High-resolution Flow Cytometry Research Articles

#1219 Extracellular vesicles from patients with AMR-induced reprogramming of human podocytes into senescent cells: role of SGLT2i

Abstract Background and Aims Extracellular vesicles (EVs) are organelles released by different cells that deliver biological information into the extracellular space and contribute to several diseases. However, their role in patients with antibody-mediated allograft rejection (AMR) remains uncertain. Our aim was to test the influence of plasma-derived EVs on human conditional immortalized podocytes (PODO) by investigating the possible role of dapagliflozin (Dapa) as a pharmacological agent. Methods Twenty-eight Rtx patients were included in the study, 14 with AMR, and 14 control TX patients (RTX). (CTRL). Plasma-derived Evs were used to stimulate PODO for 24 h, followed by exposure to 100 nM Dapa for 24 h. Differential ultracentrifugation at 110,000 g was applied, followed by counting with Nanosight and characterization by high-resolution flow cytometry according to the MISEV2018 criteria. PKH26 dye was used for the EVs-PODO incorporation study using Z-stack microscopy image acquisition. Total RNA and protein were extracted for gene expression and protein analysis, and the cells were fixed for immunostaining evaluation. Results We first found that upon activation with EV, PODO incorporated Es and shared them between cells; moreover, AMR-EVs led to an increased expression of tetraspanin marker of cell activation CD9 (p = 0.028) compared with CTRL. Importantly, the incubation of PODO with RTX-EVs induced a senescence-associated secretory phenotype (TNF alpha p = 0.0006, MCP1 p = 0.014); with cytoskeletal dysregulation and increase in gene expression and proteins involved in the epithelial to mesenchymal transition such as VIM (p = 0.026), senescence P21 (p = 0.04), inflammation CD44 (p = 0.034) with downregulation of Klotho (p = 0.002). Interestingly, incubation with Dapagliflozin significantly counteract PODO activation with reduction in P21 expression (p = 0.039) and an increase in Klotho (p = 0.047) abrogating inflammaging. Conclusions Our findings demonstrate that plasma EVs of AMR induced cellular senescence in PODO, reprogramming cellular processes such as cytoskeleton shape and composition, secretion of inflammatory cytokines, and decrease of the nephroprotective protein Klotho. This pathogenic process can be efficiently counteracted by SGLT2i.

Immune system-related plasma extracellular vesicles in healthy aging.

To identify age-related plasma extracellular vehicle (EVs) phenotypes in healthy adults. EV proteomics by high-resolution mass spectrometry to evaluate EV protein stability and discover age-associated EV proteins (n=4 with 4 serial freeze-thaws each); validation by high-resolution flow cytometry and EV cytokine quantification by multiplex ELISA (n=28 healthy donors, aged 18-83 years); quantification of WI-38 fibroblast cell proliferation response to co-culture with PKH67-labeled young and old plasma EVs. The EV samples from these plasma specimens were previously characterized for bilayer structure, intra-vesicle mitochondria and cytokines, and hematopoietic cell-related surface markers. Compared with matched exo-EVs (EV-depleted supernatants), endo-EVs (EV-associated) had higher mean TNF-α and IL-27, lower mean IL-6, IL-11, IFN-γ, and IL-17A/F, and similar mean IL-1β, IL-21, and IL-22 concentrations. Some endo-EV and exo-EV cytokine concentrations were correlated, including TNF-α, IL-27, IL-6, IL-1β, and IFN-γ, but not IL-11, IL-17A/F, IL-21 or IL-22. Endo-EV IFN-γ and exo-EV IL-17A/F and IL-21 declined with age. By proteomics and confirmed by flow cytometry, we identified age-associated decline of fibrinogen (FGA, FGB and FGG) in EVs. Age-related EV proteins indicated predominant origins in the liver and innate immune system. WI-38 cells (>95%) internalized similar amounts of young and old plasma EVs, but cells that internalized PKH67-EVs, particularly young EVs, underwent significantly greater cell proliferation. Endo-EV and exo-EV cytokines function as different biomarkers. The observed healthy aging EV phenotype reflected a downregulation of EV fibrinogen subpopulations consistent with the absence of a pro-coagulant and pro-inflammatory condition common with age-related disease.

