sivapatham, Sephane Chevrier, Jennifer Pfeil, Lisa Martin, Maria Komisarenko, Philip Dube, Susan Prendeville, Hartland Jackson, Antonio Finelli, Gary Bader, Bernd Bodenmiller, Keith A. Lawson, Laurie Ailles. Single-cell proteomic characterization of the tumor microenvironment in humanized NOG-EXL patient-derived xenograft (PDX) models [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 4215.

Abstract 3045: PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer

Abstract Peroxisome proliferator activated receptor alpha (PPARα) is a transcription factor and master regulator of fatty acid oxidation. PPARα ligands modulate the switch between co-activator and co-repressor transcription complexes at PPARα-controlled target genes. In addition, PPARα has been shown to transrepress NF-κB signaling and inhibit angiogenesis. Renal cell carcinoma (RCC) expresses high levels of PPAR-α and is a highly angiogenic cancer. Current frontline treatments for RCC include chemotherapy, anti-angiogenics, and immunotherapy but are limited by the immune suppressive state of the tumor microenvironment. TPST-1120 is a novel PPARα antagonist for the treatment of cancer patients that binds in the ligand binding domain, positioning the AF-2 activation helix in an inactive conformation. The inactive conformation has a lower affinity for co-activator motifs and reduces activation of PPARα regulated genes. In a CHO-based reporter cell line, TPST-1120 potently competed against the PPARα-specific agonist GW7647 (IC50 = 36 nM at 20 nM GW7647) and an endogenous PPARα agonist oleoylethanolamide (OEA; IC50 = 15 nM at 30 µM OEA). Furthermore, in an enzyme fragment complementation assay, we demonstrated that TPST-1120 inhibited agonist-induced binding of PPARα to MED1 by destabilizing the co-activator complex thereby stabilizing the inactive conformation of PPAR-α. In a murine model of renal cell adenocarcinoma (RENCA), TPST-1120 treatment for 12 days (30 mg/kg qd) reduced tumor growth by 52% as a monotherapy (p&amp;lt;0.0001). Consistent with these findings, TPST-1120 inhibited tumor angiogenesis and proliferation as quantified by Ki67 levels. Combination treatment with current frontline therapeutics resulted in synergistic tumor inhibition of 74% with anti-PD1 (200 ug Q3D for 12 days) and 81% with cabozantinib (15 mg/kg QD for 15 days) (p&amp;lt;.0001). Flow cytometry-based analysis of tumors demonstrated increased CD8+ T cell infiltration in the TME as well as decreases in M2 and elevations in M1 macrophages. Importantly, we demonstrate that TPST-1120 can reverse an immunosuppressive class of immune cells to promote anti-tumor immunity and anti-angiogenesis in kidney cancer in the absence of overt toxicity. These data support advancement of TPST-1120 into RCC. Citation Format: Michael Gillespie, Valerie Chen, Isabella Howard, Keira Smith, Dave Freund, Nathan Standifer, Dara Burdette, Thomas W. Dubensky, Sam Whiting, Dipak Panigrahy. PPAR-α antagonist enhances immunotherapy and anti-angiogenic therapy to inhibit murine renal cancer [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 3045.

Abstract B109: Uncovering novel vulnerabilities in ovarian cancer by studying cell death in neoplastic transformation

Abstract B109: Uncovering novel vulnerabilities in ovarian cancer by studying cell death in neoplastic transformation

Marine Polysaccharides Carrageenans Enhance Eryptosis and Alter Lipid Order of Cell Membranes in Erythrocytes.

Carrageenans of ι-, κ- and λ-types were incubated with washed erythrocytes (hematocrit 0.4%) at 0-1-5-10 g/L for either 24 h or 48 h. Incubation was followed by flow cytometry-based quantitative analysis of eryptosis parameters, including cell volume, cell membrane scrambling and reactive oxygen species (ROS) production, lipid peroxidation markers and confocal microscopy-based evaluation of intracellular Ca2+ levels, assessment of lipid order in cell membranes and the glutathione antioxidant system. Confocal microscopy was used to assess carrageenan cellular internalization using rhodamine B isothiocyanate-conjugated carrageenans. All three types of carrageenans were found to trigger eryptosis. Pro-eryptotic properties were type-dependent and λ-carrageenan had the strongest impact inducing phosphatidylserine membrane asymmetry, changes in cell volume, Ca2+ signaling and oxidative stress characterized by ROS overproduction, activation of lipid peroxidation and severe glutathione system depletion. Eryptosis induction by carrageenans does not require their uptake by erythrocytes. Changes in physicochemical properties of cell membrane were also type-dependent. No carrageenan-induced generation of superoxide and hydroxyl radicals was observed in cell-free milieu. Our findings suggest that ι-, κ- and λ-types trigger eryptosis in a type-dependent manner and indicate that carrageenans can be further investigated as potential eryptosis-regulating therapeutic agents.

