Flow Cytometric Assay Research Articles

Nimotuzumab and irinotecan synergistically induce ROS-mediated apoptosis by endoplasmic reticulum stress and mitochondrial-mediated pathway in cervical cancer.

Irinotecan (CPT-11), a chemotherapeutic agent used to treat several types of cancer, induces cytotoxic effects on healthy cells. The epidermal growth factor receptor (EGFR) plays a crucial role in various forms of cancer. Nimotuzumab (NmAb), a monoclonal antibody that targets the EGFR, is utilized in some countries to treat malignancies that have an overexpression of EGFR. Yet, there is a lack of literature on the potential anticancer properties of the CPT-11 and NmAb combination on in vitro human cervical cancer cells. This study investigates the apoptosis mode of the CPT-11 and NmAb combination on cervical HeLa cancer cells. The Annexin V/propidium iodide staining examination demonstrated that the combination of CPT-11 and NmAb resulted in a decrease in the number of viable cells and more potent induction of cell apoptosis than the effects of CPT-11 or NmAb alone in HeLa cells. Furthermore, the combined treatment resulted in elevated levels of reactive oxygen species (ROS) and Ca2+ compared to the treatment with CPT-11 or NmAb alone. Cells that were pretreated with N-acetyl-l-cysteine, a substance that scavenges ROS, and then treated with CPT-11, NmAb, or a combination of CPT-11 and NmAb exhibited higher numbers of viable cells compared to those treated with CPT-11 or NmAb alone. The combination of CPT-11 and NmAb resulted in significantly higher caspase-3, -8, and -9 activity levels than CPT-11 or NmAb alone, as measured by flow cytometer assay. The combination of CPT-11 and NmAb in HeLa cells resulted in elevated endoplasmic reticulum stress-, mitochondria-, and caspase-mediated proteins compared to treatment with CPT-11 or NmAb alone. According to these observations, NmAb enhances the effectiveness of CPT-11 in fighting cancer by stimulating cell death in the HeLa cells. Therefore, NmAb has the potential to improve the efficacy of CPT-11 as a future cervical cancer treatment in humans.

Hsa_circ_0000520 Serves as a Prognostic Biomarker for Colorectal Cancer and Promotes in the Disease Progression.

Colorectal cancer (CRC) constitutes one of the prevalent malignancies within the gastrointestinal tract and serves as a primary contributor to cancer-related mortalities. This investigation sought to investigate the expression and prognostic significance of hsa_circ_0000520 in CRC and to evaluate its impact on the onset of CRC. The levels of hsa_circ_0000520 were measured via quantitative real-time polymerase chain reaction (qRTPCR). To delve into the mechanism through which circ_0000520 impacts CRC and to assess the cellular behavior of CRC cells, a series of experiments including the CCK-8, transwell assay, flow cytometer assay, cell cloning formation, dual-luciferase reporter assay, and bioinformatics method were performed. The expression levels of hsa_circ_0000520 were markedly elevated in CRC cells and tissue specimens, and this elevation was correlated with a low survival rate. hsa_circ_0000520 affected CRC cell function via the miR-542-3p/MYH9 axis, thus exacerbating cancer progression. hsa_circ_0000520 functions as a predictive biomarker for the prognosis of CRC and participates in its progression. hsa_ circ_0000520 emerges as a new treatment strategy for CRC patients.

TXN promotes tumorigenesis by activating the ERK1/2 and ERK5 signaling pathways regulating c-Myc in non-small cell lung cancer

Lung cancer is the primary cause of cancer-related deaths worldwide, particularly for non-small cell lung cancer (NSCLC). However, the exact mechanism underlying tumor formation remains unclear. It is widely acknowledged that inflammation and oxidative stress occur in the tumor microenvironment, promoting cell malignant growth and metastasis. Thioredoxin-1 (TXN), the main regulator of oxidative stress, plays a significant role in the development of NSCLC. However, the specific tumor-promoting mechanism is still being investigated. This study aimed to examine the function and mechanism of TXN in NSCLC. The effects of knockdown or overexpression TXN on cell proliferation, invasion and apoptosis were evaluated by Cell Counting Kit-8, colony formation, wound healing, transwell, TUNEL staining, and flow cytometric assays. Western blotting was performed to analyze the regulation of TXN and downstream proteins suppressed by genes and pharmacology. TXN knockdown significantly suppressed cell proliferation, invasion and promoted apoptosis both in vitro and in vivo, whereas TXN overexpression reversed these malignant phenotypes. We found that TXN regulated c-Myc expression through ERK1/2 and ERK5 signaling pathways. Suppressing ERK1/2 led to the compensatory activation of ERK5, and simultaneously inhibiting ERK1/2 and ERK5 synergistically reduced c-Myc expression, further attenuating cell proliferation, invasion and enhanced apoptosis. Our results indicated tumor promotion of TXN in NSCLC and TXN regulated c-Myc in the interest of tumorigenesis through ERK1/2 and ERK5 signaling pathways. Targeting TXN and blocking the ERK1/2 and ERK5 pathways could potentially offer new therapeutic strategies for NSCLC.

