Dead Cells Research Articles

PROGRESSIVE MUSCLE HYPOXIA RESEARCH: A MODIFIED METHOD FOR ASSESSING CELL VIABILITY IN SKELETAL MUSCLE BIOPSIES USING VITALITY DYES AND CONFOCAL MICROSCOPY

AbstractObjectivesAcute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency and can have detrimental outcomes for patients if not treated optimally. The current problem is that there is no clear diagnostic threshold for ACS or guidance as to when fasciotomies should be performed. A new diagnostic method(s) is necessary to provide real-time information about the extent of muscle ischaemia in ACS. Given that lactic acid is produced by cells through anaerobic respiration, it may be possible to measure H+ ion concentration and to use this as a measure of ischaemia within muscle. Although we are familiar with the key biochemical metabolites involved in ischaemia; and the use of viability dyes in cell culture to distinguish between living or dead cells is well recognised; research has not been undertaken to correlate the biochemical and histological findings of ischaemia in skeletal muscle biopsies. Our primary aim was to investigate the potential for viability dyes to be used on live skeletal muscle biopsies (explants). Our secondary aim was to correlate the intramuscular pH readings with muscle biopsy viability.MethodsNine euthanised Wistar rats were used. A pH catheter was inserted into one exposed gluteus medius muscles to record real-time pH levels and muscle biopsies were taken from the contralateral gluteus medius at the start of experiment and subsequently at every 0.1 of pH unit drop. Prior to muscle biopsy, the surface of the gluteus medius was painted with a layer of 50µmol/l Brilliant blue FCF solution to facilitate biopsy orientation. A 4mm punch biopsy tool was used to take biopsies. Each muscle biopsy was placed in a base mould filled with 4% ultra-low melting point agarose. The agarose embedded tissue block was sectioned to generate 400 micron thick tissue slices with a vibratome. The tissue slices were then placed in the staining solution with Hoechst 33342, Ethidium homodimer-1 and Calcein am. The tissue slices were imaged with Zeiss LSM880 confocal microscope's Z stack function. A dead muscle control was created by adding TritonX-100 to other tissue slices. For quantitative analyses, the images were analysed in Image J using the selection tool. This permitted individual cells to be identified and the mean grey value of each channel to be defined. Using the dead control, we were able to identify the threshold value for living cells using the Calcein AM channel.ResultsViability dyes, used primarily for cell cultures, can be used with skeletal muscle explants. Our study also showed that despite a significant reduction in tissue pH concentration over time, that almost 100% of muscle cells were still viable at pH 6.0, suggesting that skeletal muscle cells are robust to hypoxic insult in the absence of reperfusion.ConclusionsViability dyes can be used on skeletal muscle biopsies. Further research investigating the likely associations between direct measured pH using a pH catheter, the concentrations of key cellular metabolic markers, and muscle tissue histology using vitality dyes in response to ischaemia, rather than hypoxia, is warranted.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Effects of Light Intensity on the Growth and Biochemical Composition in Various Microalgae Grown at High CO2 Concentrations.

In modern energy, various technologies for absorbing carbon dioxide from the atmosphere are being considered, including photosynthetic microalgae. An important task is to obtain maximum productivity at high concentrations of CO2 in gas-air mixtures. In this regard, the aim of the investigation is to study the effect of light intensity on the biomass growth and biochemical composition of five different microalgae strains: Arthrospira platensis, Chlorella ellipsoidea, Chlorella vulgaris, Gloeotila pulchra, and Elliptochloris subsphaerica. To assess the viability of microalgae cells, the method of cytochemical staining with methylene blue, which enables identifying dead cells during microscopy, was used. The microalgae were cultivated at 6% CO2 and five different intensities: 80, 120, 160, 200, and 245 μmol quanta·m-2·s-1. The maximum growth rate among all strains was obtained for C. vulgaris (0.78 g·L-1·d-1) at an illumination intensity of 245 µmol quanta·m-2·s-1. For E. subsphaerica and A. platensis, similar results (approximately 0.59 and 0.25 g·L-1·d-1 for each strain) were obtained at an illumination intensity of 160 and 245 µmol quanta·m-2·s-1. A decrease in protein content with an increase in illumination was noted for C. vulgaris (from 61.0 to 46.6%) and A. platensis (from 43.8 to 33.6%), and a slight increase in lipid content was shown by A. platensis (from 17.8 to 21.4%). The possibility of increasing microalgae biomass productivity by increasing illumination has been demonstrated. This result can also be considered as showing potential for enhanced lipid microalgae production for biodiesel applications.

