Quantify Cell Death Research Articles

DDDR-27. BMP SIGNALING MODULATES APOPTOTIC AND NON-APOPTOTIC CELL DEATH IN GLIOBLASTOMA

Abstract Recurrence occurs in approximately 90% of glioblastoma (GBM) patients, in part due to the tumors becoming resistant to cell death in response to existing therapies. These therapies often trigger apoptosis; however, apoptosis is only one of several different forms of cell death. We hypothesize that activating non-canonical death mechanisms could provide a back door to killing treatment-resistant GBM cells. To test this hypothesis, we first developed a new imaging-based high-throughput method to directly quantify cell death in 3D models. Using this method, we find patient-derived GBM spheroids to be resistant to the induction of cell death in response to various standard-of-care and experimental therapeutic compounds. To further investigate mechanisms underlying this resistance, we screened a panel of extracellular protein ligands for the modulation of cell death response. We find that bone morphogenic protein (BMP) signaling increases resistance to several apoptosis-inducing compounds, including temozolomide and tyrosine kinase inhibitors. Intriguingly, BMP signaling simultaneously enhanced sensitivity to a novel non-apoptotic cell death pathway triggered by the small molecule CIL56. Efforts are ongoing to further understand how BMP signaling governs apoptotic and non-apoptotic cell death mechanisms. Nevertheless, our results suggest a novel approach to treating GBM, centered upon the modulation of BMP signaling combined with the use of agents that induce non-apoptotic cell death.

Defining PANoptosis: Biochemical and Mechanistic Evaluation of Innate Immune Cell Death Activation.

The innate immune system is the first line of host defense. Innate immune activation utilizes pattern recognition receptors to detect pathogens, pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), and homeostatic alterations and drives inflammatory signaling pathways and regulated cell death. Cell death activation is critical to eliminate pathogens and aberrant or damaged cells, while excess activation can be linked to inflammation, tissue damage, and disease. Therefore, there is increasing interest in studying cell death mechanisms to understand the underlying biology and identify therapeutic strategies. However, there are significant technical challenges, as many cell death pathways share key molecules with each other, and genetic models where these cell death molecules are deleted remain the gold standard for evaluation. Furthermore, extensive crosstalk has been identified between the cell death pathways pyroptosis, apoptosis, necroptosis, and the more recently characterized PANoptosis, which is defined as a prominent, unique innate immune, lytic, and inflammatory cell death pathway initiated by innate immune sensors and driven by caspases and RIPKs through PANoptosomes. PANoptosomes are multi-protein complexes assembled by innate immune sensor(s) in response to pathogens, PAMPs, DAMPs, cytokines, and homeostatic changes that drive PANoptosis. In this article, we provide methods for molecularly defining distinct cell death pathways, including PANoptosis, using both genetic and chemical approaches through western blot, LDH assay, and microscopy readouts. This procedure allows for the assessment of cell death on the cell population and single-cell levels even without access to genetic models. Having this comprehensive workflow that is more accessible to all labs will improve our ability as a scientific community to accelerate discovery. Using these protocols will help identify new innate immune sensors that drive PANoptosis and define the molecular mechanisms and regulators involved to establish new targetsfor clinical translation. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction and quantification of cell death using live cell imaging Alternate Protocol 1: Quantification of cell death using LDH Alternate Protocol 2: Assessment of cell death complexes in single cells using immunofluorescence staining Basic Protocol 2: Analysis of cell death mechanisms by immunoblots (western blots).

Short-term Effects of Non-invasive Physical Plasma Treatment on Genomic Stability.