Human placenta releases extracellular vesicles carrying corticotrophin releasing hormone mRNA into the maternal blood

The human placenta releases diverse extracellular vesicles (EVs), including microvesicles (100–1000 nm) and exosomes (30–150 nm), into the maternal blood for feto-maternal communication. Exosomes and microvesicles contribute to normal pregnancy physiology and major pregnancy pathologies. Differences in miRNA expressions and protein content in placental exosomes have been reported in complicated pregnancies. During human pregnancy, Corticotropin-Releasing Hormone (CRH) is produced and released by the placenta into the maternal blood. CRH is involved in regulating gestational length and the initiation of labour. CRH mRNA levels in the maternal plasma rise with gestation. High levels of CRH mRNA are reported to be associated with preeclamptic and preterm pregnancies. However, the underlying mechanism of placental CRH mRNA secretion remains to be elucidated. We hypothesise that the placenta releases CRH mRNA packaged within extracellular vesicles (EVs) into the maternal blood. In this study, placental EVs (microvesicles and exosomes) were isolated from human term healthy placentas via villus washes and from explant culture media by differential centrifugation and purified by density gradient ultracentrifugation using a continuous sucrose gradient (0.25–2.5 M). Western blotting using placenta- and exosome-specific markers and electron microscopy confirmed exosomes and microvesicles in the placental wash and explant media samples. Real-time quantitative RT-PCR data detected CRH mRNA in placenta-derived EVs from placental washes and explants. We also sorted placenta-secreted EVs in maternal plasma samples (≥37 weeks) by high-resolution flow cytometry using a fluorescent-labelled PLAP antibody. CRH mRNA was demonstrated in placental EVs obtained from maternal blood plasma. We therefore show that human placental EVs carry CRH mRNA into the maternal blood. Our study implies that measuring CRH mRNA in placental EVs in the maternal plasma could beused for monitoring pregnancy.

GABARAPL1 is essential in extracellular vesicle cargo loading and metastasis development

Background and purposeHypoxia is a common feature of tumours, associated with poor prognosis due to increased resistance to radio- and chemotherapy and enhanced metastasis development. Previously we demonstrated that GABARAPL1 is required for the secretion of extracellular vesicles (EV) with pro-angiogenic properties during hypoxia. Here, we explored the role of GABARAPL1+ EV in the metastatic cascade. Materials and methodsGABARAPL1 deficient or control MDA-MB-231 cells were injected in murine mammary fat pads. Lungs were dissected and analysed for human cytokeratin 18. EV from control and GABARAPL1 deficient cells exposed to normoxia (21% O2) or hypoxia (O2 < 0.02%) were isolated and analysed by immunoblot, nanoparticle tracking analysis, high resolution flow cytometry, mass spectrometry and next-generation sequencing. Cellular migration and invasion were analysed using scratch assays and transwell-invasion assays, respectively. ResultsThe number of pulmonary metastases derived from GABARAPL1 deficient tumours decreased by 84%. GABARAPL1 deficient cells migrate slower but display a comparable invasive capacity. Both normoxic and hypoxic EV contain proteins and miRNAs associated with metastasis development and, in line, increase cancer cell invasiveness. Although GABARAPL1 deficiency alters EV content, it does not alter the EV-induced increase in cancer cell invasiveness. ConclusionGABARAPL1 is essential for metastasis development. This is unrelated to changes in migration and invasion and suggests that GABARAPL1 or GABARAPL1+ EV are essential in other processes related to the metastatic cascade.

Isolated Extramedulary Relapse in T-Acute Lymphoblastic Leukemia

Isolated extramedullary acute lymphoblastic leukemia is a rare phenomenon. This event is more common in acute myeloid leukemia as myeloid sarcoma. This terminology is near the lymphoblastic lymphoma. Isolated extramedullary presentations hasn’t bone marrow involvement morphologically .Submicroscopic bone marrow involvement confirmed by a high-resolution flowcytometry detection method.

Identifying cancer-associated leukocyte profiles using high-resolution flow cytometry screening and machine learning.

Machine learning (ML) is a valuable tool with the potential to aid clinical decision making. Adoption of ML to this end requires data that reliably correlates with the clinical outcome of interest; the advantage of ML is that it can model these correlations from complex multiparameter data sets that can be difficult to interpret conventionally. While currently available clinical data can be used in ML for this purpose, there exists the potential to discover new "biomarkers" that will enhance the effectiveness of ML in clinical decision making. Since the interaction of the immune system and cancer is a hallmark of tumor establishment and progression, one potential area for cancer biomarker discovery is through the investigation of cancer-related immune cell signatures. Hence, we hypothesize that blood immune cell signatures can act as a biomarker for cancer progression. To probe this, we have developed and tested a multiparameter cell-surface marker screening pipeline, using flow cytometry to obtain high-resolution systemic leukocyte population profiles that correlate with detection and characterization of several cancers in murine syngeneic tumor models. We discovered a signature of several blood leukocyte subsets, the most notable of which were monocyte subsets, that could be used to train CATboost ML models to predict the presence and type of cancer present in the animals. Our findings highlight the potential utility of a screening approach to identify robust leukocyte biomarkers for cancer detection and characterization. This pipeline can easily be adapted to screen for cancer specific leukocyte markers from the blood of cancer patient.

Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

BIO15: Inhibition of Leukocytes Decreases Thrombin Generation in a In Vitro ECMO model

Background: Despite systemic anti-coagulation, blood exposure to extracorporeal membrane oxygenation circuits (ECMO) generates excess thrombin, often resulting in thrombus formation, device failure, and life-threatening thromboembolism. Activated leukocytes and platelets promote ECMO thrombosis under controlled static and flow conditions. Previous studies document that phosphodiesterase type 4 inhibitor (PDE4) can selectively inhibit the pro-thrombotic function of leukocytes. Our hypothesis is that roflumilast (PDE4 inhibitor) decreases thrombin generation in an in vitro ECMO model using human blood. Methods: Simulated neonatal roller pump with a KIDS D100 oxygenator circulated human whole blood at a nominal flow rate (0.5 L/min) for six hours or until clot formation caused device failure. Samples were collected hourly from the ECMO model that received heparin alone or heparin plus roflumilast, a PDE4 inhibitor. Expression of pro-thrombotic proteins on leukocytes and extracellular vesicles (EV) was measured with high-resolution flow cytometry. A calibrated automated thrombogram without a trigger measured differences in time to thrombin and peak thrombin generation. Results: The addition of roflumilast to heparin sustained patency in 75% of tested circuits (3/4) after six hours, compared to 28% of circuits treated with heparin alone (2/7). Roflumilast decreased extracellular leukocytes expressing tissue factor. Roflumilast plus heparin prolonged time to thrombin generation at 13.9 minutes, compared to heparin alone at 6.8 minutes at the 3-hour time point (p<0.04). The addition of roflumilast significantly reduced peak thrombin generation compared to heparin alone (69 vs. 207 nm, p<0.02). Conclusions: Inhibition of leukocytes may be an effective adjunct to systematic anti-coagulation in extracorporeal life support.

Determination of genome size variation among varieties of Ilex cornuta (Aquifoliaceae) by fow cytometry

Ilex cornuta Lindl. &amp; Paxton is a commercially important horticultural species worldwide, and extensive cultivation and hybridization have produced many varieties. Despite the considerable breeding, selection, widespread cultivation and domestication, which may have a significant role in the composition of genomes, there are no other previous reports of intraspecific genome size variation in the different cultivars or hybrids of this species. In the present work, genome size of 12 varieties of I. cornuta was assessed and analyzed through high-resolution flow cytometry (FCM). Nuclear DNA was analyzed using nuclei isolated from young leaves, which used propidium iodide (PI) staining, with rice (Oryza sativa cv. Nipponbare) as internal reference. As a result, statistically significant differences in genome size were detected among all diploid I. cornuta varieties considered. The estimated genome size (2C value) of I. cornuta varieties ranged from 1.47 to 1.80 pg, with 1.22-fold variation and an average size of 1.65 pg. The domestication and interspecific hybridization induced variation of genome size in I. cornuta, and the genome size of hybrids exhibited a wider range of variation compared with that of cultivars. In summary, flow cytometry is a useful tool to analyze the genome size of I. cornuta. The first report of the genome sizes of varieties of this species would provide useful data for further research on I. cornuta, and enrich the C value database of Ilex L. What’s more, our findings could be the foundation in the future of I. cornuta genome sequencing and breeding programs.