Exploring the sequence diversity and surface expression of Factor H-Binding Protein among invasive serogroup B meningococcal strains from selected European countries

ABSTRACT Factor H-Binding Protein (fHbp) is a key component of meningococcal vaccines such as MenB-fHbp, licensed in the EU, UK, and other countries. Sufficient expression of fHbp on the bacterial surface is necessary for vaccine-induced antibodies to bind and exert bactericidal activity. The flow cytometric MEASURE assay quantifies fHbp expression in vitro, and previous studies have shown that strains with a Mean Fluorescence Intensity (MFI) >1000 are likely to be killed by MenB-fHbp-induced antibodies. This study assessed fHbp peptide distribution and expression among 451 invasive group B strains collected in 2016 across England, Wales, and Northern Ireland (EW&NI), Germany, Italy, and Spain. We found that 92% of the strains expressed fHbp above the MFI 1000 threshold. The strain distribution across EW&NI, Germany, and Italy was similar, with coverage ranging from 92.1% to 94.6%, dominated by a small number of clonal complexes and fHbp peptides. Although, the Spanish subset had a higher proportion of lower-expressing strains, particularly clonal complex 213, resulting in a lower predicted coverage for Spain (84%). These results, along with other published MEASURE data, can provide a basis for genotypic MenB-fHbp coverage predictions, however, inclusion of the upstream intergenic sequence of the fHbp gene in the prediction improved its accuracy by distinguishing between low- and high-expressing strains. Future MEASURE analyses of strains with less common fHbp variants would serve to further refine vaccine coverage predictions.

TMIC-63. DUAL IMMUNE CHECKPOINT BLOCKADE IN A NOVEL EGFR-DRIVEN MURINE GLIOMA MODEL INCREASES PERIPHERAL T CELL PROLIFERATION BUT FAILS TO PROLONG SURVIVAL

Abstract Patients with glioblastoma (GBM) survive only 15-18 months following standard-of-care surgical resection and radio-chemotherapy. Immune checkpoint blockade (ICB), effective in extracranial cancers, failed to improve survival in patients with recurrent GBM in a multi-center phase III clinical trial. As such, we are interested in the molecular mechanisms that drive ICB resistance. Oncogenic EGFR activation is observed in ~50% of GBMs and is associated with immunosuppression; however, whether EGFR signaling impairs T cell function in GBM resulting in limited ICB efficacy remains unclear. Therefore, we leveraged the novel MADR-mEGFRvIII glioma model, which expresses murine EGFRvIII under a tetracycline-off system, to investigate the relationship between EGFR activation and responsiveness to dual anti-PD-1 and anti-CTLA-4 (10mg/kg) in vivo. Mice were bled prior to enrollment and weekly thereafter, which confirmed anti-PD-1-PD-1 binding via competitive flow cytometric assay. Following two weeks on treatment, animals were euthanized for immunophenotypic analyses of tumors and tumor-draining lymph nodes. These analyses revealed a significantly increased prevalence of peripheral CD11b+ myeloid cells and enhanced peripheral T cell proliferation, indicated by Ki67 positivity, after treatment with dual ICB compared to isotype control. However, these shifts did not translate to effective ICB-mediated recruitment of peripheral T cells to the tumor microenvironment and no significant survival benefit was observed (p=0.68, n=15/treatment arm). Given our previous findings that EGFR ablation via doxycycline enhanced intratumoral T cell infiltration, we hypothesize the rational combination of EGFR-targeted therapy and ICB will have a synergistic effect; thus, future directions include a study combining the two treatment regimens consisting of a lead-in with doxycycline to increase intratumoral T cell trafficking, followed by treatment with dual ICB. This work will provide insight into the interplay between oncogenic signaling and immunosuppression, contributing to the development of novel combination therapies in treating this lethal disease.