In situ detection and viability assessment of target microorganisms

Rapid detection and viability assessment of pathogenic microorganisms, without the need for pre-enrichment steps, is critical in clinical microbiology, food safety, environmental quality assessment, and biosecurity. We demonstrate a powerful analytical concept and the related platform that enable in situ rapid detection, separation, sensitive quantification, and viability assessment of targeted microorganisms (bacteria and fungi) from minimally processed samples. This is based on a novel integration of magneto-affine selection and electrical impedance assay. The entire process, from capture to measurement, is executed using controlled magnetic fields to manipulate magnetic particles (MPs)-microbe affinity-based clusters, in a compact, portable setup equipped with cost-effective, single-use chambers. The system was tested for non-invasive in situ evaluation of model bacteria (Escherichia coli) and fungi (Saccharomyces cerevisiae) within clinically relevant concentration ranges, and it was demonstrated amenable for both commercial and custom MPs, proving its high versatility. The high capture efficiency, the ability to provide analytic results within 30 min directly from unprocessed samples (buffer and synthetic urine), and the high sensitivity in distinguishing live and dead cells in dynamic exposures represent significant advancements over existing assays and recommend the system as a screening tool for pathogen presence and antimicrobial susceptibility in clinical and environmental samples.

Engineering alginate/carboxymethylcellulose scaffolds to establish liver cancer spheroids: Evaluation of molecular variances between 2D and 3D models

Natural polymeric hydrogels represent an optimal framework for 3D culture development. This study demonstrates a freeze-thaw-based ionic crosslinking technique for fabricating alginate/carboxymethylcellulose scaffold for culturing human hepatocellular carcinoma, Huh-7 cells to generate 3D spheroids. Consolidating morphological and biomechanical characterization of Alg/CMC scaffolds shows the formation of uniform hydrogels with significant crosslinking (ATR-FTIR), multiscale pores (FE-SEM), swelling/water absorbance, softer texture, viscoelasticity (rheology), spreading nature (contact angle), and degradation rate optimal for 3D culture establishment. The influence of cell seeding density and time with spheroid formation reveals a maximal size of 250–300 μm on day 7. Calcein AM and Propidium iodide staining confirm that a culmination of viable and dead cells generates spheroidal heterogeneity. RT-qPCR in 3D culture against RPL-13 and 2D culture controls indicate an upregulation of E-cadherin, N-cadherin, fibronectin, and integrin α9/β6. Further, western blotting and immunofluorescence confirm the collective display of cellular interactions in 3D spheroids. Thus, the expression profile signifies the role of key genes during the assembly and formation of 3D spheroids in 1%Alg/1%CMC scaffolds with a profound epithelial characteristic. In the future, this study will bring a 3D spheroid model in a platter for elucidating epithelial to mesenchymal transition of cells during in vitro disease modeling.

New Steroidal Selenides as Proapoptotic Factors.

Cytostatic and pro-apoptotic effects of selenium steroid derivatives against HeLa cells were determined. The highest cytostatic activity was shown by derivative 4 (GI50 25.0 µM, almost complete growth inhibition after three days of culture, and over 97% of apoptotic and dead cells at 200 µM). The results of our study (cell number measurements, apoptosis profile, relative expression of apoptosis-related APAF1, BID, and mevalonate pathway-involved HMGCR, SQLE, CYP51A1, and PDHB genes, and computational chemistry data) support the hypothesis that tested selenosteroids induce the extrinsic pathway of apoptosis by affecting the cell membrane as cholesterol antimetabolites. An additional mechanism of action is possible through a direct action of derivative 4 to inhibit PDHB expression in a way similar to steroid hormones.

BIOM-27. CELL-FREE DNA-BASED LIQUID BIOPSIES FOR THE REAL-TIME TRACKING OF TUMOR BURDEN AND TREATMENT RESPONSE IN GLIOBLASTOMA

Abstract Cell-free DNA (cfDNA) are short fragments of DNA that are released by apoptotic or necrotic cells into bodily fluids. It has emerged as a potential diagnostic tool allowing for the detection and analysis of tumor-derived biomarkers in cancer. Despite the detailed view provided by clinical imaging, its inability to quantify tumor burden and distinguish between pseudo-progression and pseudo-response poses a challenge for patients. In this study, we sought to investigate the effectiveness of cfDNA as a liquid biopsy tool to track tumor burden and disease progression in real time in different glioblastoma models and through different types of biofluids. To examine the feasibility of cfDNA-based tumor tracking we cultivated several patient-derived glioblastoma lines, purified cfDNA and performed sequencing. Further analysis revealed that dead cells were associated with an elevated cfDNA yield and altered fragment size profiles. Genomic sequencing revealed consistent variants shared across serial cfDNA timepoints, strengthening the suggestion that cfDNA can be used as a non-invasive biomarker. In order to demonstrate the clinical implications of our methodology, we collected a preliminary batch of n = 43 longitudinal intraventricular cerebrospinal fluid (CSF) samples from a patient that underwent CAR-T treatment over the span of two years. We observed a marked increase in cfDNA yield post-treatment compared to samples taken prior to treatment administration. Furthermore, cfDNA-based estimates of tumor burden measured as allele fraction specific to the tumor, tracked with clinical imaging-based tumor size estimates. These preliminary findings provide compelling evidence supporting the clinical utility of cfDNA analysis in monitoring the effectiveness and dynamics of CAR-T treatment. By tracking cfDNA levels in CSF, we can identify patterns reflecting treatment response and aid in assessing the therapeutic efficiency of CAR-T treatment in glioma patients. Further investigations with larger patient cohorts and more comprehensive data analysis to validate these findings are actively underway.