PD17-09 CD70-POSITIVE EXTRACELLULAR VESICLES FOR DETECTION OF CLEAR-CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023PD17-09 CD70-POSITIVE EXTRACELLULAR VESICLES FOR DETECTION OF CLEAR-CELL RENAL CELL CARCINOMA Rodrigo R. Pessoa, Reza Nabavizadeh, Ali Arafa, Roxane Lavoie, Yohan Kim, Liguo Wang, Jeanette Eckel-Passow, Bradley C. Leibovich, and Fabrice Lucien Rodrigo R. PessoaRodrigo R. Pessoa More articles by this author , Reza NabavizadehReza Nabavizadeh More articles by this author , Ali ArafaAli Arafa More articles by this author , Roxane LavoieRoxane Lavoie More articles by this author , Yohan KimYohan Kim More articles by this author , Liguo WangLiguo Wang More articles by this author , Jeanette Eckel-PassowJeanette Eckel-Passow More articles by this author , Bradley C. LeibovichBradley C. Leibovich More articles by this author , and Fabrice LucienFabrice Lucien More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: One third of patients treated for localized clear-cell renal cell carcinoma (ccRCC) will eventually develop recurrent disease after surgery. Identification of patients who may benefit from intensification of care with adjuvant therapy is an important unmet need. Tumor-derived extracellular vesicles (EVs), microscopic particles released by tumor cells, have emerged as a potential biomarker in other malignancies but have not been studied in kidney cancer. Herein, we sought to characterize tumor specific EVs levels among patients undergoing radical nephrectomy for ccRCC. METHODS: We employed in silico multi-omic analysis of ccRCC tumors using TCGA KIRC (N=537 patients), CPTAC (N= 103 patients) and GTEx (N= 948 donors) to identify CD70, an integral membrane protein, as the top marker candidate for ccRCC-derived EVs. Expression of CD70+ in ccRCC-derived EVs was confirmed using freshly isolated tumor specimens. Blood levels of circulating CD70+-EVs were measured using high resolution flow cytometry in a cohort of age-matched healthy donors (HD) (N=15) and ccRCC patients (N=29) treated with radical nephrectomy. EVs levels between HD and RCC patients were compared using ANOVA test. Association between patient characteristics and EVs levels was analyzed using Pearson’s correlation and Fisher’s exact test. RESULTS: Blood levels of CD70+-EVs were significantly elevated in patients with pT1-T2 (median: 4.3×106 per mL, p<0.01) and pT3-T4 (median: 6.0×106, p<0.001) ccRCC compared with HD (median: 0.4×106, Figure 1A). High CD70+-EVs levels were associated with pT3-T4 stage (Figure 1B, p<0.0001).There was a strong positive correlation between CD70+-EVs at diagnosis and tumor size (Pearson r=0.76, p<0.0001, Figure 1C). Radical nephrectomy was associated with reduction in CD70+-EVs levels in 83.8% of patients (Figure 1D, p=0.0076). CONCLUSIONS: Patients with ccRCC, in particular those with larger and more advanced stage tumors, have high levels of circulating CD70+-EVs, which decreases significantly after surgery. CD70+-EVs levels are a marker of tumor burden in ccRCC and could be further explored as a biomarker for risk-stratification and selection of patients for additional treatment after radical nephrectomy for ccRCC. Source of Funding: Eagles 5th District Cancer Telethon - Cancer Research Fund (FL) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e499 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rodrigo R. Pessoa More articles by this author Reza Nabavizadeh More articles by this author Ali Arafa More articles by this author Roxane Lavoie More articles by this author Yohan Kim More articles by this author Liguo Wang More articles by this author Jeanette Eckel-Passow More articles by this author Bradley C. Leibovich More articles by this author Fabrice Lucien More articles by this author Expand All Advertisement PDF downloadLoading ...

OxLDL sensitizes platelets for increased formation of extracellular vesicles capable of finetuning macrophage gene expression

Platelet extracellular vesicles (PEVs) generated upon platelet activation may play a role in inflammatory pathologies such as atherosclerosis. Oxidized low-density lipoprotein (oxLDL), a well-known contributor to atherogenesis, activates platelets and presensitizes them for activation by other agonists. We studied the effect of oxLDL on the secretion, composition, and inflammatory functions of PEVs using contemporary EV analytics. Platelets were activated by co-stimulation with thrombin (T) and collagen (C) ± oxLDL and characterized by high-resolution flow cytometry, nanoparticle tracking analysis, proximity extension assay, western blot, and electron microscopy. The effect of PEVs on macrophage differentiation and functionality was examined by analyzing macrophage surface markers, cytokine secretion, and transcriptome. OxLDL upregulated TC-induced formation of CD61+, P-selectin+ and phosphatidylserine+ PEVs. Blocking the scavenger receptor CD36 significantly suppressed the oxLDL+TC-induced PEV formation, and HDL caused a slight but detectable suppression. The inflammatory protein cargo differed between the PEVs from stimulated and unstimulated platelets. Both oxLDL+TC- and TC-induced PEVs enhanced macrophage HLA-DR and CD86 expression and decreased CD11c expression as well as secretion of several cytokines. Pathways related to cell cycle and regulation of gene expression, and immune system signaling were overrepresented in the differentially expressed genes between TC PEV -treated vs. control macrophages and oxLDL+TC PEV -treated vs. control macrophages, respectively. In conclusion, we speculate that oxLDL and activated platelets contribute to proatherogenic processes by increasing the number of PEVs that provide an adhesive and procoagulant surface, contain inflammatory mediators, and subtly finetune the macrophage gene expression.