New Indole-Based Phenylthiazolyl-2,4-dihydropyrazolones as Tubulin polymerization inhibitors: Multicomponent Synthesis, Cytotoxicity Evaluation, and in silico Studies.

A facile multicomponent synthesis of new indole-based phenylthiazolyl-dihydropyrazolone hybrids, their structural characterization, biological evaluation, and in silico investigations as anticancer agents are reported. Lead molecule 5 i of the series showed potent activity against MCF-7 breast cancer cells with an IC50 of 3.92±0.01 μM while showing minimal toxicity to normal human lung cells (IC50=69.85±3.95 μM). Further studies show that the compound exhibits antiproliferative activity by inducing apoptosis in MCF-7 cancer cells. The wound healing assay indicated impaired cell migration under the concentration-dependent dosage. The lead molecule 5 i also successfully inhibited the tubulin polymerase enzyme with an IC50 of 4.16±0.18 μM. A flow cytometric assay indicated compound 5 i induced apoptosis through G0 phase cell cycle arrest. The binding mode and interactions of the compound with the tubulin were predicted by molecular modelling and calculating binding free energies. These findings explain the current series as a new class of microtubule polymerization inhibitors with anticancer activity suitable for developing anticancer agents targeting tubulin.

RAD51 Degradation Triggered By MALAT1 Silencing Confers Synthetic Lethality to PARPi in AML

Introduction Acute myeloid leukemia (AML) critically relies on the activity of RAD51, the central eukaryotic recombinase essential for the homologous recombination (HR) repair of double-strand DNA breaks. PARP inhibitors (PARPi), the first clinically approved drugs designed to exploit the concept of synthetic lethality, selectively target cancer cells with HR deficiencies, particularly those harboring BRCA1/BRCA2 mutations, while sparing normal cell. However, the clinical efficacy of PARPi in AML has been limited, largely due to the infrequency of mutations in DDR genes within these malignancies. Despite this, AML cells exhibit a high accumulation of DNA lesions compared to normal cells, implicating their heavy dependence on DDR pathways, particularly RAD51. Therefore, targeting RAD51 in combination with PARPi in AML cells, even without DDR gene mutations, may exploit synthetic lethality to selectively eradicate leukemic cells while sparing normal hematopoietic cells. Despite the fact that AML cell survival relies on RAD51 overactivation, the regulatory mechanisms governing RAD51 activity in AML remain insufficiently understood. MALAT1, a highly conserved nuclear long non-coding RNA (lncRNA), stands out due to its exceptional evolutionary conservation and extraordinary abundance among lncRNAs. It plays a crucial oncogenic role and is highly expressed across various types of cancer, making it an attractive target for cancer therapy. Methods RNA sequencing was performed on bone marrow samples from seven AML patients and four healthy individuals to identify the genes crucially associated with the progression of AML. Further analyzed MALAT1 expression using two publicly available microarray datasets. Tumorigenicity assays were performed to determine the effects of MALAT1 on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on leukemogenesis or survival of NSG mice xenografted with AML cells (in vivo). Meanwhile, impacts of MALAT1 on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. RNA sequencing to analyze and compare the expression profiles of mRNAs from K562 control and MALAT1 knockout cells. Concurrently, protein mass spectrometry (MS) was conducted to identify differentially expressed proteins between MALAT1 knockout and control cells. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of MALAT1 and RAD51, followed by RNA-pulldown and CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, RAD51/EMI1 interaction and RAD51 ubiquitination in cells in which MALAT1 expression was manipulated. Xenograft mouse models were established by injecting human AML cells into NOG mice via tail vein injection to evaluate the role of MALAT1 and PARPi in the malignant progression of AML in vivo. Results MALAT1 expression positively associates with poor prognosis in AML. Knockout of total MALAT1 resulted in decreased colony formation and proliferation, and increased apoptosis. And cells in G0/G1 phase increased significantly, while the cells in the S phase and G2/M phase decreasedMALAT1 knockout results in decreased. MALAT1 silencing triggers homologous recombination deficiency (HRD) in AML cells. RAD51 protein levels without significant changes in RAD51 mRNA levels, suggesting post-translational regulation may play a key role in its stability. Further investigations demonstrated that MALAT1 directly interacts with RAD51, preventing its ubiquitination and thus stabilizing the protein to promote leukemogenesis. Co-IP experiments suggested that MALAT1 regulates RAD51 ubiquitination by inhibiting the interaction between EMI1 and RAD51. MALAT1 silencing inhibits the progression of AML and triggers PARPi synthetic lethality in AML cells and in vivo. Conclusions MALAT1 modulates RAD51 activity by preventing its degradation, and silencing MALAT1 can induce synthetic lethality in AML cells when combined with PARP inhibitors. This strategy offers a novel therapeutic avenue, though careful management of specificity, toxicity, and resistance is essential for successful clinical application.