The application of non-invasive physical plasma (NIPP) generates reactive oxygen species. These can lead to chemical oxidation of cellular molecules including DNA. On the other hand, NIPP can induce therapeutically intended apoptosis, which also leads to DNA fragmentation in the late phase. Therefore, to assess unwanted genotoxic effects, the formation of DNA damage was investigated in this study in discrimination from apoptotic processes. Mutation events after NIPP application were analyzed in CCL-93 fibroblast cells using the hypoxanthine phosphoribosyl transferase assay. Additionally, DNA single-strand breaks (SSB) and double-strand breaks (DSB) were quantified by performing the alkaline comet assay, and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. DSBs were quantified by phospho-histone 2AX-p53-binding protein 1 co-localization DSB focus assay. The data were compared with cell death quantification by the caspase-3/7 apoptosis assay. Treatment with NIPP led to exceedingly rapid damage to genomic DNA and the appearance of DNA SSBs and DSBs in the initial 4 h. However, damage decreased again within the first 4-8 h, then the late phase began, characterized by DNA DSB and increasing caspase-3/7 activation. Although NIPP treatment leads to extremely rapid damage to genomic DNA, this damage is reversed very quickly by efficient DNA-repair processes. As a consequence, only those cells whose genome damage can be repaired actually survive and proliferate. Persistent genotoxic effects were not observed in the cell system used.

Cabozantinib in neuroendocrine tumors: tackling drug activity and resistance mechanisms.

Neuroendocrine tumors (NETs) are highly vascularized malignancies in which angiogenesis may entail cell proliferation and survival. Among the emerging compounds with antivascular properties, cabozantinib (CAB) appeared promising. We analyzed the antitumor activity of CAB against NETs utilizing in vitro and in vivo models. For cell cultures, we used BON-1, NCI-H727 and NCI-H720 cell lines. Cell viability was assessed by manual count coupled with quantification of cell death, performed through fluorescence-activated cell sorting analysis as propidium iodide exclusion assay. In addition, we investigated the modulation of the antiapoptotic myeloid cell leukemia 1 protein under CAB exposure, as a putative adaptive pro-survival mechanism, and compared the responses with sunitinib. The activity of CAB was also tested in mouse and zebrafish xenograft tumor models. Cabozantinib showed a dose-dependent and time-dependent effect on cell viability and proliferation in human NET cultures, besides a halting of cell cycle progression for endoduplication, never reported for other tyrosine kinase inhibitors. In a transplantable zebrafish model, CAB drastically inhibited NET-induced angiogenesis and migration of implanted cells through the embryo body. CAB showed encouraging activity in NETs, both in vitro and in vivo models. On this basis, we envisage future research to further investigate along these promising lines.

Label-free identification of cell death mechanism using scattering-based microscopy and deep learning

The detection of cell death and identification of its mechanism underpins many of the biological and medical sciences. A scattering microscopy based method is presented here for quantifying cell motility and identifying cell death in breast cancer cells using a label-free approach. We identify apoptotic and necrotic pathways by analyzing the temporal changes in morphological features of the cells. Moreover, a neural network was trained to identify the cellular morphological changes and classify cell death mechanisms automatically, with an accuracy of over 95%. A pre-trained network was tested on images of cancer cells treated with a different chemotherapeutic drug, which was not used for training, and it correctly identified cell death mechanism with ∼100% accuracy. This automated method will allow for quantification during the incubation steps without the need for additional steps, typically associated with conventional technique like fluorescence microscopy, western blot and ELISA. As a result, this technique will be faster and cost effective.

1722-P: RIPK1 Promotes Thapsigargin-Induced ß-Cell Death Independent of Caspase 3/7 Activity In Vitro