Thyroid cells from normal and autoimmune thyroid glands suppress T lymphocytes proliferation upon contact revealing a new regulatory inhibitory type of interaction independent of PD1/PDL1

Immune Checkpoint Receptors include a number of inhibitory receptors that limit tissue damage during immune responses; blocking PD-1/PD-L1 checkpoint receptor axis led to a paradigm shift in cancer immunotherapy but also to autoimmune adverse effects, prominently thyroid autoimmunity. Although PD-L1 is known to be expressed on thyroid follicular cells (TFCs) of autoimmune glands the role on PD-1/PD-L1 in the interaction between T cells and thyroid cells in the tissue has not been investigated. Here we report that autologous primary TFCs, but not transformed TFCs, inhibit CD4 and CD8 T cell proliferation but no cytokine production. This effect is not, however, mediated by PD-1/PD-L1 nor locally produced cytokines. Beta galactosidase analysis excluded culture-induced senescence as an explanation. High resolution flow cytometry demonstrated that autologous TFC/T cells co-culture induced the expansion of several clusters of double negative (DN) T cells characterized by high expression of activation markers and negative immune checkpoints. Single cell transcriptomic profiling demonstrated that dissociated TFC express numerous candidate molecules for mediating this suppressive activity, including CD40, E-Cadherin and TIGIT ligands. These ligands directly or through the generation of a suppressor population of DN T cells, and not the PD-1/PD-L1 axis, are most likely the responsible of TFC immunosuppressive activity. These results contribute to reveal the complex network of inhibitory mechanism that operate at the tissue level to restrain autoimmunity but also point to pathways, other that PD-1/PD-L1, that can contribute to tumor evasion.

Human Nanoplatelets as Living Vehicles for Tumor-Targeted Endocytosis In Vitro and Imaging In Vivo

Recent studies have shown human platelets can access the tumor microenvironment by passive diffusion across capillaries or via activated immune cells. In a previous study, we exploited this affinity of platelets for tumor cells as part of a new approach to target tumors with modified platelets. Therefore, the engineering of human nanoplatelets as living vehicles for in vivo tumor-targeted near-infra-red fluorescence (NIRF) imaging and the delivery of cytotoxins to tumor cells by endocytosis are described in this study. Nanoplatelets with an average diameter of 200 nm were prepared by mild sonication of kabiramide C (KabC)-loaded human platelets. The sealed plasma membrane of the nanoplatelets allows them to accumulate and retain membrane-permeable chemicals, such as epidoxorubicin (EPI) and KabC. Tumor-targeted imaging functionalities were engineered on the nanoplatelets by surface-coupling transferrin, Cy5 and Cy7. High-resolution fluorescence imaging and flow cytometry analyses showed that the nanoplatelets loaded with EPI and Cy5 targeted human myeloma cells (RPMI8226 cells) that over-expressed the transferrin receptor. The endocytosis of the nanoplatelets by RPMI8226 cells was transferrin-dependent and induced apoptosis. The test results also showed that the nanoplatelets functionalized with transferrin and Cy7 and injected in mice bearing RPMI8226 cells-derived myeloma xenotransplants accumulated in the tumor tissue and could be used for high-contrast in vivo NIRF imaging of early-stage tumors. Nanoplatelets represent a new class of living nano-vehicles that may efficiently target and deliver therapeutic agents and imaging probes to diseased tissues including tumors.

Extracellular Vesicles from Ocular Melanoma Have Pro-Fibrotic and Pro-Angiogenic Properties on the Tumor Microenvironment

Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells.

Teloxantron inhibits the processivity of telomerase with preferential DNA damage on telomeres

Telomerase reactivation is one of the hallmarks of cancer, which plays an important role in cellular immortalization and the development and progression of the tumor. Chemical telomerase inhibitors have been shown to trigger replicative senescence and apoptotic cell death both in vitro and in vivo. Due to its upregulation in various cancers, telomerase is considered a potential target in cancer therapy. In this study, we identified potent, small-molecule telomerase inhibitors using a telomerase repeat amplification protocol assay. The results of the assay are the first evidence of telomerase inhibition by anthraquinone derivatives that do not exhibit G-quadruplex-stabilizing properties. The stability of telomerase in the presence of its inhibitor was evaluated under nearly physiological conditions using a cellular thermal shift assay. Our data showed that the compound induced aggregation of the catalytic subunit (hTERT) of human telomerase, and molecular studies confirmed the binding of the hit compound with the active site of the enzyme. The ability of new derivatives to activate DNA double-strand breaks (DSBs) was determined by high-resolution microscopy and flow cytometry in tumor cell lines differing in telomere elongation mechanism. The compounds triggered DSBs in TERT-positive A549 and H460 lung cancer cell lines, but not in TERT-negative NHBE normal human bronchial epithelial and ALT-positive U2OS osteosarcoma cell lines, which indicates that the induction of DSBs was dependent on telomerase inhibition. The observed DNA damage activated DNA damage response pathways involving ATM/Chk2 and ATR/Chk1 cascades. Additionally, the compounds induced apoptotic cell death through extrinsic and intrinsic pathways in lung cancer cells. Taken together, our study demonstrated that anthraquinone derivatives can be further developed into novel telomerase-related anticancer agents.