Ly6E on tumor cells impairs anti-tumor T-cell responses: a novel mechanism of tumor-induced immune exclusion

BackgroundLymphocyte antigen 6 complex, locus E (Ly6E) has been initially demonstrated to involve in T cell activity and impair viral infectivity. Recently, high expression levels of Ly6E have been reported in tumor microenvironment (TME) of various types of cancers. However, the immunoregulatory mechanism of Ly6E manipulating TME remains unknown.MethodsTCGA database and Kaplan–Meier plotter database were used to evaluate the correlation between Ly6E expression levels and cancer patient survival. After analyzing Ly6E expression levels in human breast cancer tissues and tumor cell lines, we generated Ly6E knockout (KO) and overexpression (OE) mouse cell lines. Cell proliferation ability in vitro and the ability of growth and metastasis in mouse tumor models were compared between KO/OE and wild-type tumor cells. On day 7 after tumor implantation, tumor tissues were separated for flow cytometric assay, bulk RNA sequencing and single-cell RNA sequencing (ScRNA-seq). The role of Ly6E-expressing tumor cell on macrophage was analyzed in vitro.ResultsOur result surprisingly found that high Ly6E expression levels were associated with CD8+ T cell exclusion in tumor tissues and resistance to immunotherapy. Our data showed that knockout of Ly6E in tumor cells prompts tumor regression and inhibits tumor metastases, and Ly6E-OE tumor cells vice versa. The enhanced anti-tumor effect of Ly6E knockout in tumor cells was dependent on T cell response and formed long-lasting memory. The increase in the CD8+ T-cell infiltration into the tumor islet of Ly6E-KO tumors confirmed the role of Ly6E on T cell exclusion. ScRNA-seq analysis showed that M2 macrophages are particularly abundant in the Ly6E-expressing tumor tissues, especially M2-4 macrophage cluster identified by high levels of Arg-1, indicates that Ly6E-expressing tumor cells might restrict T cell infiltration via M2 macrophages. Moreover, in vitro assay showed that cell culture media derived from Ly6E-positive tumor cells promoted macrophage migration and M2 polarization.ConclusionOur study illuminated that Ly6E-expressing tumor cells facilitated the accumulation of M2 macrophages in TME, which contributes to CD8+ T cell exclusion and provides new insights for improving efficacy of cancer immunotherapy.

Combined Treatment of Caffeic Acid Phenethyl Ester With Docetaxel Inhibits Survival of Non-small-cell Lung Cancer Cells via Suppression of c-MYC.

Non-small-cell lung cancer (NSCLC) comprises approximately 85% of lung cancer. Treatment with docetaxel prolongs the survival of patients with NSCLC. However, the development of resistance to docetaxel has compromised its efficacy. Caffeic acid phenethyl ester (CAPE) has been reported to suppress survival and radiotherapy resistance in lung cancer cells. We determined in this study if combination treatment of docetaxel with CAPE suppresses the proliferation and the survival of NSCLC cells more effectively. Proliferation, viability, flow cytometric and comet assays were used to examine the difference in anticancer effects of combined treatment as compared to docetaxel treatment alone. Western blot and gene overexpression were used to unravel the underlying molecular mechanism. Treatment with docetaxel or CAPE alone dose-dependently suppressed the proliferation and survival of H1299 and A549 cells. Combined treatment of docetaxel with CAPE caused greater inhibition of survival of H1299 and A549 cells. Docetaxel alone and the combined treatment both dose-dependently increased apoptosis of H1299 cells; however, combined treatment induced much more apoptosis than docetaxel alone. Combined treatment suppressed the protein expression of phospho-protein kinase B (AKT, Ser 473), S-phase protein 2 (SKP2), MYC proto-oncogene bHLH transcription factor (c-MYC), epidermal growth factor receptor (EGFR), phospho-EGFR (Tyr 1045, and Tyr 992) but increased levels of cleaved caspase 3 and cytochrome c proteins in H1299 and A549 cells. The inhibition of expression of SKP2, c-MYC, phospho-EGFR (Tyr 992) proteins by combined treatment was significantly greater than that with treatment using either CAPE or docetaxel alone. Overexpression of c-MYC in rescued proliferation of H1299 cells under combination treatment. Our study revealed that the combination of CAPE with docetaxel is more effective at reducing the proliferation and survival of NSCLC cells, and this is via inhibition of c-MYC. Combined therapy of docetaxel and CAPE may benefit patients with NSCLC.