Endoplasmic reticulum (ER) stress promotes β-cell dysfunction and death in diabetes. Although studies of ER stress-induced β-cell death have focused on mechanisms of apoptosis, we recently showed that β cells are also susceptible to a form of programmed cell death called necroptosis. Necroptosis occurs downstream of receptor interacting protein kinase 1 (RIPK1), does not require caspase activation, and results in lytic cell death. Studies in non-islet cell types have linked ER stress-induced cell death to necroptosis. Thus, we hypothesized that RIPK1 promotes ER stress-induced β-cell death in vitro. To test this hypothesis, we quantified cell death and caspase 3/7 activity in NIT-1 β cells, human iPSC-derived β-like cells and intact mouse islets following treatment with thapsigargin (a SERCA2b inhibitor and ER stress-inducing agent, 50 nM or 500 nM) and zVAD-FMK (a pan-caspase inhibitor, 40 μM). After quantifying cell death hourly for 48 hrs, we found that thapsigargin significantly increased cell death in NIT-1 cells, β-like human iPSCs and intact mouse islets, and that this occurred in conjunction with increased caspase 3/7 activity. We next asked whether co-treatment with zVAD was sufficient to prevent thapsigargin-induced cell death. In each cell type evaluated, we found that zVAD inhibited caspase 3/7 activity but failed to protect from thapsigargin-induced cell death. To establish the role of RIPK1 in this process, we next evaluated cell death in gene-edited NIT-1 control (non-targeting gRNA) and NIT-1 Ripk1Δ (gRNA targeting Ripk1 exons 2-3) β cells. After 24 hrs, we found that NIT-1 Ripk1Δ cells were strongly protected from thapsigargin-induced cell death both when caspases were active (n=6, p<0.001) and when they were inhibited (n=6, p<0.001). These data indicate that RIPK1 regulates thapsigargin-induced β-cell death independent of caspase 3/7 activation. Future studies will examine the mechanisms by which RIPK1 regulates ER stress-induced β-cell death in vitro and in vivo. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

P039 The disappearance of cell death brakes in inflammatory bowel disease: a step towards molecular healing

Abstract Background The core mechanistic driver underpinning the relapsing and progressive nature of inflammatory bowel disease (IBD) remains unknown with current therapies targeting immune or inflammatory pathways. Our in-depth analysis of regulated death pathways remarkably reveals that cell death brakes are removed in intestinal IBD tissue, indicating cells remain “primed” to die regardless of endoscopic and histologic healing. Characterisation of cell death is essential to confirm our hypothesis that disinhibition of programmed cell death is a rudimentary molecular defect in IBD. Restoration of cell death brakes may be intrinsic to molecular healing and novel drug development. Methods Intestinal biopsies are collected from inflamed, marginal and non-inflamed regions during endoscopy for patients with IBD and matched with those without IBD (“controls”). Tissue is processed to quantify cell death signals and changes to the local microbiome using immunoblotting, immunohistochemistry, immunofluorescence, organoid preparation and RNA sequencing (Fig. 1). Laboratory data are correlated with clinical, biochemical, endoscopic and histologic parameters. Results To date, 59 patients have been recruited: 29 controls; 31 IBD (13 Crohn’s disease, 16 ulcerative colitis, 2 pouchitis). Interim analyses of 20 patients show loss of cell death brakes (cIAP1, cIAP2, cFLIP) suggesting disinhibition of cell death even in endoscopically and histologically normal IBD tissue (Fig. 2). Concurrently, there is increased apoptosis (characterised by cleaved caspase-3) in IBD intestinal tissue regardless of inflammatory activity (Fig. 3). Necroptosis, a programmed lytic form of cell death, is active in steady state conditions in controls. Additionally, necroptosis appears markedly elevated in stricturing and penetrating Crohn’s disease compared to other subsets of IBD (Fig. 2). Conclusion This study demonstrates for the first time that cellular death brakes are removed in intestinal tissue in IBD, despite deep remission with seemingly effective biologic therapy. Disinhibition of programmed cell death is likely intrinsic to the inflammatory sequence underlying disease recurrence and progression. Restoration of cell death brakes may hence be key to achieving long-term, relapse-free remission and should be considered an aspirational goal for both drug development and achievement of molecular healing. Our results challenge current dogma based on murine data that necroptosis occurs in the absence of apoptosis and that necroptosis is universally increased in all forms of IBD. Instead, we propose that a balance of apoptosis and necroptosis is essential in intestinal homeostasis with endotyping of disease based on cell death profile a feasible goal towards precision medicine in IBD.

Quantifying Cell Death Induced by the NLRC4 Inflammasome.

