Flow Cytometric Analysis Research Articles

Flow cytometry for meningeal infiltration in B acute lymphoblastic leukemia in a low middle income country.

Meningeal infiltration in children with B acute lymphoblastic leukemia is one of the most serious complications. Timely diagnosis not only significantly enhances treatment efficacy but also leads to improve patient outcome and reduce risk of relapse. This is particularly crucial in low to middle income countries facing health constraints, where optimizing resources is essential. Conventional cytology (CC) study of cerebrospinal fluid (CSF) is considered in different countries to be the Gold-standard despite its low sensitivity (< 50%). The study of CSF by multiparametric flow cytometry (MFC) appears to be an alternative. The aim of our study was to assess MFC analytical performance compared with CC. Our cross sectional study was conducted over a six-month period in the biological hematology department. CSF samples underwent analysis for the presence of blasts using both CC and MFC. Cytological slides of the CSF were prepared by cytocentrifugation in a Shandon Cytospin 4™. Flow cytometric analysis was performed on the BD FACSLyric™ flow cytometer. All statistical analyses were performed using SPSS version 21.0 (SPSS Inc.). Agreement between the two methods was made using the Kappa index and χ2 test. This study was approved by the local ethics committee. Sixty CSF samples from 39 children with B acute lymphoblastic leukemia were analyzed. Meningeal infiltration was detected respectively in 20% of cases by MFC and 5% of cases by CC, with a significant difference p = 0.006. Comparing the two methods, the Kappa coefficient was 0.35, indicating weak agreement between the two methods. Moreover, MFC positivity was higher even for hypocellular samples. Of the 51 hypocellular samples, eight were positive by MFC while they were negative by CC. MFC shows better sensitivity while retaining good specificity for the detection of meningeal involvement. MFC could therefore be a complementary method to CC for detecting blast cells in the central nervous system.

A prospective study of posttransplant cyclophosphamide for unrelated donor peripheral blood stem cell transplant with special attention to graft content and the impact of a higher γδ T cell dose.

Posttransplant cyclophosphamide (PtCy) has been shown to decrease post-hematopoietic stem cell transplant acute and chronic graft-versus-host disease (GVHD). In this study, PtCy was used in 44 patients along with mycophenolate and tacrolimus with HLA matched (29) and mismatched (15) unrelated donors to determine the impact of graft content on outcome; thus, all patients had flow cytometric analysis of their graft content including the number of B cells, NK cells, and various T cell subsets. Higher γδ T cell dose was associated with the development of acute GVHD (p = .0038). For PtCy, further studies of the cell product along with further graft manipulation, such as selective γδ T cell depletion, could potentially improve outcomes.

Simple, fast, cost-efficient, reliable, and highly automated DNA content analysis of cells in adherent cultures.

The most commonly used flow cytometric (FCM) analysis of cellular DNA content relies on ethanol fixation followed by RNA digestion and propidium iodide (PI) intercalation into double-stranded DNA. This is a laborious and time-consuming procedure that is subject to systematic errors due to centrifugation and washing steps associated with sample preparation. It can adversely affect the reliability of the results. Here, we present a modified concept of DNA quantification in adherent cell lines by FCM that involves neither ethanol fixation nor any washing and cell transferring steps. Our high throughput assay of adherent cell lines reduces sample-processing time, requires minimal workload, provides a possibility for automation, and, if needed, also allows a significant reduction in the size of individual samples. Working with a well-proven commercial tool-The BD Cycletest™ Plus DNA Reagent Kit-primarily designed for cell cycle analysis and aneuploidy determination in experimental and clinical samples, we suggest a novel, very efficient, and robust approach for DNA research in adherent cell cultures.