Retracted] Activating autophagy and ferroptosis of 3‑Chloropropane‑1,2‑diol induces injury of human umbilical vein endothelial cells via AMPK/mTOR/ULK1.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the flow cytometric assay data shown in Fig. 2C on p. 5, certain of the fluorescence microscopy data shown in Fig. 4B on p. 7, the FerroOrange‑stained cellular data in Fig. 5B and the C11‑BODIPY581/591‑stained cellular data in Fig. 5C on p. 8, and the cell autophagic data in Fig. 6E and G on p. 9 were strikingly similar to data that had already been submitted for publication in different form in different articles written by different authors at different research institutes (a few of which have now been retracted). Owing to the fact that the contentious data in the above article had already been published, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a satisfactory reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 27: 76, 2023; DOI: 10.3892/mmr.2023.12963].

Development of β‐Carboline‐Coumarin Based Hybrids as Potential Cytotoxic and Topoisomerase IIα Inhibitors

AbstractCancer remains a global health concern, prompting extensive efforts to develop novel inhibitors targeting the enzyme topoisomerase IIα as potential anti‐cancer agents. Herein, we strategically combined the pharmacophores from natural (β‐carboline) and (coumarin) sources, diversifying them at two distinct points. The in vitro evaluation of cytotoxic properties were estimated on human cancer cell lines, reveals that among the tested compounds, 13 r (isopropyl‐substituted β‐carboline) exhibited remarkable potency with IC50 value of 4.37 μM in the HCT116 cell line. Its selectivity for cancer cells was validated against normal keratinocyte cells, establishing a favourable selectivity index. Further, SAR studies indicate that no substitution (13 r) at C‐6 and C‐7 of β‐carboline, enhance anticancer activity. Assays confirmed its ability to intercalate with DNA, inhibit topoisomerase IIα, induce apoptosis, and disrupt mitochondrial membrane potential in HCT116 cells. Flow cytometric assays gauged the induction of apoptosis using Annexin‐V/PI dual staining and the disruption of mitochondrial membrane potential through JC staining. Results demonstrated that compound 13 r induced apoptosis and dose‐dependent depolarization of the mitochondrial membrane in HCT116 cells. Molecular docking studies further validated that compound 13 r binds within the active site of DNA when complexed with topo IIα, and this binding was stabilized through interactions with DNA base pairs and amino acid residues.

Integration of a fully automated flow cytometry system with high robustness into a Screening Station

In recent years, there has been an increasing demand for the detection of rare cells in drug discovery research, such as cells that have differentiated off-purpose or are required for immunogenicity evaluation. Since detection and quantification limits depend on the robustness of the experiment, inter-human differences in technique have a significant impact on the performance of the assay system. Here, we integrated flow cytometry into a cell experiment platform, Screening Station, to construct a robust assay system, examined each step of the flow cytometric pretreatment using Jurkat cells, and finally evaluated the overall assay performance. Cell detection rate when the experiment was performed manually was 48.8 % ± 5.7 % (CV=11.6 %) versus 73.7 %±2.0 % (CV=2.8 %) with the automated method. To further clarify the analytical performance of the automated method, 1–100 PD-1 expressing Jurkat cells were spiked with 1 × 105 Jurkat cells, and the lower limit of detection, linearity, and CV% were evaluated. Average detection rate was 69 %, decision count was 0.985, and lower limit of detection was 4 cells (0.004 %). We evaluated the CV% value of the number of detected cells per spiked cell and found our system to be highly robust, approximating a binomial distribution with a 69 % recovery rate. In conclusion, we have integrated the Novocyte flow cytometry system into an automated experimental platform, Screening Station, to create a fully automated flow cytometric assay system with high robustness. Our platform can fulfill the technology needs of drug discovery for rare cell detection, which have intensified in recent years.

Synthesis and Antiproliferative Activity of Cisplatin-3-Chloropiperidine Conjugates.