IMMU-14. SINGLE CELL RNASEQ OF PBMCS FROM NEWLY-DIAGNOSED GLIOBLASTOMA PATIENTS

Abstract INTRODUCTION A multitude of recently published papers have found immunosuppression caused by glioblastoma (GBM) is not limited to the tumor microenvironment and impacts the peripheral immune system. Our group has recently reported an expansion of granulocyte macrophage precursors, a subset of hematopoietic stem and progenitor populations (HSPCs), in glioma-bearing mice relative to healthy, age-matched controls. Thus we sought to determine if GBM causes cell fate dysfunction in CD34+ HSPCs in patients. METHODS CD34+ cells were magnetically isolated from peripheral blood of newly-diagnosed GBM patients and healthy age-matched donors and processed for single-cell RNA sequencing. CD34+ cells were magnetically isolated from peripheral blood of newly-diagnosed GBM patients and healthy age-matched donors and processed for single-cell RNA sequencing using the 10X Genomics platform. CellRanger was used for primary analysis with default parameters. After quality control removal of doublets, dead, and low quality cells automated cell type annotation was performed using the Azimuth Hubmap Consortium human bone marrow reference. The annotated single cell object was then read into R and Seurat was used for differential expression analysis and visualization. RESULTS AND CONCLUSIONS There we no significant expansions of stem and progenitor populations. GBM patient CD34+ samples had upregulation of VWF, a marker of myeloid-bias cell subset. Within the mature immune cell compartments, we observed a significant expansion of NK cells in GBM patients including 1,997 genes differentially expressed in the NK cell cluster between cohorts. This includes downregulation of SLAMF6, which triggers cytolytic activity in NK cells, in GBM patients. Upregulation of early NK cell activation markers CD69 and JUN were seen in GBM patient samples. These results strongly suggest NK are likely activated in the context of GBM, but may not be functional.

Relationship between antimicrobial peptides-induced cell membrane damage and bactericidal activity

Most antimicrobial peptides (AMPs) act by killing bacterial cells. However, there is little information regarding the required interaction time between AMPs and bacterial cells to exert the bactericidal activity. One of the causes of the bactericidal activity is considered to be cell membrane damage, although little direct evidence is available. Here, we investigated the relationship between AMP-induced cell membrane damage in Escherichia coli and AMP-induced cell death at the single-cell level. Magainin 2, lactoferricin B, and PGLa were selected as the AMPs. First, we examined the interaction time (t) of AMPs with cells required to induce cell death using the single-cell analysis. The fraction of microcolonies containing only a single cell, Psingle (t), which indicates the fraction of dead cells, increased with time to reach ∼1 in a short time (≤5 min). Then, we examined the interaction between AMPs and single cells using confocal laser scanning microscopy in the presence of membrane-impermeable SYTOX green. Within a short time interaction, the fluorescence intensity of the cells due to SYTOX green increased, indicating that AMPs induced cell membrane damage through which the dye entered the cytoplasm. The fraction of cells in which SYTOX green entered the cytoplasm among all examined cells after the interaction time (t), Pentry (t), increased with time, reaching ∼1 in a short time (≤5 min). The values of Psingle (t) and Pentry (t) were similar at t ≥ 3 min for all AMPs. The bindings of AMPs to cells were largely reversible, whereas the AMP-induced cell membrane damages were largely irreversible because SYTOX green entered the cells after dilution of AMP concentration. Based on these results, we conclude that the rapid, substantial membrane permeabilization of cytoplasmic contents after a short interaction time with AMPs and the residual damage after dilution induce cell death.

CNSC-39. GENERATION AND CHARACTERIZATION OF LIVING ORGANOTYPIC BRAIN SLICE CULTURES AS A NOVEL PRE-CLINICAL MODEL FOR BRAIN CANCER

Abstract A challenge in cancer research is developing reproducible, reliable, and practical models which can capture the complexity of cancer development and treatment. The development of functional precision medicine platforms is emerging as a promising strategy for improving pre-clinical drug testing and guiding clinical decisions. We have developed an organotypic brain slice culture (OBSC) technology composed of intact multicellular tissue which can be rapidly used for spatiotemporal drug response testing. OBSCs are reproducibly generated from Sprague-Dawley rat pups and used as living tissue substrates to culture treat different tumor cell lines and uncultured patient brain tumor resection tissue. We evaluated OBSC quality and reproducibility throughout the study by using a propidium iodide nuclear permeability assay. This technique enabled a broad dynamic range to distinguish between healthy and unhealthy slices. In these studies, the rat pup age at the time of generation had an impact on OBSC quality and quantity, indicating that OBSCs should be generated from eight-day-old pups. We also found that optimal OBSCs were generated using improved methods for brain dissection and OBSC culture conditions, establishing our robust, standardized procedure for OBSC generation. Following this optimization period, we continued to conduct quality control analysis with 6 OBSCs per batch for reproducibility. We also conducted immunohistochemistry analysis immediately after slicing and concluded that morphology of neurons in OBSCs remained unchanged between day 0 and 4. In addition, the activation of astrocytes attenuates by day 4 but persists in macrophages/microglia, suggesting that the myeloid cells can phagocytose dead cells and debris in OBSCs. In summary, these results indicate that the OBSC platform may be an effective model that accelerates preclinical drug testing and directs drug development towards clinical evaluation.