Abstract PO4-28-07: SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism

Abstract Background: The disturbance of lipid metabolism in the microenvironment plays an important role in the occurrence and development of triple-negative breast cancer(TNBC). SLC26A9, a member of the SLC26A family of anion transporters, is involved in breast oncogenesis. However, the effect of SLC26A9 in triple-negative breast cancer on lipid metabolism in the tumor microenvironment remains unclear. Methods: Bioinformatics, tissue microarrays and TNBC cell lines were used to detect the expression of SLC26A9 and its clinical significance. By altering the expression of SLC26A9 gene in TNBC cells, the effects of SLC26A9 on the regulation of lipid metabolism, cell biological behavior and its related molecular mechanisms in the TNBC microenvironment were explored using adoptive staining techniques, flow cytometric analysis and animal models. Results: We found by tissue microarray (TNBC=105 cases, Nor=67 cases) that the expression of SLC26A9 was significantly up-regulated in BC compared to paracancerous tissues, where SLC26A9 expression was higher in TNBC, and the same results were obtained in BC cell lines. Secondly, we found that SCL26A9 was positively correlated with lipid metabolism status in TNBC microenvironment and possessed a close association with P53 mutation by analyzing GEO and TCGA databases. Therefore, we used two cell lines (MDA-MB-231, MDA-MB-468) in which SCL26A9 was highly expressed in TNBC cell lines. It was demonstrated by oil red O staining and lipid fluorescence staining that knockdown/silencing of SLC26A9 diminished the ability of MDA-MB-231 and MDA-MB-468 cells to uptake and utilize lipids, especially for metabolites such as neutral lipids: triglycerides, lipid droplets, and steroidal substances. Then we demonstrated by using fatty acid flow cytometric analysis that knockdown/silencing of SLC26A9 significantly reduced the utilization and production of fatty acids in MDA-MB-231 and MDA-MB-468 cells, which are the key metabolic indicators of cancer cells as well as important energy-providing substances for cancer progression. Therefore, we further verified functionally that cell proliferation, migration, invasion and anti-apoptosis were significantly inhibited in in vitro after knockdown/silencing of SLC26A9, and in vivo tumorigenesis was significantly inhibited in BALB/c nude mice in in vivo. In addition, the expression of markers of lipid metabolic activity (FASN, ACLY) was reduced after knockdown/silencing of SLC26A9 in MDA-MB-231 cells by Western Blot, and the reduced expression of FASN and ACLY mainly inhibited the ab initio synthesis, uptake and utilization of fatty acids in TNBC cells, which in turn regulated the proliferation and invasion of TNBC. Mechanistically, SLC26A9 silencing/knockdown inhibited the synthesis, uptake, and utilization of multiple metabolites of lipids in TNBC, which combined with the TCGA database results revealed the activation of the key signaling pathway, the JAK-STAT3 signaling pathway, to promote the progression of TNBC. Conclusion(s): SLC26A9 is involved in the disturbance of lipid metabolism in the TNBC microenvironment and regulates the progression of TNBC. The search for new SLC26A9 inhibitors is expected to provide a new direction for the treatment of TNBC, but the exact molecular mechanisms need to be further explored. SLC26A9 upregulation in TNBC leads to a poor prognosis by referencing lipid metabolism in the TNBC microenvironment Wench. SLC26A9 is involved in lipid metabolism disorders and cell biological functions in the TNBC microenvironment in vivo. SLC26A9 is involved in disordered lipid metabolism in the TNBC microenvironment. Citation Format: Yingming Zhou, Hu Wang, Zhengxing Zhou, Xuemei Liu, Biguang Tuo, Taolang Li. SLC26A9 promotes triple-negative breast cancer progression by regulating lipid metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-07.

Synthesized Gold Nanoparticles with Moringa Oleifera leaf Extract Induce Mitotic Arrest (G2/M phase) and Apoptosis in Dalton's Lymphoma Cells.

The therapeutic potential of chemically synthesized AuNPs has been demonstrated in various types of cancer. However, gold nanoparticles (AuNPs) synthesized using typical chemical methods have concerns regarding their environmental safety and adverse impact on human well-being. To overcome this issue, we used an environmentally friendly approach in which gold nanoparticles were synthesized using Moringa oleifera leaf extract (MLE). The present research was mainly focused on the biosynthesis and characterization of gold nanoparticles (AuNPs) using Moringa oleifera leaf extract (MLE-AuNPs) and explore its anticancer potential against Dalton's Lymphoma (DL) cells. Characterization of the MLE-AuNPs was conducted using UV-Vis Spectroscopy to confirm the reduction process, FTIR analysis to ascertain the presence of functional groups, and XRD analysis to confirm the crystallinity. SEM and TEM images were used to examine size and morphology. After characterization, MLE-AuNPs were evaluated for their cytotoxic effects on Dalton's lymphoma cells, and the results showed an IC50 value of 75 ± 2.31 µg/mL; however, there was no discernible cytotoxicity towards normal murine thymocytes. Furthermore, flow cytometric analysis revealed G2/M phase cell cycle arrest mediated by the downregulation of cyclin B1 and Cdc2 and upregulation of p21. Additionally, apoptosis induction was evidenced by Annexin V Staining, accompanied by modulation of apoptosis-related genes including decreased Bcl-2 expression and increased expression of Bax, Cyt-c, and Caspase-3 at both the mRNA and protein levels. Collectively, our findings underscore the promising anti-cancer properties of MLE-AuNPs, advocating their potential as a novel therapeutic avenue for Dalton's lymphoma.