We report the synthesis and characterization of two novel cisplatin- alkylating agents conjugates. Combining a platinum based cytostatic agent with a sterically demanding alkylating agent could potentially induce further DNA damage, block cell repair mechanisms and keep the substrate active against resistant tumor cell lines. The 3-chloropiperidines utilized as ligands in this work are cyclic representatives of the N-mustard family and were not able to coordinate platinum on their own. The introduction of a second coordination site, in form of a pyridine moiety, led to the isolation of the desired conjugates. They were characterized with HRMS, CHN-analyses and XRD. We concluded this work by examining the cytotoxicity of the ligands and the obtained complexes with MTT assays in human cancer cell lines. While the ligands showed hardly any activity, the novel conjugates both displayed a high antiproliferative and cytotoxic potency in a panel of three cell lines. Moreover, both complexes were able to largely circumvent the acquired cisplatin resistance of A2780cisR ovarian cancer cells, both in the MTT assay and a flow-cytometric apoptosis assay.

Cisplatin induces acute liver injury by triggering caspase-3/GSDME-mediated cell pyroptosis

BackgroundCisplatin triggers Gasdermin E (GSDME) cleavage, causing membrane bubble formation, content release, and inflammation. Caspase-3 activation initiates GSDME cleavage, and thus inhibiting this pathway mitigates cisplatin-induced pyroptosis in hepatocytes. This study aimed to delve into how cisplatin induces liver injury via pyroptosis. MethodsFor animal experiments, C57BL/6J mice were divided into three groups: control, liver injury model group, and Ac-DMLD-CMK (caspase-3 inhibitor) intervention group. The liver histology was evaluated by hematoxylin and eosin staining, immunohistochemistry, immunofluorescence and TUNEL staining. The mRNA and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot analysis. For in vitro experiments, HL-7702 cells were treated with cisplatin or GSDME siRNA. Cell pyroptosis was determined via cellular morphology, cytotoxicity and viability detection, flow cytometric assay, and Western blot detection for the expression of pyroptosis-related proteins. ResultsCisplatin-induced distinct liver morphological changes, hepatocellular injury, and inflammation in mice, along with elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and increased pro-inflammatory cytokine expression. Heightened macrophage infiltration and hepatocellular death indicated cisplatin-induced hepatotoxicity. Cisplatin upregulated GSDME activation, along with Bax-mediated caspase-3 cleavage both in vivo and in vitro, implicating caspase-3/GSDME-dependent pyroptosis in liver injury. Treatment with Ac-DMLD-CMK ameliorated cisplatin-induced liver injury, reducing hepatocellular lesions, serum ALT and AST levels, cytokine expression, macrophage infiltration, and hepatocyte death. Ac-DMLD-CMK also attenuated GSDME-dependent pyroptosis post-cisplatin induction, as evidenced by decreased GSDME expression, Bax upregulation, and cleaved caspase-3 activation. For HL-7702 cells, GSDME siRNA transfection reduced GSDME expression, attenuated typical signs of cisplatin-induced pyroptosis, partially restored cell viability, and significantly inhibited cytotoxicity and a decrease in the proportion of propidium iodide-positive cells, indicating protection against cisplatin-induced hepatocyte pyroptosis. ConclusionOur study underscores the role of the caspase-3/GSDME signaling pathway in mediating cisplatin-induced hepatotoxicity, particularly in cases of excessive or cumulative cisplatin exposure. These findings suggest that targeting GSDME could represent a promising therapeutic approach to mitigate cisplatin-induced liver damage.

Flow cytometric analysis for Ki67 assessment in formalin-fixed paraffin-embedded breast cancer tissue

BackgroundPathologists commonly employ the Ki67 immunohistochemistry labelling index (LI) when deciding appropriate therapeutic strategies for patients with breast cancer. However, despite several attempts at standardizing the Ki67 LI, inter-observer and inter-laboratory bias remain problematic. We developed a flow cytometric assay that employed tissue dissociation, enzymatic treatment and a gating process to analyse Ki67 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue.ResultsWe demonstrated that mechanical homogenizations combined with thrombin treatment can be used to recover efficiently intact single-cell nuclei from FFPE breast cancer tissue. Ki67 in the recovered cell nuclei retained reactivity against the MIB-1 antibody, which has been widely used in clinical settings. Additionally, since the method did not alter the nucleoskeletal structure of tissues, the nuclei of cancer cells can be enriched in data analysis based on differences in size and complexity of nuclei of lymphocytes and normal mammary cells. In a clinical study using the developed protocol, Ki67 positivity was correlated with the Ki67 LI obtained by hot spot analysis by a pathologist in Japan (rho = 0.756, P < 0.0001). The number of cancer cell nuclei subjected to the analysis in our assay was more than twice the number routinely checked by pathologists in clinical settings.ConclusionsThe findings of this study showed the application of this new flow cytometry method could potentially be used to standardize Ki67 assessments in breast cancer.