The Nod-like Receptor (NLR) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that sense cytosolic bacterial proteins. NAIP ligation induces its association with NLRC4, leading to the assembly of the NAIP/NLRC4 inflammasome, which induces the activation of the caspase-1 protease. Caspase-1 then cleaves pro-interleukin (IL)-1β, pro-IL-18, and gasdermin D and induces a form of pro-inflammatory cell death, pyroptosis. These processes culminate in host defense against bacterial infection. Here we describe methods for activating NAIP/NLRC4 inflammasome signalling in human and murine macrophages and quantifying inflammasome-induced cell death.

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

LANCE: a Label-Free Live Apoptotic and Necrotic Cell Explorer Using Convolutional Neural Network Image Analysis.

Identifying and quantifying cell death is the basis for all cell death research. Current methods for obtaining these quantitative measurements rely on established biomarkers, yet the marker-based approach suffers from limited marker specificity, high cost of reagents, lengthy sample preparation, and fluorescence imaging. Based on the morphological difference, we developed a Live, Apoptotic, and Necrotic Cell Explorer (LANCE) to categorize cell death status in a label-free manner, by incorporating machine learning and image processing. The LANCE workflow includes cropping individual cells from microscopic images having hundreds of cells, formation of an image database of around 5000 events, training and validation of the convolutional neural network models using multiple cell lines, and treatment conditions. With LANCE, we precisely categorized live, apoptotic, and necrotic cells with a high accuracy of 96.3 ± 0.5%. More importantly, the nondestructive label-free LANCE method allows for tracking time dynamics of the cell death process, which enhances the understanding of subtle cell death regulation at the molecular level. Hence, LANCE is a fast, low-cost, and nondestructive label-free method to distinguish cell status, which can be applied to cell death studies as well as many other biomedical applications.

Designing Organoid Models to Monitor Cancer Progression, Plasticity and Resistance: The Right Set Up for the Right Question.

Simple SummaryIn this article, we describe a 3D model that supports the maintenance of cell polarity in cancer and normal cells by growing them on a collagen-coated microsupport. Instead of the spheroids model, the cells are directly positioned to adopt a basal/luminal organization, favoring differentiation and migration in the surrounding matrix. This model can be enriched with other components of the microenvironment such as fibroblasts and immune cells. For the proof-of-concept experiments, we treated mouse and human cell lines, then primary tumor cells from PDX, co-cultured with fibroblasts or immune cells. We monitored cell viability, proliferation and cytotoxicity using several light emission-based methods to obtain significant and reliable results, validating the method.The recent trend in 3D cell modeling has fostered the emergence of a wide range of models, addressing very distinct goals ranging from the fundamental exploration of cell–cell interactions to preclinical assays for personalized medicine. It is clear that no single model will recapitulate the complexity and dynamics of in vivo situations. The key is to define the critical points, achieve a specific goal and design a model where they can be validated. In this report, we focused on cancer progression. We describe our model which is designed to emulate breast carcinoma progression during the invasive phase. We chose to provide topological clues to the target cells by growing them on microsupports, favoring a polarized epithelial organization before they are embedded in a 3D matrix. We then watched for cell organization and differentiation for these models, adding stroma cells then immune cells to follow and quantify cell responses to drug treatment, including quantifying cell death and viability, as well as morphogenic and invasive properties. We used model cell lines including Comma Dβ, MCF7 and MCF10A mammary epithelial cells as well as primary breast cancer cells from patient-derived xenografts (PDX). We found that fibroblasts impacted cell response to Docetaxel and Palbociclib. We also found that NK92 immune cells could target breast cancer cells within the 3D configuration, providing quantitative monitoring of cell cytotoxicity. We also tested several sources for the extracellular matrix and selected a hyaluronan-based matrix as a promising alternative to mouse tumor basement membrane extracts for primary human cancer cells. Overall, we validated a new 3D model designed for breast cancer for preclinical use in personalized medicine.