Abstract PO5-24-12: IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells

Abstract Research Objectives and Rationale: Previously, RARγ agonism has been shown to play an essential role in CD8 T-cell-mediated immunity to infectious pathogens. However, we found no previous reports on effects of RARγ agonists on in vivo tumor growth of triple negative or Her2+ breast cancers. Here we explored whether RARγ could play a critical role in T-cell-mediated immunity in these breast cancers, using potent highly selective novel RARγ agonist compounds. Experimental methods and results: We synthesized and screened compounds for selective transactivation of RAR γ in reporter assays. Three highly potent compounds, demonstrating highly selective RARγ transactivation at less than one nanomolar concentrations, were selected for further evaluation of potential anti-cancer activities in vitro. None of the three evaluated compounds demonstrated significant inhibitory effects on in vitro growth of EMT-6 triple negative breast cancer cells at concentrations of 20-100 millimolar, indicating that the three compounds have insignificant direct inhibitory effects on EMT-6 cancer cell growth in vitro. We then examined the effects of one of the compounds in vivo in a syngeneic mouse model of EMT-6 triple negative breast cancer. Treatment with the most potent of the three compounds, IRX5010, demonstrated dose response inhibition of in vivo growth of EMT-6 tumors. Inhibition of tumor growth at day 17 relative to vehicle was 59% and 69% respectively in mice treated with IRX5010 at 10 or 25 mg/kg/day. A cohort treated with 50 mg/kg/day was sacrificed early on day 12 due to excessive weight loss and deaths of some of the animals. This cohort experienced a 73% inhibition of tumor growth relative to vehicle. Gross necropsies of animals in the 25 and 50 mg/kg/day dose groups did not show observable pathologic findings in any major organ. Flow cytometry was performed on freshly excised tumors on the day of termination of the in-life portion of the study in the 10 and 25 mg/kg/day dose groups. Tumors from mice treated with IRX5010 had increased intratumoral numbers of Total CD45+ cells; Total CD3+, Total CD4+, and Total CD8+ T-cells per mg of tumor tissue, relative to vehicle controls. IRX5010 treated mice also had increased intratumoral CD4+IL-17+ T-helper cells; and increased CD4+CD62L- and CD8+CD62L- T-effector memory T-cells per mg of tumor tissue relative to vehicle-treated control mice. We also conducted an experiment of treatment with IRX5010 in mice transplanted with human immune cells (to provide a human immune system) to examine its effects on xenografted human Her2+ JIMT-1 cancer cells. In this model, treatment with IRX5010 at 10 mg/kg/day resulted in a 17% inhibition of tumor growth relative to vehicle control over 26 days of treatment. Flow cytometric analysis showed that tumors from the IRX5010 treated mice contained increased numbers per mg of tumor tissue of intratumoral human CD8+ T-cells, most of which were CD8+ CD7- CD45RA+ terminally differentiated effector memory phenotype T-cells. Conclusions: Treatment with IRX5010, a potent and highly selective RARγ nuclear receptor agonist compound demonstrated substantial treatment effects relative to vehicle control on inhibition of growth of murine triple negative breast cancer in vivo, despite having essentially no growth inhibitory effect on these cells in vitro. Treatment of xenografted human Her2+ tumors in mice with humanized immune systems resulted in a moderate inhibition of Her2+ breast cancer growth relative to vehicle controls. In both models, treatment with IRX5010 induced tumor infiltrating T-lymphocytes of largely effector memory phenotypes, demonstrating an effect on the tumor immune microenvironment. These data identify RARγ and RARγ agonists as a new treatment target and novel treatment modality for breast cancers, especially for the highly unmet need for new and better treatments for triple negative breast cancer. Citation Format: Martin Sanders, Mary Topalovski, Vidyasagar Vuligonda. IRX5010, a Highly Selective RARγ Nuclear Receptor Agonist Compound Inhibits In Vivo Growth of Murine EMT-6 Triple Negative Breast Cancer and Human JIMT-1Her2+ Breast Cancer By Induction of Tumor-Infiltrating Effector Memory T-cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-12.