Increased infiltration of CD4+ T cell in the complement deficient lymphedema model

BackgroundLymphedema is an intractable disease that can be caused by injury to lymphatic vessels, such as by surgical treatments for cancer. It can lead to impaired joint mobility in the extremities and reduced quality of life. Chronic inflammation due to infiltration of various immune cells in an area of lymphedema is thought to lead to local fibrosis, but the molecular pathogenesis of lymphedema remains unclear. Development of effective therapies requires elucidation of the immunological mechanisms involved in the progression of lymphedema. The complement system is part of the innate immune system which has a central role in the elimination of invading microbes and acts as a scavenger of altered host cells, such as apoptotic and necrotic cells and cellular debris. Complement-targeted therapies have recently been clinically applied to various diseases caused by complement overactivation. In this context, we aimed to determine whether complement activation is involved in the development of lymphedema.ResultsOur mouse tail lymphedema models showed increased expression of C3, and that the classical or lectin pathway was locally activated. Complement activation was suggested to be involved in the progression of lymphedema. In comparison of the C3 knockout (KO) mouse lymphedema model and wild-type mice, there was no difference in the degree of edema at three weeks postoperatively, but the C3 KO mice had a significant increase of TUNEL+ necrotic cells and CD4+ T cells. Infiltration of macrophages and granulocytes was not significantly elevated in C3 KO or C5 KO mice compared with in wild-type mice. Impaired opsonization and decreased migration of macrophages and granulocytes due to C3 deficiency should therefore induce the accumulation of dead cells and may lead to increased infiltration of CD4+ T cells.ConclusionsVigilance for exacerbation of lymphedema is necessary when surgical treatments have the potential to injure lymphatic vessels in patients undergoing complement-targeted therapies or with complement deficiency. Future studies should aim to elucidate the molecular mechanism of CD4+ T cell infiltration by accumulated dead cells.

A novel degradable PCL/PLLA strapping band for internal fixation of fracture

Early fracture fixation is the critical factor in fracture healing. Common internal fracture implants are made of metallic materials, which often affects the imaging quality of CT and MRI. Most patients will choose secondary surgery to remove the internal fixation implants, which causes secondary damage to them. The development of new degradable internal fracture implants has attracted more and more attention from orthopedic surgeons and researchers. Based on these problems, we improved the various properties of medical grade polycaprolactone (PCL) by adding poly(L-lactide) (PLLA). We produced PCL/PLLA strapping bands with different mass ratios by injection molding. We compared the mechanical properties, degradation properties, cell biocompatibility, bone marrow mesenchymal stem cells (BMSCs) adhesion, proliferation, osteogenic differentiation and fracture fixation effect of these strapping bands. The results showed that the tensile strength and yield force of the strapping bands increased with the increase of the content of PLLA. The addition of PLLA could significantly improve the mechanical strength in the early stage and accelerate the degradation rate of the strapping band. PCL/PLLA (80/20) strapping band had no significant cytotoxicity toward rBMSCs and could promote osteogenic differentiation of rBMSCs. The strapping band could ensure femoral fracture healing of beagles in 3 months and didn’t cause damage to the surrounding tissues and main organs. This study will provide some new insights into the biodegradable products of PCL/PLLA blends for internal fixation of fracture.Graphical We produced novel degradable PCL/PLLA strapping bands with different mass ratios by injection molding. We tested the biological safety of the prepared internal fixation strapping bands for fracture, such as cell experiment in vitro and animal experiment, and studied the degradation behavior in vitro. The strapping bands could ensure femoral fracture healing of beagles. This study will provide some new insights into the biodegradable products of PCL/PLLA blends for internal fixation of fracture. A Immunofluorescence staining of rBMSCs (live cells: green; dead cells: red). B Young’s modulus change curve during strapping bands degradation. C The implantation process of strapping bands. D Micro-CT images of the beagle’s fracture recovery after the operation.

Modulatory effect of pomegranate peel extract on key regulators of ovarian cellular processes in vitro.

In this study, response of ovarian cells (human granulosa cell line HGL5, and human adenocarcinoma cell line OVCAR-3) to short-term pomegranate peel extract (PPE) treatment (for 24 hours in cell culture) was evaluated in vitro. Quantitative and qualitative screening of polyphenols revealed punicalagins α and β as major polyphenolic components. Total phenolic content (TPC) was 93.76 mg GAE/g d.w. with a high antioxidant activity of 95.30 mg TEAC/g d.w. In OVCAR-3, PPE treatment inhibited the metabolic activity, and increased cyclin-dependent kinase 1 (CDKN1A, p21) level at the highest dose, but not in HGL5. Flow cytometry analysis could not detect any significant difference between proportions of live, dead, and apoptotic cells in both cell lines. Reactive oxygen species (ROS) revealed an antioxidant effect on HGL5, and a prooxidant effect by stimulating ROS generation in OVCAR-3 cells at the higher doses of PPE. However, in contrast to HGL5, PPE treatment decreased release of growth factors - TGF-β2 and EGF at the highest dose, as well as their receptors TGFBR2 and EGFR in OVCAR-3 cells. PPE also influenced steroidogenesis in granulosa cells HGL5 by stimulating 17β-estradiol secretion at higher doses. In conclusion, the present study highlighted the bioactive compounds in pomegranate peels and the possible mechanisms of action of PPE, shedding light on its promising role in ovarian cancer (chemo)prevention and/or management.