Low-dose IL-2 reduces IL-21+ T cell frequency and induces anti-inflammatory gene expression in type 1 diabetes

Despite early clinical successes, the mechanisms of action of low-dose interleukin-2 (LD-IL-2) immunotherapy remain only partly understood. Here we examine the effects of interval administration of low-dose recombinant IL-2 (iLD-IL-2) in type 1 diabetes using high-resolution single-cell multiomics and flow cytometry on longitudinally-collected peripheral blood samples. Our results confirm that iLD-IL-2 selectively expands thymic-derived FOXP3+HELIOS+ regulatory T cells and CD56bright NK cells, and show that the treatment reduces the frequency of IL-21-producing CD4+ T cells and of two innate-like mucosal-associated invariant T and Vγ9Vδ2 CD8+ T cell subsets. The cellular changes induced by iLD-IL-2 associate with an anti-inflammatory gene expression signature, which remains detectable in all T and NK cell subsets analysed one month after treatment. These findings warrant investigations into the potential longer-term clinical benefits of iLD-IL-2 in immunotherapy.

Comprehensive Exploration of NK Cell Education in ADCC Triggered By a Monoclonal Antibody or a Bispecific Killer Engager Molecule (BiKE)

The responsiveness of NK cells to malignant cells that lack self-HLA class I (HLA-I) expression is controlled by NK cell education governed by the interaction between selected inhibitory NK cell receptors and their cognate HLA-I ligands. However, it remains unclear to what extent education impacts the ability of NK cells to perform antibody-dependent cellular cytotoxicity (ADCC) as previous studies show inconsistent results and are limited in resolution. Moreover, no study has yet addressed if NK cell education impacts ADCC triggered by novel CD16-binding therapeutics such as bispecific killer engagers (BiKEs). In this study we systematically characterized NK cell degranulation against a panel of tumor cell lines with defined HLA-I expression in the presence of either the monoclonal antibody rituximab or a recently developed CD34xCD16 BiKE. Using high-resolution flow cytometry, our data revealed that NK cells educated by one or two KIRs did not have significantly enhanced ADCC capacity over uneducated KIR+ or KIR- NK cells, especially when increasing the antibody concentration. Contrasting co-education by KIRs, NKG2A was consistently associated with improved responsiveness and superior ADCC. Surprisingly, these results were despite that KIR-education correlated with higher CD16 expression that was markedly lower on NKG2A+ NK cells compared to both KIR-educated and uneducated subsets. An extended analysis uncovered that the inhibitory KIR2DL1-C245 receptor, reported to mediate weak NK cell education compared to the KIR2DL1-R245 variant, further repressed the ADCC capacity of NKG2A+ NK cells from donors expressing its ligand HLA C2. This highlights that also inhibitory KIRs can dampen the ADCC potential of NK cells similar to that of activating KIRs which also were discovered to restrict ADCC even when stimulated by its ligand. Finally, our study unveiled that BiKE-triggered ADCC has a similar relationship to NK cell education and its hierarchies as that triggered by a monoclonal antibody. In conclusion, the ADCC capacity of an NK cell is more regulated by restrictive mechanisms of education conferred by activating or selected inhibitory KIRs in individuals carrying the cognate KIR ligand than that of inhibitory KIRs conferring higher education potential. Hence, this study establishes that NK cell education does not enhance the ADCC capacity of an NK cell per se while providing new insights that also inhibitory KIRs can dampen the ADCC potential even when the target cell lacks its ligand. Our results add to the fundamental understanding of NK cell biology as well as to avenues of therapeutic applications for NK cells.