Batf3+ DCs and the 4-1BB/4-1BBL axis are required at the effector phase in the tumor microenvironment for PD-1/PD-L1 blockade efficacy

The cellular source of positive signals that reinvigorate Tcells within the tumor microenvironment (TME) for the therapeutic efficacy of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade has not been clearly defined. We now show that Batf3-lineage dendritic cells (DCs) are essential in this process. Flow cytometric analysis, gene-targeted mice, and blocking antibody studies revealed that 4-1BBL is a major positive co-stimulatory signal provided by these DCs within the TME that translates to CD8+ Tcell functional reinvigoration and tumor regression. Immunofluorescence and spatial transcriptomics on human tumor samples revealed clustering of Batf3+ DCs and CD8+ Tcells, which correlates with anti-PD-1 efficacy. In addition, proximity to Batf3+ DCs within the TME is associated with CD8+ Tcell transcriptional states linked to anti-PD-1 response. Our results demonstrate that Batf3+ DCs within the TME are critical for PD-1/PD-L1 blockade efficacy and indicate a major role for the 4-1BB/4-1BB ligand (4-1BBL) axis during this process.

Structural characterization of the polysaccharide from the black crystal region of Inonotus obliquus and its effect on AsPC-1 and SW1990 pancreatic cancer cell apoptosis

In this study, one water soluble polysaccharide (IOP1–1) with a weight average molecular weight of 6886 Da was obtained from the black crystal region of Inonotus obliquus by hot water extraction, DEAE-52 cellulose extraction and Sephadex-100 column chromatography purification. Structural analysis indicated that IOP1–1 was a glucan with a main chain composed of α-Glcp-(1 → 4)-α-Glcp-(1 → 4)-β-Glcp-(1 → 4)-β-Glcp-(1 → 4)-α-Glcp-(1 → 6)-β-Glcp-(1 → 4)-α-Glcp-(1 → 3)-β-Glcp-(1→. The CCK-8 assay results showed that IOP1–1 inhibited AsPC-1 and SW1990 pancreatic cancer cell proliferation in a concentration-dependent manner. Flow cytometric analysis revealed that IOP1–1 induced cell cycle arrest in AsPC-1 and SW1990 cells. Hoechst 33342 staining and Annexin V-FITC/PI double staining analysis showed that IOP1–1 could induce apoptosis in AsPC-1 and SW1990 cells. Furthermore, western blot analysis confirmed that IOP1–1 could induce apoptosis in AsPC-1 and SW1990 pancreatic cancer cells through three pathways: the mitochondrial pathway, the death receptor pathway, and endoplasmic reticulum stress. According to these research data, IOP1–1 may be utilized as an adjuvant treatment to anticancer medications, opening up new application prospects and opportunities.

Flow cytometric analysis of immune cell populations in the bronchial and mesenteric lymph nodes of the dromedary camel