Illicium verum anticancer activity against MDA-MB-231 cell line

Objective: To evaluate the anticancer activity of methanolic extract of Illicium verum against triple-negative breast cancer MDA-MB-231 cell line.
 Methods: A cell culture experimental study was carried out at Pharmacology department of Bahria University Medical and Dental College (January to June 2021) in collaboration with Aga Khan University, Karachi, Pakistan. Cell viability and proliferation assays were used to quantify dead and alive cells by utilizing a tetrazolium assay and an enzyme immunosorbent plate reader was used to calculate their absorbance. For the apoptosis initiation assay, these cells were dyed with a fluorescent stain and observed for fluorescence and apoptosis. During cell viability testing, various I. verum methanolic extract doses (0.125, 0.25, 0.5, 1, 3, 6, 12, and 25µg/ml) were employed to treat MDA-MB-231 cells, while the IC50 dose of 2.8µg/ml was used for both the cell proliferation and apoptosis initiation assays.
 Results: In the cell viability assay, all I. verum methanolic extract doses exhibited a substantial decrease in the viability of MDA-MB-231 cells (less than 0.01 p-value). In cell proliferation assay and apoptosis initiation, the IC50 dose of 2.8µg/ml of I. verum methanolic extract also exhibited a substantial decrease in cell division (less than 0.01 p-value) and the initiation of apoptosis in MDA-MB-231 cells.
 Conclusion: Illicium verum methanolic extract have strong anticancer activity against triple-negative breast cancer MDA-MB-231 cell line through cytotoxicity, proliferation reduction, and apoptosis initiation mechanisms.
 doi: https://doi.org/10.12669/pjms.40.1.7860
 How to cite this: Pahore AK, Khan S, Karim N. Illicium verum anticancer activity against MDA-MB-231 cell line. Pak J Med Sci. 2024;40(1):---------. doi: https://doi.org/10.12669/pjms.40.1.7860
 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Comparison of Live Cell Enrichment Methods for Single-Cell Rnaseq

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for investigating cellular diversity and gene expression profiles at the individual cell level. However, to ensure accurate analysis of scRNA-seq data, it is crucial to generate high-quality single-cell suspensions by effectively removing dead cells, debris, and ambient RNA. This study aims to assess and compare the performance of five commercially available methods for dead cell removal: Fluorescence activated cell sorting (FACS), Akadeum Microbubble kit, StemCell EasySep kit, Miltenyi Biotec Dead Cell Removal Kit, and LevitasBio Levicell Live Cell Enrichment. Among the 5 methods we evaluated, FACS is considered the gold standard technique due to its remarkable efficiency and versatility. However, the high-pressure nature of FACS can exert mechanical stress on cells during the sorting process, potentially affecting cellular viability and gene expression profiles. On the other hand, the remaining four methods, while less versatile, utilize milder conditions, which may better preserve fragile cell populations. To evaluate those 5 methods, we used two tissues requiring only no to mild dissociation process: lymph node and cerebrospinal fluid (CSF). Viability of cells from each tissue was assessed using trypan blue staining after dissociation. The cell suspensions were then subjected to the five dead cell removal methods following the manufacturer's instructions. For each cell suspension, 10,000 cells were targeted using the 10x Genomics 3' v3.1 kit. The evaluation criteria encompassed hands-on time, required equipment, cost, improvement in live cell percentage, cell type recovery, gene expression profile, and cellular stress response. We hope these findings will provide valuable guidance for researchers in selecting the most suitable dead cell removal method based on their experimental requirements, thereby facilitating efficient, cost-effective, and high-quality scRNA-seq analysis.

Synergy of Ruxolitinib and Carfilzomib in Targeting the PAX5::JAK2 fusion I n Vitro: Potential Therapeutic Advantage for a Subset of Ph-like Acute Lymphoblastic Leukemia