Rare PNH Cells May be Considered As Genuine PNH Clone: Insights from Analysis of the 5-Year French Nation-Wide Multicenter Observational Study

Introduction The successive International Guidelines (Borowitz. et al. Cytometry B Clin Cytom 2010; Illingworth. et al. Cytometry B Clin Cytom 2018) recommend both detection and follow-up of rare PNH cells using high-resolution flow cytometry (FCM) analysis. These rare PNH cells, defined as GPI-deficient cells ranging from 0.01% to <0.1%, are distinguished from minor PNH clone, ranging from 0.1% to 1%. However, clinical relevance as well as long-term evolution of such rare PNH cells are still unknown. Objective We aimed to compare clinical and biological presentation of patients with rare PNH cells or a minor PNH clone along with long-term follow-up using data provided by the 5-year French nation-wide multicenter observational study, the Observatory of PNH Clones. Methods All patients of any age with a newly or previously diagnosed PNH clone or GPI-deficient cells ≥0.01%, detected in France by FCM from February 29th, 2016 to February 28th, 2021 were eligible for inclusion in this Observatory, provided that the center had participated in the inter-laboratory comparison program ongoing since 2013 (Debliquis et. al. Am J Clin Pathol 2015). For each patient, the baseline assessment was always considered as the initial PNH clone detection, even if it occurred before the initiation of the Observatory. Thus, this strategy allowed the collection of cases with long-term follow-up. A referent cytometrist at each center included patients in the e-CRF available on the CytHem-HPN website providing clinical and biological information as well as FCM raw data files. This study was approved by the national research ethics board. Results As of February 28th, 2021, 41 registered flow cytometry laboratories across France enrolled 637 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. Selection of patients displaying rare PNH cells (i.e., o.o1% to <0.1%) or a minor PNH clone (i.e., 0.1% to 1%) on neutrophils led to the re-examination of 163 patient FCM files (provided by 26 centers) by the two principal investigators. FCM data and the e-CRF filling were both thoroughly reviewed leading to the update of roughly half of the submitted files. This enabled the validation of 138 patients at diagnosis and the exclusion of 18 (11%) patients who did not display a genuine PNH clone according to the international recommendations (at least 20 clustered events). For 7 of 163 selected patients, exchanges with centers are still ongoing for FCM data validation or due to missing clinical or biological data. Sixty-nine of the 138 validated patients displayed at least one follow-up point (Figure 1). Thus, we were able to compare 49 patients with rare PNH cells and 89 patients with a minor PNH clone with a significantly different clone size on the 3 lineages (median size on neutrophils: 0.03 [0.01 - 0.09] vs 0.34 [0.1 - 0.97] ; p<0.0001, on monocytes: 0.03 [0 - 7.88 ] vs 0.50 [0 - 7.08] ; p<0.0001, on red blood cells: 0.01 [0 - 1.36 ] vs 0.08 [0 - 1.24]; p= 0.0096). The patient median age at diagnosis was similar for both groups, 59 years [6 - 91] with 4 pediatric cases (<18y) for "rare PNH cells” group vs 49 years [10 - 87] with 9 pediatric cases for "minor PNH clone” group and a M/F sex ratio of 1.45 vs 0.98. Diagnoses were made between 2010 and 2021 with clinical information available in 98% of cases (n=47 and 88). Ninety-two to 94% of patients from both groups displayed BM failure including mainly aplastic anemia (n=30/47 vs 61/88), myelodysplastic syndrome (n=2 vs 6) and unexplained cytopenia(s) (n=11 vs 16). Only 4 cases of thrombosis for "rare PNH cells” group vs 3 for "minor PNH clone” group were observed, and the 2 cases of hemolytic anemia belong to the latter group. A more severe thrombopenia was observed in the "minor PNH clone” (68 [2-356] vs 44 [2-308] G/l) while all other biological parameters showed no significant difference. Long-term follow-up up to 7.0 years showed that one third of patients from both groups displayed an increase in clone size (above 0.1% for "rare PNH cells” and above 1% for "minor PNH clone", see Figure 2), most remained stable and a few decreased, the disappearance being always due to BM transplantation. Conclusion This multicenter study based on robust FCM analysis showed no differences between the clinical and biological presentation of patients with either rare PNH cells or a minor PNH clone. Long-term follow-up showed that rare PNH cells can evolve into minor PNH clone, suggesting that these cases may be considered as genuine minor clones. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Evolutionary Portrait of Adult Core-Binding Factor Leukemia Patients Treated with a Continuation Therapy with Midostaurin: Preliminary Results