Flow cytometry-based measurable residual disease (MRD) analysis identifies AML patients who may benefit from allogeneic hematopoietic stem cell transplantation.

Measurable residual disease (MRD) monitoring independently predicts long-term outcomes in patients with acute myeloid leukemia (AML). Of the various modalities available, multiparameter flow cytometry-based MRD analysis is widely used and relevant for patients without molecular targets. In the transplant (HCT) setting, the presence of MRD pre-HCT is associated with adverse outcomes. MRD-negative remission status pre-HCT was also associated with longer overall (OS) and progression-free survival and a lower risk of relapse. We hypothesize that the combination of disease risk and MRD at the time of first complete remission (CR1) could identify patients according to the benefit gained from HCT, especially for intermediate-risk patients. We performed a retrospective analysis comparing the outcomes of HCT versus non-HCT therapies based on MRD status in AML patients who achieved CR1. Time-dependent analysis was applied considering time-to-HCT as a time-dependent covariate and compared HCT versus non-HCT outcomes according to MRD status at CR1. Among 336 patients assessed at CR1, 35.1% were MRD positive (MRDpos) post-induction. MRDpos patients benefitted from HCT with improved OS and relapse-free survival (RFS), while no benefit was observed in MRDneg patients. In adverse-risk patients, HCT improved OS (HR for OS 0.55; p = 0.05). In intermediate-risk patients, HCT benefit was not significant for OS and RFS. Intermediate-risk MRDpos patients were found to have benefit from HCT with improved OS (HR 0.45, p = 0.04), RFS (HR 0.46, p = 0.02), and CIR (HR 0.41, p = 0.02). Our data underscore the benefit of HCT in adverse risk and MRDpos intermediate-risk AML patients.

Isolation of Murine Adipose-Derived Stromal/Stem Cells for Adipogenic and Osteogenic Differentiation or Flow Cytometry-Based Analysis.

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells, along with protocols for inducing adipogenesis to white or beige adipocytes in this cell population and osteogenic differentiation. Isolation of the adipose stromal vascular fraction cells for flow cytometric analysis is also described.

Dual TTK/PLK1 inhibition has potent anticancer activity in TNBC as monotherapy and in combination.

Threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) are common essential kinases that collaborate in activating the spindle assembly checkpoint (SAC) at the kinetochore, ensuring appropriate chromosome alignment and segregation prior to mitotic exit. Targeting of either TTK or PLK1 has been clinically evaluated in cancer patients; however, dual inhibitors have not yet been pursued. Here we present the in vitro and in vivo characterization of a first in class, dual TTK/PLK1 inhibitor (BAL0891). Mechanism of action studies utilized biochemical kinase and proteomics-based target-engagement assays. Cellular end-point assays included immunoblot- and flow cytometry-based cell cycle analyses and SAC integrity evaluation using immunoprecipitation and immunofluorescence approaches. Anticancer activity was assessed in vitro using cell growth assays and efficacy was evaluated, alone and in combination with paclitaxel and carboplatin, using mouse models of triple negative breast cancer (TNBC). BAL0891 elicits a prolonged effect on TTK, with a transient activity on PLK1. This unique profile potentiates SAC disruption, forcing tumor cells to aberrantly exit mitosis with faster kinetics than observed with a TTK-specific inhibitor. Broad anti-proliferative activity was demonstrated across solid tumor cell lines in vitro. Moreover, intermittent intravenous single-agent BAL0891 treatment of the MDA-MB-231 mouse model of TNBC induced profound tumor regressions associated with prolonged TTK and transient PLK1 in-tumor target occupancy. Furthermore, differential tumor responses across a panel of thirteen TNBC patient-derived xenograft models indicated profound anticancer activity in a subset (~40%). Using a flexible dosing approach, pathologically confirmed cures were observed in combination with paclitaxel, whereas synergy with carboplatin was schedule dependent. Dual TTK/PLK1 inhibition represents a novel approach for the treatment of human cancer, including TNBC patients, with a potential for potent anticancer activity and a favorable therapeutic index. Moreover, combination approaches may provide an avenue to expand responsive patient populations.