Dromedary camel is an important livestock species with special economic value in arid and semi-arid regions of the world. Given the limited data on detailed immune cell composition and cell marker expression in the dromedary camel lymph node tissue, the present study was undertaken to investigate the immune cell composition of bronchial and mesenteric lymph nodes from healthy dromedary camels using flow cytometry. In this study, we applied flow cytometry and multicolor immuno-fluorescence to phenotype the main populations of immune cells in the bronchial and mesenteric camel lymph nodes and compared them with separated peripheral blood mononuclear cells and granulocytes. We used antibodies to detect several cell surface molecules associated with camel T cells (CD4, WC1), B cells (MHCII, BAQ44A), monocytes/macrophages (CD172a, CD14, CD163), in addition to the pan-leukocyte marker CD45 and the cell adhesion molecules CD44 and CD18. Compared to blood mononuclear cells, camel lymph node cells contained a higher percentage of lymphoid cells with only a minor fraction of myeloid cells. In addition, the lower expression of CD44 and CD18 on lymph node lymphocytes compared to lymphocytes from peripheral blood indicates higher frequency of naïve lymphocytes in the lymph nodes. The frequency of CD4+ T cells, B cells and γδ T cells within camel lymph node lymphocytes compared to blood indicates a similar tissue distribution pattern of lymphocyte subsets in camel and bovine and supports previous reports on the similarity between the camel immune system and the immune system of other ruminants. Lymph node neutrophils were identified as CD45++ CD172a++, CD14+, MHCIIlow, BAQ44A+, CD44++, CD18++ cells. In conclusion, the present study is describing the employment of flow cytometric single-cell analysis and immunostaining for the analysis of the immune cell composition in the camel lymph node.

The Evolving Landscape of Flowcytometric Minimal Residual Disease Monitoring in B-Cell Precursor Acute Lymphoblastic Leukemia

Detection of minimal residual disease (MRD) is a major independent prognostic marker in the clinical management of pediatric and adult B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), and risk stratification nowadays heavily relies on MRD diagnostics. MRD can be detected using flow cytometry based on aberrant expression of markers (antigens) during malignant B-cell maturation. Recent advances highlight the significance of novel markers (e.g., CD58, CD81, CD304, CD73, CD66c, and CD123), improving MRD identification. Second and next-generation flow cytometry, such as the EuroFlow consortium’s eight-color protocol, can achieve sensitivities down to 10−5 (comparable with the PCR-based method) if sufficient cells are acquired. The introduction of targeted therapies (especially those targeting CD19, such as blinatumomab or CAR-T19) introduces several challenges for flow cytometric MRD analysis, such as the occurrence of CD19-negative relapses. Therefore, innovative flow cytometry panels, including alternative B-cell markers (e.g., CD22 and CD24), have been designed. (Semi-)automated MRD assessment, employing machine learning algorithms and clustering tools, shows promise but does not yet allow robust and sensitive automated analysis of MRD. Future directions involve integrating artificial intelligence, further automation, and exploring multicolor spectral flow cytometry to standardize MRD assessment and enhance diagnostic and prognostic robustness of MRD diagnostics in BCP-ALL.

Modulation of Human Bone Marrow Mesenchymal Stem Cells (BMMSC) by Nigella Sativa and Trigona Honey: An in vitro Study

Maintenance of bone marrow mesenchymal stem cells (BMMSC) numbers is crucial for tissue repair and regeneration since adult stem cells are often limited in number, expansion capacity and lifespan. It is believed that certain types of foods are beneficial in the preservation and stimulation of stem cells throughout life. Black seeds and stingless bee honey are among the widely investigated functional food with general health promoting benefit. We aim to explore the proliferative, anti-apoptosis and anti-senescence effect of Nigella sativa and Trigona honey on BMMSC. Cell proliferation was evaluated using MTT assay. We performed flow cytometric analysis to verify stem cell surface markers while quantitative PCR was done to measure the relative expression of target genes. Results showed favourable concentration dependent enhancement of cell proliferation by 1 to 10 ug/mL Nigella sativa and 0.097% to 0.195% Trigona honey. Observation on BMMSC’s morphology and surface markers expression revealed no alteration on BMMSC stemness properties. In addition, gene expression analysis supported that there was a significant (P&lt;0.05) increase in proliferation related gene, β-Catenin, and lower expression of apoptotic- and senescence-related gene, BAX and p21, respectively. These preliminary findings suggest a promising effect of Nigella sativaand Trigona honey on stem cell proliferation.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis

Abstract Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection is critical for rigorous evaluation of in vivo metastasis models. Clinical data shows that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high-throughput, broadly applicable and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

Increased tumor-infiltrating plasmacytoid dendritic cells express high levels of PD-L2 and affect CD8+ T lymphocyte infiltration in human laryngeal squamous cell carcinoma