INTRODUCTION PAX5::JAK2 is a gene fusion that drives a subset of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) and confers aggressive disease, poor prognosis and poor response to treatment. PAX5::JAK2 is present in approximately 12% of Ph-like ALL cases. Targeted therapy is urgently required to improve outcomes. We evaluated the efficacy of JAK inhibitor ruxolitinib (RUX) and proteosome inhibitors carfilzomib (CFZ) and bortezomib (BTZ) as monotherapy and in combination against PAX5::JAK2 transfected Ba/F3 cells in vitro. Downregulation of proteosome genes via reduction in STAT3 phosphorylation is a shared mechanism of RUX and proteosome inhibitors. Given that the PAX5::JAK2 fusion results in increased STAT3 phosphorylation driving proliferation, it was proposed that the combination of proteosome inhibitors and RUX would demonstrate synergy in inhibiting proliferation of PAX5::JAK2 driven leukemic cells. METHODS Mouse derived Ba/F3 cells, an IL-3 dependent B-ALL model, were transfected with the PAX5::JAK2 fusion and demonstrated IL-3 independence. We applied, as single treatments, differing concentrations of RUX and the proteosome inhibitors CFZ and BTZ to PAX5::JAK2 transfected Ba/F3 cells and two control cell lines - IL-3 dependent empty vector Ba/F3 cells and an IL-3 independent KG1a myeloid cell line. Differing concentrations of CFZ and 200nM of RUX were then applied in combination. 200nM of RUX was selected because it is a dose at which the drug has minimal effect as a single agent. Inhibitors were applied to cells for 72 hours then percentage of live and dead cells at each concentration was ascertained by flow cytometry using Annexin V and LIVE/DEAD fixable aqua dead cell stain. Downstream STAT5 and STAT3 activation in basal state, and as a response to treatment, was assessed using phosphoflow. We demonstrated mechanisms of growth inhibition via reduction in STAT5 and STAT3 phosphorylation by the inhibitors. RESULTS RUX, CFZ and BTZ were all effective against PAX5::JAK2 transfected Ba/F3 cells with a median lethal dose (LD50) of 514nM (CI 498-530nM) for RUX, 38nM (CI 36-39nM) for CFZ and 19nM (CI 18-20nM) for BTZ. RUX had a variable effect on IL-3 dependent empty vector control cells which also demonstrate JAK/STAT activation, but no effect on the IL-3 independent KG1a cells. CFZ had a lesser effect on empty vector control cells with a LD50 of 59nM (CI 58-60nM) and no effect on KG1a control cells. BTZ had a significant effect on both control cell lines, predicting off-target toxicity and consequently was not applied in combination with RUX. RUX and CFZ in combination demonstrated an LD50 of 21nM (CI 18-25nM) (see figure 1) which is significantly lower than the LD50s for either agent used as monotherapy. Combination index for RUX and CFZ at 45nM was 0.80 and at 50nM was 0.49, suggesting moderate synergy. Phosphoflow demonstrated reduced pSTAT5 with the application of RUX and CFZ to PAX5::JAK2 transfected Ba/F3 cells with mean fluorescence intensity (MFI) reducing from 351 to 285 in inhibitor treated cells, while for STAT3 MFI reduced from 396 to 249. (see figure 2) There was no effect on MFI for control cell lines. This demonstrates that the mechanism for growth inhibition is inhibition of JAK/STAT signaling due to the reduction in pSTAT3 and pSTAT5. CONCLUSIONS We showed in vitro that RUX, CFZ and BTZ are all potent against the high risk PAX5::JAK2 fusion. RUX and CFZ in combination demonstrated moderate synergy against PAX5::JAK2 and inhibited proliferation through their effect on JAK/STAT pathways. The RUX/CFZ combination, drugs widely available in clinical practice, is particularly promising given the apparent synergistic effect on limiting leukemic cell growth. Such treatments warrant clinical trials for management of the poor prognostic group of Ph-like ALL with PAX5::JAK2, given that they may enhance efficacy and lessen toxicity. Clinical trials have already demonstrated efficacy and safety of RUX and CFZ as single agents in combination with chemotherapy for the treatment of B-ALL which indicates the feasibility of combining the two agents clinically in Ph-like ALL.

Drug Response Profiling Informs Personalized Bridging to Cell Therapy for Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia

Introduction Treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL) remains challenging in spite of recent progress with immunotherapy and targeted small-molecule combinations. Growing awareness of the molecular complexity of the disease with subtype-specific cancer dependencies positions functional drug testing as a promising precision tool to improve the prediction of clinical drug efficacy and adapt treatment. We leverage automated, image-based Drug Response Profiling (DRP) and machine learning to deliver individualized therapeutic options in real time for ALL patients with urgent medical needs. Here we report on the DRP experience of 307 primary samples from 278 patients with BCP-ALL, T-ALL/T-LBL and mixed phenotypic acute leukemia (MPAL) registered from 2016 to 2022 in a non-interventional setting. Methods We have generated a reference set of 141 diagnostic samples from patients with high-risk (75%) or standard/medium-risk (25%) disease as defined by the clinical trial AIEOP-BFM ALL 2017, alongside 122 samples with first and 44 with more advanced relapse. We assayed cell viability at single-cell level from viably frozen bone marrow (BM) samples, co-cultured with hTERT-immortalized mesenchymal stromal cells (MSC) and following a 72h drug incubation with a library of up to 110 compounds. We quantified cell counts at five concentrations in duplicates by segmenting and classifying MSC, live and dead leukemia cells using a modular image processing pipeline deployed on cloud infrastructure. We longitudinally confirmed assay consistency with PDX models and monitored integrity of the stromal layer on a per-sample basis. In a subset of cases (15-20%), relevant stromal detachment or low leukemia survival led to samples not meeting all quality control standards. Drugs were ranked by the area under the dose response curve (AUC) and referenced to all previous samples. This patient-specific ex vivo drug fingerprint was reported to the treating physician with a typical turn-around time of less than two weeks. Results Dexamethasone sensitivities measured ex vivo in BM samples from pediatric front-line patients, who were treated on a one-week cytoreductive steroid pre-phase followed by a four-drug induction regimen, correlated significantly with clinical responses at d8 (PGR vs. PPR, p<0.001), d15 (flow MRD below 10%, p<0.001) and d33 (PCR MRD below E-2, p<0.001). By integrating dose-response profiles from all patients, we observed differential drug activities across cytogenetic groups and identified exceptional responders to Venetoclax in very high-risk subtypes including TCF3::HLF (5 patients, AUC percent rank: 0.03 - 0.19) as well as refractory T-ALL. In 77 r/r T-ALL patients from 12 different countries, DRP supported selection of a personalized bridging regimen prior to consolidation with cellular therapy. Top-scoring drug sensitivities (10th percentile of AUC) were found for a variety of agents including Imatinib, Dasatinib, Venetoclax, Bortezomib, Selinexor and different nucleoside analogs. Eleven T-ALL and 5 BCP-ALL patients received a DRP-guided bridging element involving compounds with high ex vivo drug efficacy and complemented by chemotherapy (Table 1). The other 66 r/r patients were treated according to physician's choice incorporating information on drug resistance detected by DRP in real time. Bridging strategies implementing DRP-guided therapy blocks stabilized or reduced disease burden in 15/16 patients allowing HSCT or infusion of CAR-T cells in 12 patients with 8 patients still being in remission at last follow-up. Conclusion We describe an array of pharmacological sensitivity and resistance patterns across a heterogeneous, high-risk cohort of BCP- and T-ALL patients. We identified individual susceptibilities to targeted agents of different classes including BH3-mimetics, proteasome, tyrosine kinase and exportin inhibitors as well as standard-of-care chemotherapeutics. Prospective recording of drug response profiles offers opportunities to systematically correlate ex vivo and clinical responses and inform personalized therapies. These data strongly advocate inclusion of DRP into randomized, early and late phase clinical trials for high-risk pediatric ALL, comprehensively integrating molecular and functional precision information to improve patient stratification and long-term outcome after HSCT and CAR-T.

Fatty acid modification of casein bioactive peptides nano-assemblies, synthesis, characterization and anticarcinogenic effect

In this study, the nano-assemblies of bovine casein hydrolyzed peptides (HP) modified by fatty acids with various alkyl chain lengths (C8, C10, C12 and C14) were synthesized. The physicochemical properties of HP-C8-HP-C14 nano-assemblies were characterized using spectra, laser particle size analyzer, contact angle meter, scanning electron microscope (SEM) and cryo-transmission electron microscope (Cryo-TEM). HP-C8 and HP-C10 self-assembled into a hollow cube cage with an average size of ~500 nm, and the assembly of HP-C12 showed a flower-shaped morphology with more dispersed behavior, and droplet size was observed as ~20 nm. The in vitro cytotoxicity against human breast cancer cells MCF-7 was tested using CCK-8 assay and flow cytometry analysis. HP-C12 showed the highest cytotoxicity for MCF-7 cells with an inhibition rate of 66.03 % ± 0.35 % with an IC50 value of 7.4 μM among HP-Cn. HP-C8, HP-C10 and HP-C12 significantly affected on the migration, invasion and apoptosis of MCF-7 cells. The apoptosis mechanism may depend on the upregulation of anti-apoptotic protein Bcl-2 as well as pro-apoptotic proteins Bax and caspase-8. The dead MCF-7 cells were analyzed with UHPLC-MS/MS using untargeted metabolomics, revealing key metabolic pathways.

Activation of cannabinoid receptors 2 alleviates myocardial damage in cecal ligation and puncture-induced sepsis by inhibiting pyroptosis

BackgroundIt has been reported that cannabinoid receptors 2 (CB2 receptors) play an important role in the pathophysiological process of sepsis, which may also be associated with the regulation of pyroptosis, an inflammatory programmed cell death. The present study aimed to investigate the protective effect of CB2 receptors on myocardial damage in a model of septic mice by inhibiting pyroptosis. MethodsThe C57BL/6 mice underwent cecal ligation and puncture (CLP) to induce sepsis. All mice were randomly divided into the sham, CLP, or CLP+HU308 group. Blood and heart tissue samples were collected 12 h after surgery. Hematoxylin and eosin staining was used for analyzing histopathological results. Creatine kinase isoenzymes (CK-MB) and IL-1β were measured using ELISA, while lactate dehydrogenase (LDH) level was determined using photoelectric colorimetry. The expression levels of CB2 receptors and pyroptosis-associated proteins (NLRP3, caspase-1, and GSDMD) were measured using western blotting. The location and distribution of CB2 receptors and caspase-1 in myocardial tissues were assessed by immunofluorescence. TUNEL staining was used to quantify the number of dead cells in myocardial tissues. ResultsThe CLP procedure increased CB2 receptor expression in mice. CB2 receptors were located in myocardial macrophages. Activating CB2 receptors decreased the levels of myocardial damage mediator LDH, CK-MB, and inflammatory cytokine IL-1β. The results also showed that CLP increased the pyroptosis in myocardial tissues, while CB2 agonist HU308 inhibited pyroptosis by decreasing the level of NLRP3 and activating caspase-1 and GSDMD. ConclusionsCB2 receptor activation has a protective effect on the myocardium of mice with sepsis by inhibiting pyroptosis.