Synthesis and evaluation of tetrahydropyrrolo[1,2-a]quinolin-1(2H)-ones as new tubulin polymerization inhibitors.

Here we explored new 1,5-disubstituted pyrrolidin-2-ones 1, 2 and 5-aryl-3,3a,4,5-tetrahydropyrrolo[1,2-a]quinoline-1(2H)-ones 3 as inhibitors of tubulin polymerization. We evaluated their effects on microtubule dynamics in vitro and on the proliferation of A549 cells, using flow cytometry-based cell cycle analysis. The results were verified with phase-contrast microscopy in three cancer cell lines: A549, HeLa and MCF-7. Guided by molecular modeling of the interactions between tubulin and the most active of the identified compounds, we designed, synthesized, and tested the 3-hydroxyphenyl-substituted compound 3c. This compound was further shown to bind to the colchicine site of tubulin and reduce microtubule growth rates in vitro. Moreover, compound 3c arrested division of the A549 cells in the low micromolar range (IC50 = 5.9 μM) and exhibited cytotoxicity against four different cell lines in the MTT assay for cell proliferation. Our findings demonstrate that 5-aryltetrahydropyrrolo[1,2-a]quinoline-1(2H)-one is a promising scaffold for the development of novel tubulin polymerization inhibitors.

Single-Cell RNA-Seq Reveals Intermediate Cell States and Identifies Features Defining Cellular Heterogeneity in Inv(16) Acute Myeloid Leukemia (AML)

AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. One recurrent chromosomal translocation common in AML is chromosome 16 inversion, inv(16)(p13.1q22), which results in the leukemogenic fusion gene CBFB- MYH11. Using a conditional Cbfb-MYH11 (CM) knock-in mouse model to mimic the somatic expression of the CM fusion gene, we demonstrated that CM expression leads to impaired hematopoietic differentiation as well as accumulation of phenotypic hematopoietic stem cells (HSC) and pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitors, which are predisposed to leukemic transformation. Flow cytometry-based phenotypic analysis revealed that the transformed leukemic blasts are predominantly characterized as pre-granulocyte-macrophage progenitor (pre-GM) (Lin −cKit +Sca1 −CD16/32 -/loCD150 −CD105 −). However, cell type characterization via conventional surface markers is limited by known cell markers and is incapable of resolving pathologic heterogeneities. We previously reported that modeling AML in a continuum of differentiation states using single-cell RNA-seq (scRNA-seq) data predicts intermediate states of differentiation in AML. Herein, we utilized scRNA-seq to characterize CM leukemic blasts, and identified transcriptomic alternations and features that defined the leukemic cell states and heterogeneities. CM expression was induced by poly (I:C) and mice (n=3) were monitored for leukemia progression. At the leukemic end-stage (detection of &amp;gt;50% of circulating cKit + cells), we collected unfractionated and ckit + sorted peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells. Similarly poly(I:C)More than 105,000 cells harvested from the WT and CM leukemic mice were analyzed by scRNA-seq. We applied uniform manifold approximation and projection (UMAP) for dimensionality reduction and performed cell type annotation by SingleR. First, we mapped mature hematopoietic cell types in the WT PB and hematopoietic stem/progenitor cell populations (HSPC) in the WT BM, and in contrast, leukemic PB and BM were highly similar and showed distinct cell clusters compared to WT. To characterize HSPC subpopulations, we focused on cKit+ sorted WT and leukemic BM cells, visualized them via UMAP, and performed clustering and cell type annotation. The leukemic cKit+ cells were mostly identified as megakaryocytic-erythroid progenitor (MEP)-like HSC, revealing a discrepancy between flow cytometry-based phenotypic characterization and transcriptome-based cell type assignment. These leukemic MEP-like HSCs showed up-regulation of erythroid differentiation genes, including Hba-a1, Hba-a2, Hbb-bs, Hbb-bt and Sox6, together with decreased expression of granulocytic markers such as Elane, Prtn3, S100a8, S100a9 and Mpo. Comparison of leukemic MEP-like HSCs vs WT MEP clusters via gene set enrichment analysis (GSEA) with a threshold of nominal p-value ≤ 0.05 and FDR ≤ 0.05 in 50 hallmark gene sets (MSigDB) indicated dysregulated pathways, including up-regulation of “TNFα signaling pathway via NFκB”, “inflammatory response”, and “UV responsive downregulated genes”; and down-regulation of “MYC target V1”, “MYC target V2”, “E2F targets”, “G2M checkpoint”, and “oxidative phosphorylation”. Similarly, GSEA in Gene Ontology (GO) showed downregulation of pathways relevant to mitochondrial protein translation and ribosomal biogenesis/assembly in leukemic MEP-like HSCs, compared to WT MEP. In addition, cKit+ BM cells can be separated into 31 clusters (C0-C30), of which 9 leukemic clusters were identified. Through gene expression-based cell cycle scoring, single sample gene set enrichment (ssGSEA), and marker identification, these 9 leukemic clusters can be defined by several features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). Altogether, these data reveal intermediate differentiation cell states in inv(16) leukemic cells which are highly heterogeneous populations with distinct cell cycle stages, metabolic activities, and marker expression.