The infiltration and prognostic significance of tumor-infiltrating plasmacytoid dendritic cells (TI-pDC) have been elucidated in various human solid cancers. However, the infiltrating patterns and functional importance of TI-pDC in laryngeal squamous cell carcinoma (LSCC) remain unknown. In this study, flow cytometric analyses were conducted to characterize the infiltration of dendritic cells and T lymphocytes, along with their respective subgroups in tumor tissues (TT), para-carcinoma tissues (PT), and peripheral blood (PB) from LSCC patients. Immunohistochemical staining for CD4 and CD8, as well as immunofluorescence staining for CD123, were performed on serial tissue sections to investigate the co-localization of TI-pDC and tumor-infiltrating T lymphocytes (TIL) within the tumor microenvironment (TME). Our results demonstrated significantly lower percentages of all three DC subsets in PB compared to TT and PT. Notably, the pDC percentage was markedly higher in TT than in PT. Moreover, TI-pDC percentage was significantly elevated in N+ stage patients compared to those with N0 stage. The results of survival analysis consistently demonstrated that high levels of TI-pDC infiltration were indicative of a poor prognosis. Further investigation revealed a significant negative correlation between TI-pDC and CD8+ TILs; notably, pDCs expressed an inhibitory surface molecule PD-L2 rather than PD-L1 within PT. Collectively, our findings suggest that increased TI-pDC is associated with adverse outcomes in LSCC patients while exhibiting an inhibitory phenotype that may play a crucial role in suppressing CD8+ TILs within LSCC tumors. These results highlight the potential therapeutic strategy targeting PD-L2+ pDCs for immunotherapies against LSCC.

Fabrication and in vitro–in vivo evaluation of ligand appended isoniazid loaded nanoparticulate systems for the treatment of tuberculosis

Isoniazid (INH) is amongst the first-line antibiotics that have been employed for the treatment of Tuberculosis (TB). Despite its potent anti-tubercular action, susceptibility to rapid hepatic first-pass metabolism and elimination largely limits its oral bioavailability, and has been associated with the induction of drug resistance and adverse effects. This presents study aims at the development and evaluation of unique mannosylated INH loaded solid lipid nanoparticles (Mn-INH-SLNs) for the treatment of TB. The Mn-INH-SLNs demonstrated a particle size of 466 ± 11 nm, which was acceptable for macrophage targeting and had % entrapment efficiency of 80.41 ± 1.37% (n = 6). The dissolution studies depicted a dual-phase drug release profile, i.e., burst release followed by a sustained release, revealing the best fit with the Korsmeyer-Peppas model. The MTT assay (cytotoxicity study) performed utilizing J774A.1 cells with optimized Mn-INH-SLNs deemed them to be safe and nontoxic. Mannosylated SLNs tagged with coumarin-6 (C6 – an established fluorescent marker) showed a substantially high intracellular internalization (1.11-folds) when analyzed through flow cytometric analysis (FACS). The in vivo pharmacokinetic evaluation following per-oral administration in rats demonstrated a significant rise in relative bioavailability (∼5.5-folds) with Mn-INH-SLNs compared to pure drug solution. The bio-distribution (drug disposition) studies exhibited greater lung accumulation of Mn-INH-SLNs (2.13-folds) in comparison to the unconjugated INH-SLNs (Un-INH-SLNs). The promising results of the study depict that the targeted-optimized Mn-INH-SLNs may be a propitious and potent tool to fight TB.

Dried storage of sperm at supra-zero temperatures: An alternative for flow cytometric analysis under field conditions

In biotechnological processes such as chromosomal manipulation studies, semen has become a reference in the ploidy verification of the evaluated material. However, the use of fresh samples is limited to the use at field conditions because the analysis is performed under laboratory conditions. Thus, this study aimed to develop a simpler procedure for storing dry semen at 28 °C to reduce cold storage costs. For this, semen samples were evaluated according to established quality semen parameters, a protocol for dry, and 3 sterilization treatments of dry semen were applied to the store. The integrity of the DNA was evaluated every two months, using fresh semen, dry semen (untreated), and particles 3C to compare the peaks by flow cytometry. The results indicated that all samples evaluated before and after drying showed no significant difference in the DNA content. UV-treated semen showed a 1C peak in the histogram up to 180 days of storage and a non-significant difference (P > 0.05) from fresh control in the number of DNA particles up to 120 days and untreated only showed a 1C peak up to 120 days. The developed method may become an interesting procedure to serve as a reference peak for practical flow cytometric analysis, not only in the field of fish biology but also in biomedical and agricultural sciences. Furthermore, dried semen can become a tool for the preservation of genetic material and is a promising low-cost storage technique for biobanking.

SiRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression

BackgroundBreast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets.MethodsTranscriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target.ResultsIntegrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours.ConclusionOur findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Immunological Characteristics of Hepatic Dendritic Cells in Patients and Mouse Model with Liver Echinococcus multilocularis Infection

The cestode Echinococcus multilocularis, which mainly dwells in the liver, leads to a serious parasitic liver disease called alveolar echinococcosis (AE). Despite the increased attention drawn to the immunosuppressive microenvironment formed by hepatic AE tissue, the immunological characteristics of hepatic dendritic cells (DCs) in the AE liver microenvironment have not been fully elucidated. Here, we profiled the immunophenotypic characteristics of hepatic DC subsets in both clinical AE patients and a mouse model. Single-cell RNA sequencing (scRNA-Seq) analysis of four AE patient specimens revealed that greater DC numbers were present within perilesional liver tissues and that the distributions of cDC and pDC subsets in the liver and periphery were different. cDCs highly expressed the costimulatory molecule CD86, the immune checkpoint molecule CD244, LAG3, CTLA4, and the checkpoint ligand CD48, while pDCs expressed these genes at low frequencies. Flow cytometric analysis of hepatic DC subsets in an E. multilocularis infection mouse model demonstrated that the number of cDCs significantly increased after parasite infection, and a tolerogenic phenotype characterized by a decrease in CD40 and CD80 expression levels was observed at an early stage, whereas an activated phenotype characterized by an increase in CD86 expression levels was observed at a late stage. Moreover, the expression profiles of major immune checkpoint molecules (CD244 and LAG3) and ligands (CD48) on hepatic DC subsets in a mouse model exhibited the same pattern as those in AE patients. Notably, the cDC and pDC subsets in the E. multilocularis infection group exhibited higher expression levels of PD-L1 and CD155 than those in the control group, suggesting the potential of these subsets to impair T cell function. These findings may provide valuable information for investigating the role of hepatic DC subsets in the AE microenvironment and guiding DC targeting treatments for AE.

Differentiation of neural stem cells from human olfactory mucosa into dopaminergic neuron-like cells.

The aim of this study was to develop an alternative treatment method for neurodegenerative diseases with dopaminergic neuron loss such as Parkinson's disease by differentiating cells obtained from human olfactory mucosa-derived neural stem cells (hOM-NSCs) with neurotrophic agents in vitro. hOM-NSCs were isolated and subjected to immunophenotypic and MTT analyses. These hOM-NSCs were then cultured in a 3D environment to form neurospheres. The neurospheres were subjected to immunophenotypic analysis and neuronal differentiation assays. Furthermore, hOM-NSCs were differentiated into dopaminergic neuron-like cells in vitro. After differentiation, the dopaminergic neuron-like cells were subjected to immunophenotypic (TH, MAP2) and genotypic (DAT, PITX3, NURR1, TH) characterization. Flow cytometric analysis showed that NSCs were positive for cell surface markers (CD56, CD133). Immunofluorescence analysis showed that NSCs were positive for markers with neuronal and glial cell characteristics (SOX2, NESTIN, TUBB3, GFAP and NG2). Immunofluorescence analysis after differentiation of hOM-NSCs into dopaminergic neuron-like cells in vitro showed that they were positive for a protein specific for dopaminergic neurons (TH). qRT-PCR analysis showed that the expression of dopaminergic neuron-specific genes (DAT, TH, PITX3, NURR1) was significantly increased. It was concluded that hOM-NSCs may be a source of neural stem cells that can be used for cell replacement therapies in neurodegenerative diseases such as Parkinson's disease, are resistant to cell culture, can differentiate into neuronal and glial lineage, are easy to obtain and are cost effective.