Diosmetin-7-O-β-D-glucopyranoside from Pogostemonis Herba alleviated SARS-CoV-2-induced pneumonia by reshaping macrophage polarization and limiting viral replication

Ethnopharmacological relevanceViral pneumonia is the leading cause of death after SARS-CoV-2 infection. Despite effective at early stage, long-term treatment with glucocorticoids can lead to a variety of adverse effects and limited benefits. The Chinese traditional herb Pogostemonis Herba is the aerial part of Pogostemon Cablin (Blanco) Benth., which has potent antiviral, antibacterial, anti-inflammatory, and anticancer effects. It was used widely for treating various throat and respiratory diseases, including COVID-19, viral infection, cough, allergic asthma, acute lung injury and lung cancer. Aim of the studyTo investigate the antiviral and anti-inflammatory effects of chemical compounds from Pogostemonis Herba in SARS-CoV-2-infected hACE2-overexpressing mouse macrophage RAW264.7 cells and hACE2 transgenic mice. Materials and methodsThe hACE2-overexpressing RAW264.7 cells were exposed with SARS-CoV-2. The cell viability was detected by CCK8 assay and cell apoptotic rate was by flow cytometric assay. The expressions of macrophage M1 phenotype markers (TNF-α and IL-6) and M2 markers (IL-10 and Arg-1) as well as the viral loads were detected by qPCR. The mice were inoculated intranasally with SARS-CoV-2 omicron variant to induce viral pneumonia. The levels of macrophages, neutrophils, and T cells in the lung tissues of infected mice were analyzed by full spectrum flow cytometry. The expressions of key proteins were detected by Western blot assay. ResultsDiosmetin-7-O-β-D-glucopyranoside (DG) presented the strongest anti-SARS-CoV-2 activity. Intervention with DG at the concentrations of 0.625–2.5 μM not only reduced the viral replication, cell apoptosis, and the productions of inflammatory cytokines (IL-6 and TNF-α) in SARS-CoV-2-infected RAW264.7 cells, but also reversed macrophage polarity from M1 to M2 phenotype. Furthermore, treatment with DG (25–100 mg/kg) alleviated acute lung injury, and reduced macrophage infiltration in SARS-COV-2-infected mice. Mechanistically, DG inhibited SARS-COV-2 gene expression and HK3 translation via targeting YTHDF1, resulting in the inactivation of glycolysis-mediated NF-κB pathway. ConclusionsDG exerted the potent antiviral and anti-inflammatory activities. It reduced pneumonia in SARS-COV-2-infected mice via inhibiting the viral replication and accelerating M2 macrophage polarization via targeting YTHDF1, indicating its potential for COVID-19 treatment.

Implementation of flow cytometry testing on rare matrix samples: Special considerations and best practices when the sample is unique or difficult to obtain.

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

Additive antitumor effect of arsenic trioxide with exposure to ionizing radiation to human acute promyelocytic leukemia HL‑60 cells.

Arsenic trioxide (ATO) is expected to be a chemical drug with antitumor activity against acute promyelocytic leukemia (APL), a type of acute myeloid leukemia. In Japan, its antitumor effects were confirmed in clinical trials for APL, and it has been approved in various countries around the world. However, there have been no reports on ATO's antitumor effects on radioresistant leukemia cells, which can be developed during radiotherapy and in combination with therapeutic radiation beams. The present study sought to clarify the antitumor effect of ATO on APL cells with radiation resistance and determine its efficacy when combined with ionizing radiation (IR). The radiation‑resistant HL60 (Res‑HL60) cell line was generated by subjecting the native cells to 4‑Gy irradiation every week for 4 weeks. The half‑maximal inhibitory concentration (IC50) for cell proliferation by ATO on native cell was 0.87 µM (R2=0.67), while the IC50 for cell proliferation by ATO on Res‑HL60 was 2.24 µM (R2=0.91). IR exposure increased the sub‑G1 and G2/M phase ratios in both cell lines. The addition of ATO resulted in a higher population of G2/M after 24 h rather than 48 h. When the rate of change in the sub‑G1 phase was examined in greater detail, the sub‑G1 phase in both control cells without ATO significantly increased by exposure to IR at 24 h, but only under the condition of 2 Gy irradiation, it had continued to increase at 48 h. Res‑HL60 supplemented with ATO showed a higher rate of sub‑G1 change at 24 h; however, 2 Gy irradiation resulted in a decrease compared with the control. There was a significant increase in the ratio of the G2/M phase in cells after incubation with ATO for 24 h, and exposure to 2 Gy irradiation caused an even greater increase. To determine whether the inhibition of cell proliferation and cell cycle disruptions is related to reactive oxygen species (ROS) activity, intracellular ROS levels were measured with a flow cytometric assay. Although the ROS levels of Res‑HL60 were higher than those of native cells in the absence of irradiation, they did not change after 0.5 or 2 Gy irradiation. Furthermore, adding ATO to Res‑HL60 reduced intracellular ROS levels. These findings provide important information that radioresistant leukemia cells respond differently to the antitumor effect of ATO and the combined effect of IR.