Genetic and Clinical Background The clinical outcome of Core Binding Factor Leukemia (CBFL) has been reported to be influenced by KIT mutations, that was frequent in CBFL, and by co-occurring different spectra of cooperating mutations. In addition, gene expression studies found KIT to be highly expressed in CBFL regardless of its mutational status. In vitro experiments revealed that Midostaurin (Mido) is effective in inhibiting both wild type (WT) and a range of KIT mutants. In vivo studies demonstrate the efficacy in KIT-positive malignancies such as Aggressive Systemic Mastocytosis, Mast Cell Leukemia, and SM with Associated Hematological Neoplasm. Therefore, we designed a Phase II trial to evaluate the safety and efficacy of Mido, in association with Intensive Chemotherapy (IC), in CBFL regardless of KIT mutational status. (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345). 34 adult patients with t(8;21) (n=16) and inv(16) (n=18) were enrolled between December 2018 to July 2022. Method Patients with de-novo CBFL received standard induction therapy with an anthracycline containing regimen ("7+3"-like) + Mido, three cycles of post-remission consolidation chemotherapy with high-dose cytarabine + Mido, and 12 months of Mido as Maintenance. At diagnosis, 32 out of 34 patients were studied by a comprehensive NGS panel targeting 40 DNA genes and 29 RNA fusion driver genes associated with major myeloid disorders (Oncomine Myeloid Research Assay). Aim To study the clonal evolution of CBFLs, NGS and MRD status (performed by qPCR and high-resolution multicolor flow cytometry) were assessed at established check-points during consolidation, maintenance therapy, and at relapse. ResultsKIT was the most frequently mutated gene (28%) and there was an enrichment of FLT3 and NRAS (Figure 1). Additionally, the spectrum of recurrent mutations was different, as codon 816 KIT mutations and FLT3-ITD were common only in RUNX1::RUNX1T1 cases. Overall, the CR rate was 97%. At a median follow-up of 18 months (range 2-43 months), we recorded a RI of 37.5 %, an OS of 85 %, and a DFS of 60%. We performed targeted next generation sequencing for six out of 12 relapsed samples in the cohort t(8;21) (n=5) and inv(16) (n=1), showing loss, retention or gain of somatic mutations (Figure 2). Three out of six relapsed patients showed at diagnosis a CEBPA mutation (Gly104del) co-occurring with KIT in RUNX1::RUNX1T1 cases and with NRAS in CBFB::MYH11. Conclusion In patients with CBFL, we found FLT3 and/or KIT mutations actionable by a regimen consisting of intensive chemotherapy and Mido in 47% of cases. In two out of three relapsed cases the KIT mutation is maintained, although frequency of the mutant allele is lower at relapse, while all FLT3 and NRAS mutated patients lose the mutation upon disease relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Selected Articles from This Issue

The overall aims of this study were to describe the association between body composition measures obtained from pre-HCT CT images and early-onset. In this cohort of adults who underwent allogeneic HCT, Bhandari and colleagues found significant associations between body composition measurements obtained from pre-HCT CT imaging studies and early-onset glucose intolerance after HCT. The components of body composition that 10 increased risk for glucose intolerance differed by BMI; in non-overweight patients, skeletal muscle loss increased this risk, while visceral adiposity was associated with increased risk in overweight/obese patients. These findings suggest that multiple mechanisms underlie the development of glucose intolerance and/or DM after HCT and highlight the importance of improving DM risk characterization according to pre-HCT body composition phenotype.Fortunato, Huber, and colleagues, performed peripheral blood high resolution flow cytometry of lung cancer screening volunteers to define an immune classifier discriminating early-stage lung cancer patients from controls. In parallel, a gene signature encompassing the most relevant immune cell subsets defined by the classifier was selected to identify a final diagnostic model. Globally, this immune classifier comprises phenotypic and molecular features of key immune cellular elements such as monocytic and PMN MDSC, intermediate monocytes and activated cytotoxic T cells. By applying a chained-rule simulation approach, the immune classifier was estimated to reduce unnecessary procedures in the 48% of screening participants with a false positive CT result.Menstrual and reproductive factors affected the gender disparity in liver cancer incidence, but the evidence was inconsistent. This study by Tuo and colleagues was the first population-based cohort evaluating the associations between menstrual and reproductive factors and liver cancer risk in Chinese women. The authors found that HRT and injective contraceptives were positively associated with liver cancer risk, while live births' number, menopausal age and reproductive period reduced the risk. The findings suggested that female sex hormones could play roles on liver carcinogenesis, which would be important for the prevention and control of liver cancer.The complex nature of oropharyngeal cancer (OPC), implying a different epidemiological pattern of the subtypes, requires an accurate and timely assessment of the burden of the disease. Accordingly, this study by Lorenzoni and colleagues report an updated overview of the incidence and mortality of OPC worldwide based on GLOBOCAN-2020 estimates. Age-standardized incidence and mortality rates, male-to-female ratios, and incidence-to-mortality ratios were estimated and compared between genders, by country and across world regions. Results highlighted substantial geographical and gender variability, probably related to the different distribution of OPC subtypes. This overview might help guide future studies, support planning and prioritization of prevention and treatment programs.