Skp2 Inhibition Diminishes Essential Cell Survival Markers c-Maf and c-Myc, Enhancing Therapeutic Efficacy Against Drug Resistant Multiple Myeloma

Multiple Myeloma (MM) presents challenges as a hematologic malignancy, with drug resistance leading to limited curative options and disease relapse despite advancements in therapy. Understanding the molecular mechanisms driving MM's development and progression is vital for identifying new treatment approaches. Clinical and experimental evidence supports the role of S-phase kinase-associated protein 2 (Skp2) in driving the progression of several cancers, including MM. We interrogated a public microarray dataset derived from clinical specimens of MM patients and observed a significantly higher expression of Skp2 within the relapsed patient cohort (N=28) compared to the newly diagnosed group (N=63) (P=0.0041). Additionally, we observed elevated Skp2 levels and decreased P27 Kip1 levels in drug-resistant MM cell lines (MM.1R and RPMI 8226.R5) compared to their parental cell lines (MM.1S and RPMI 8226). Hence, a comprehensive understanding of Skp2's molecular functions in drug-resistant MM becomes paramount for facilitating the development of precision-targeted interventions. We investigated Skp2's molecular interactions in MM by performing a targeted knockdown with siRNA in drug-resistant and sensitive cell lines (OPM1, RPMI.8226, MM.1S, and MM.1R). Immunoblot analysis showed reduced STAT3 activation and decreased levels of cell survival markers c-Maf and c-Myc. Treatment with 10uM of the small molecule inhibitor SkpinC1 also inhibited STAT3 activation and led to significant reductions in c-Maf and c-Myc protein levels compared to the vehicle-treated groups. These findings hold immense significance as c-Maf and c-Myc are intricately associated with MM progression and poorer outcomes, although there is limited information on their regulation by the Skp2/P27 axis. Functionally, treatment of the MM cell lines with increasing dosages of SkpinC1 showed a progressive decrease in MM cell line survival, with IC50 values spanning 5-15uM, signifying the broad applicability of the inhibitor among the tested cell lines. Moreover, flow cytometry-based analysis of the surviving cell population demonstrated that SkpinC1 based inhibition of the Skp2/P27 axis considerably decreased the percentage of proliferating M-phase cells, while elevating the proportion of quiescent and apoptotic cells relative to the control group. At the molecular level, targeted inhibition of the Skp2/P27 axis by SkpinC1 caused the accumulation of cell cycle inhibitors P27 Kip1, P21 (Cip1/Waf1), and P57 Kip2, activation of the tumor suppressive TP53 protein with a simultaneous decrease in phospho-MDM2 levels in addition to an increase in the levels of apoptotic markers cleaved PARP and Caspase 3. From a clinical perspective, we also probed the applicability of Skp2 inhibition using SkpinC1 as a potential strategy for managing therapeutic resistance in MM. We treated multiple MM cell lines (OPM1, RPMI.8226, MM.1S and MM.1R) using a combination of Lenalidomide (an immune modulator) or Bortezomib (a proteasomal inhibitor) with SkpinC1 and compared it with single agent treatments. Both Lenalidomide/SkpinC1 as well as Bortezomib/SkpinC1 showed a strong dose dependent decrease in the percentage of surviving cells relative to the single agent treatments, thereby highlighting the remarkable potential of targeting the Skp2/P27 axis for clinical therapy in MM. To gain deeper insights into the molecular roles of Skp2/P27 axis inhibition by SkpinC1, we subjected drug-resistant MM.1R cells to 10uM SkpinC1 treatment for 24 hours, followed by single-cell transcriptome sequencing. This approach illuminated considerable heterogeneity between the drug-treated and control cells, accompanied by global changes in various signaling pathways associated with cell survival. In conclusion, the inhibition of Skp2 in MM cells exerts a significant impact on crucial cell survival pathways, notably c-Maf and c-Myc, thereby promoting apoptosis and offering a promising therapeutic avenue to combat drug resistance in MM. The precise targeting of Skp2 using small molecule inhibitors, such as SkpinC1, demonstrates substantial therapeutic potential, enhancing the efficacy of combinatorial treatment approaches for more effective interventions in MM.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