Evidence of Perivascular Mesenchymal Bone Marrow Microenvironment Affection in Poor Graft Function Post Allogenic Stem Cell Transplantation

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is regarded as a curative treatment option for a variety of hematological disorders. The prognosis of allogenic (HSCT) patients is dependent on a variety of factors that affect the bone marrow microenvironment. One of these factors is the number and function of CD146+ perivascular mesenchymal cells that support the hematopoietic stem cells in the bone marrow and help their proliferation. Aim of the Study The aim of this study was to assess how the bone marrow microenvironment is being affected in patients with poor graft function post allogenic stem cell transplantation targeting the perivascular mesenchymal cells using multiparameter flow cytometric analysis of CD146 antibody. Patients and Methods Forty patients were enrolled in this study with both benign and malignant hematological diseases who underwent allogenic HSCT from matched related donors. Patients were divided into 2 equal groups: poor graft function (PGF) group and good graft function group (GGF). Patients were subjected to full history recording, clinical examination, full laboratory and radiological work-up, tissue typing and chemotherapeutic conditioning before transplantation. After transplant, bone marrow aspirate was obtained and analyzed for CD146+ perivascular mesenchymal cells using Quantitative tri-color Flow cytometric assay. Results PGF group showed significantly lower counts of WBCs, platelets and hemoglobin concentration compared to GGF group. In addition, CD34 dose showed significantly lower concentration in PGF group compared to GGF group. CD146 expression intensity on non-stem cells was significantly higher in PGF group compared to the GGF group to increase the function of these cells in supporting the proliferation of stem cells in the bone marrow. Conclusion CD146 can have a potential therapeutic role if CD146+ selected mesenchymal cells are infused in patients with refractory poor graft function to enhance the proliferation of stem cells and help in recovery of patients with PGF however further studies are warranted to document its suggested therapeutic role.

Evidence of Endothelial Bone Marrow Microenvironment Affection in Poor Graft Function Post Allogenic Stem Cell Transplanration

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is considered a curative option for several hematological disorders. The prognosis of patients undergoing allogenic (HSCT) is dependable on various factors that affect bone marrow microenvironment and poor prognosis can results from poor graft function. On of these factors is the numbers of CD31+ endothelial cells that support the hematopoietic stem cells. Aim of the Study The aim of this study was to assess how the bone marrow microenvironment is being affected in patients with poor graft function post allogenic stem cell transplantation targeting the endothelial cells using multiparameter flow cytometric analysis of CD31 antibody. Patients and Methods Forty patients were enrolled in this study with both benign and malignant hematological diseases who underwent allogenic HSCT from matched related donors. Patients were divided into 2 equal groups: poor graft function (PGF) group and good graft function group (GGF). Patients were subjected to full history recording, clinical examination, full laboratory and radiological work-up, tissue typing and chemotherapeutic conditioning before transplantation. After transplant, bone marrow aspirate was obtained and analyzed for CD31+ endothelial cells using Quantitative tri-color Flow cytometric assay. Results PGF group showed significantly lower counts of WBCs, platelets and hemoglobin concentration compared to GGF group. In addition, CD34 dose showed significantly lower concentration in PGF group compared to GGF group. PGF group had statistically significant lower percentage of stem cells total count compared to GGF group in addition, PGF Group had a significantly higher mean fluorescence intensity (MFI) of CD31 on non-stem cells compared to GGF group. Conclusion PGF had significant increase in MFI of CD31+ve on non-stem cells compared to GGF group. In addition, PGF group had statistically significant lower percentage of CD31+ stem cells total count compared to GGF group.