Shenshuaifu Granule Attenuates Acute Kidney Injury by Inhibiting Ferroptosis Mediated by p53/SLC7A11/GPX4 Pathway.

Acute kidney injury (AKI) is a common clinical condition resulting in a rapid decline in renal function, and requires improvement in effective preventive measures. Ferroptosis, a novel form of cell death, is closely related to AKI. Shenshuaifu granule (SSF) has been demonstrated to prevent AKI through suppressing inflammation and apoptosis. This study aimed to explore whether SSF can inhibit ferroptosis in AKI. Active ingredients in SSF were detected through HPLC-MS/MS, and their binding abilities with ferroptosis were evaluated by molecular docking. Then, male C57/BL/6J mice were randomly divided into control, cisplatin, and cisplatin+SSF groups. In the latter two groups, mice were intraperitoneally injected with 20 mg/kg of cisplatin. For five consecutive days prior to cisplatin injection, mice in the cisplatin+SSF group were gavaged with 5.2 g/kg of SSF per day.72 h after cisplatin injection, the mice were sacrificed. Serum creatinine (SCr) and blood urea nitrogen (BUN) were measured to evaluate renal function. H&E and PAS staining were used to observe pathological damage of kidney. Cell death was observed by TUNEL staining, and iron accumulation in kidneys of mice was detected by Prussian blue staining. Western blotting, immunohistochemistry, and immunofluorescence were used to investigate the presence of inflammation, oxidative stress, mitochondrial dysfunction, iron deposition, and lipid peroxidation in mouse kidneys. Active ingredients in SSF had strong affinities with ferroptosis. SSF reduced SCr (p<0.01) and BUN (p<0.0001) levels, pathological damage (p<0.0001), dead cells in the tubular epithelium (p<0.0001) and iron deposition (p<0.01) in mice with cisplatin induced AKI. And SSF downregulated macrophage infiltration (p<0.01), the expressions of high mobility group box 1 (HMGB1, p<0.05) and interleukin (IL)-17 (p<0.05), upregulated superoxide dismutase (SOD) 1 and 2 (p<0.01), and catalase (CAT, p<0.05), and alleviated mitochondrial dysfunction (p<0.05). More importantly, SSF regulated iron transport and intracellular iron overload and reduced the expression of ferritin (p<0.05). Moreover, it downregulated the expressions of cyclo-oxygenase-2 (Cox-2, p<0.001), acid CoA ligase 4 (ACSL4, p<0.05), and solute carrier family 7, member 11 (SLC7A11, p<001), upregulated glutathione peroxidase 4 (GPX4, p<0.01) and p53 (p<0.01), and decreased 4-hydroxynonenal (4-HNE) level (p<0.001). SSF attenuates AKI by inhibiting ferroptosis mediated by p53/SLC7A11/GPX4 pathway.

Real-time monitoring of chlorination treatment in Microcystis cells by simultaneously measuring the polarized light scattering and fluorescence

Chlorination treatment is widely used in cyanobacterial blooms to reduce harmful impacts on aquatic systems. However, it is hard to timely monitor the effect of treatment and formation of disinfection by-products (DBPs) produced by intracellular organic matter (IOM) may cause second damage to the environment. In this work, we used a conceptual setup to simultaneously measure the polarization and fluorescence parameters (PFPs) from single Microcystis cells for monitoring a continuous process of chlorination. Both the cultured samples and the field-collected samples were firstly treated with different sodium hypochlorite (NaClO) concentrations, and then they were respectively measured by the setup. Results showed that PFPs changed significantly during the chlorination treatment, which had a strong correlation with the concentration of NaClO solution. Based on the machine learning model, the proportions of dead cultured cells in different concentrations (1, 5, and 10 mg/L) grew to 35.59%, 57.10%, and 84.54% respectively after 10 min. Besides, the proportion of dead field-collected cells under chlorination (10 mg/L) for 10 min was 75.11%. Graphs from SEM and TEM revealed that cell membrane damage commonly occurred with a concentration of 10 mg/L NaClO during the same period. The optical density at 680 nm obtained by spectrophotometer decreased as increasing chlorine concentrations, which agreed with changes in fluorescence intensity at low concentrations. This paper demonstrates the feasibility of this setup and PFPs for real-time monitoring of cyanobacterial bloom chlorination treatment in aquatic environments.