The Impact of the Peptide Drug Conjugate Melflufen on the Myeloma Tumour Microenvironment

Introduction Melphalan flufenamide (melflufen), is a peptide-drug conjugate that delivers alkylating agents in tumors upon cleavage by aminopeptidases, and recently approved by the EMA for the treatment of relapsed/refractory multiple myeloma (MM). Although melflufen's MoA on tumor cells had been extensively studied, little is known about the drug impact on the tumor microenvironment. By applying single cell, multi-parametric, flow cytometry-based analysis to bone marrow mononuclear cells (BM-MNC) from MM patient samples treated ex vivo, we investigated the effect of melflufen on immune cell populations to assess for potential immune modulatory effects and to identify potential immune-based biomarkers of response. Methods Viably frozen BM-MNC from 26 samples of patients diagnosed with MM were used for multi-parametric flow cytometry-based drug sensitivity and resistance testing evaluation to melflufen after written informed consent following approved protocols in compliance with the Declaration of Helsinki. Drug sensitivity was assessed using five different concentrations of melflufen ranging between 0.5-5000 nM and cells incubated with drug or DMSO control for 72 hrs incubation. For analysis, we used antibody panels allowing us to identify and phenotype T and B lymphocytes, NK, progenitor, and malignant plasma cells (PC). Based on drug sensitivity scores (DSS) of gated PCs from drug treated and controls samples, we divided the samples into two groups - high sensitivity (HS, Q3 of PC-DSS) and low sensitivity (LS, Q1 of PC-DSS). Samples with no PCs detected (n=11) were excluded from PC subset analysis. Results The MM samples were divided based on the sensitivity of the PC population to melflufen with DSS &amp;gt; 21.25 defining a highly sensitive group and DSS &amp;lt; 15.9 defining a less sensitive group. We found that HS samples have higher abundance of mature NK and iNKT cells, although significance was low, likely due to the small cohort size. In addition, we discovered that effector memory (EM) and terminal effector (TE) T cells have higher sensitivity to melflufen, compared to naïve and central memory (CM) phenotypes (Fig. 1). More mature NK cells (Stage 5 and 6) also showed higher sensitivity than NK cells of earlier stages of maturation. Importantly, we observed that in many of the tested samples, progenitor-like CD34+CD38- cells either were not sensitive to the cytotoxic effect of the drug, or even proliferated in higher drug concentrations (Fig. 2). Interestingly, HS samples had significantly higher baseline abundance of this cell population (p&amp;lt;0.05). At the same time, response profiles of healthy B and malignant PCs were similar. Finally, in our tests, PCs in samples with trisomy 11 abnormality showed lower sensitivity to melflufen (p&amp;lt;0.05). Conclusion In a previous study, we found that effector-like cells could be associated with better response to melflufen. In this study, we observed the same trends and confirm our earlier findings demonstrating an association of NK and NKT cells with higher sensitivity to melflufen. Moreover, we identified differential response of CD4, CD8 and NK cell populations comprising the tumor microenvironment to melflufen suggesting that in addition to direct tumor cell killing, melflufen may have modulating effects on different tumor associated immune cell populations. Finally, we observed proliferation of CD34+CD38- progenitor cells, which might be relevant for lymphocyte proliferation and function, and additionally supporting potential modulating effect. Further analyses are being done to validate these findings.

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.