P179 CD19hi B cells express T-bet and display activated metabolism and function in rheumatoid arthritis and systemic lupus erythematosus patient samples

Abstract Background/Aims CD19, a co-receptor of the B cell receptor, fine-tunes the threshold for B-cell activation with implications for cell metabolism and function. Activating receptors such as CD40, IFNγR and TLRs also influence B cell activation. B cell subpopulation, double negative (DN, IgD-CD27-) T-bet+ B cells, is considered pathogenic in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Here, we explored whether CD19hi B cells differed from CD19lo B cells with regards to proliferation, metabolism, and function. Methods Peripheral blood samples from nine healthy participants, 16 RA and 20 SLE patients were analysed. Subgroup analysis of rituximab-treated (RTX-T), n = 6 versus rituximab naïve (RTX-N), n = 6, was performed. B cells or peripheral blood mononuclear cells were stimulated with anti-IgG, anti-IgM and anti-human CD40 antibodies and a TLR9 agonist for 20 hours prior to flow cytometric analysis of B cell expression of phosphorylated AMPK (pAMPK, the master regulator of metabolism), intracellular expression of Ki67 (proliferation marker), and cytokines IL-6 and IL-10. Statistical analysis was performed using GraphPad Prism v10.0.1. Results Compared to CD19+CD20+ B cells, CD19+CD20- B cells: 1) were predominantly composed of DN (55% versus 19%, p &amp;lt; 0.01) and IgD-CD27+ memory subpopulations (37% versus 25%, p = 0.1094); 2) were detectable at a hierarchical frequency, RTX-T&amp;gt;RTX-N&amp;gt;HC cohorts; 3) had a greater frequency of Ki67+ cells (38% versus 5%, p &amp;lt; 0.01); 4) displayed higher CD38 MFI (41478.0 versus 10589.0, p &amp;lt; 0.01). Approximately 50% of CD19hi B cells were T-bet+, compared to 28% of CD19lo B cells (n = 33, p &amp;lt; 0.0001). Compared to CD19lo, CD19hi B cells 1) expressed (MFI) significantly greater TLR7, TLR9, IFNγR and pAMPK (n = 7 for all except pAMPK, n = 4), and 2) had greater frequency of IL6+ and IL10+ B cells (n = 7, p &amp;lt; 0.05) (Table). Conclusion CD20- B cell subpopulations with potent proliferative capacity express CD19. Further, CD19hi B cells: 1) upregulate the TLR-IFNγ-T-bet pathway; 2) have significantly greater expression of pAMPK; 3) have higher cytokine expression compared to CD19lo B cells. These data suggest the activation pathway, metabolic activity, and functional profile of CD19hi cells. Therefore, the potential of CD19hi cells to promote inflammation provides strong rationale for targeting CD19 in autoimmune disease with emerging therapies such as CAR-T cells and T cell engagers. Disclosure K. Shah: Grants/research support; KS has received funding for research from Roche. M.J. Leandro: Consultancies; MJL acted as a consultant for Roche and Genentech. Other; MJL received funds less than $10000 and support to attend educational conferences. M.S. Cragg: Consultancies; Consultant for BioInvent International, Boehringer Ingelheim, GSK, Radiant, iteos, Surrozen, Hanall and Mestag. Honoraria; He has received honoraria/speakers fees from Boehringer Ingelheim, Amgen, Roche and Symphogen. Grants/research support; BioInvent International, GSK and UCB. F. Kollert: Corporate appointments; FK is employed by Roche. Shareholder/stock ownership; FK has stock ownership at Roche. F. Schuler: Corporate appointments; FS is employed by Roche. Shareholder/stock ownership; FS has patents and stock ownership at Roche. D.A. Isenberg: None. A. Akbar: None. C. Klein: Corporate appointments; CK is employed by Roche. Shareholder/stock ownership; CK has patents and stock ownership at Roche. V. Reddy: Grants/research support; VR has received a research grant from Roche.

Enhancing standard of care chemotherapy efficacy using DNA-dependent protein kinase (DNA-PK) inhibition in preclinical models of Ewing sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on non-homologous end joining (NHEJ) to repair DNA damage. In this study, we investigated if pharmacological inhibition of the enzyme responsible for NHEJ, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe Additivity Model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single agent TOP2 poisons induced G2M arrest and little apoptotic response, while adding DNA-PKi mediated apoptosis. In vivo, the combination of AZD-7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.