The Ying and the Yang: Compensatory UPR Signaling Responses Observed In An In‐Vitro Model Expressing Clinical Mutant Surfactant Protein C Isoforms

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by alveolar type II (AT2) cell dysfunction. Mutations in surfactant protein C (SP‐C) are a high effect size, recognized etiological cause of IPF. The SP‐C proprotein contains an ~100 KDa Bri2 chondromodulin‐1 and prosurfactant protein C (BRICHOS) domain that acts as a molecular chaperone preventing protein aggregation. The presence of misfolded proteins in the ER can be sensed by three transmembrane sentinel proteins – activating transcription factor 6 (ATF6), protein kinase R‐like ER kinase (PERK), and inositol‐requiring enzyme 1α (IRE1α) – which trigger an intracellular signaling pathway called the unfolded protein response (UPR). Previous characterization of our in‐vitro and in‐vivo models has demonstrated that BRICHOS mutants activate all 3 arms of the UPR, activate a proinflammatory cascade, and drive AT2 cell death. In‐vivo, their expression initiates pathological fibroproliferative lung remodeling, characteristic of human IPF. Using our in‐vitro models of BRICHOS‐driven ER stress, we explore potential IPF therapeutic intervention targeting two upstream sensors of the UPR, PERK and IRE‐1.Mouse lung epithelial (MLE‐12) cells were transfected with wildtype (WT) or BRICHOS mutant (C121G) SP‐C plasmids in media containing inhibitors of either PERK signaling or IRE‐1 RNase activity. Pathway readouts included: quantification of cell death via LDH cytotoxicity and flow cytometry, protein immunoblots to detect UPR activation and apoptotic signaling, QPCR of spliced X‐Box Binding Protein 1 (XBP1) and UPR transcriptional targets, and high‐resolution microscopy to observe altered SP‐C trafficking.Within 48 hours of transfection of MLE‐12 cells with SP‐CC121G we observed activation of the 3 arms of the UPR, upregulated downstream targets, and initiation of cell death. Inhibition of PERK resulted in down‐regulation of PERK pathway proteins and transcriptional targets, increased SP‐C protein expression, and promoted increased XBP1 splicing. Inhibition of IRE1 RNase inhibited splicing of XBP1, reduced expression of XBP1 transcriptional targets, and increased JNK phosphorylation. Inhibition of either pathway alone did not reduce cell death, though partial reduction of cell death was noted with either small molecule targeted caspase 3/8 or necroptosis inhibition.Our data suggests that while intervention directed at one arm of the UPR may block proinflammatory signaling or transcriptomic reprogramming, this strategy may not be sufficient to completely inhibit cell death. These findings highlight the interconnectivity and redundancy of the cell quality control machinery and the emerging role of necroptosis due to chronic UPR stress‐mediated cell death. Further characterization of this pathway is needed to improve IPF therapeutic outcomes.

Investigating the Three-Way Interaction between Gastric Epithelial Cells, Human Neutrophils, and Helicobacter pylori

Abstract Neutrophils are the most abundant white blood cells in humans and are the first line of defense against pathogens with respect to innate immunity. Helicobacter pylori are gram-negative bacteria that infect the gastric epithelium and play an important role in the development of ulcers and gastric cancer. Large numbers of neutrophils are recruited to the gastric epithelium where they become heavily infected. We demonstrated previously that when neutrophils are infected by H. pylori it extends their short lifespan. In addition, these cells display unique characteristic such as nuclear hypersegmentation that are indicative of N1-like subtype differentiation which may increase bacterial virulence. Studies by other groups have examined H. pylori-epithelial cell interactions but notably absent are three-way co-cultures that examine simultaneous interactions of neutrophils with bacteria and gastric epithelial cells. Addressing this knowledge gap is the focus of my project. Specifically, I am investigating the dynamics of infection in these co-cultures and determining whether the infected PMNs alter epithelial cell function or viability. Using immunofluorescence I am determining bacterial localization, analyzing PMN nuclear morphology, and determining the extent of changes in epithelial cell junctions and the actin cytoskeleton over 24 hours. Additionally, I am using flow cytometry, lactate dehydrogenase release assays, western blotting and microscopy to quantify cell death, including apoptosis, to test the hypothesis that infected PMNs are more toxic to the epithelium than the bacteria alone. Ultimately, our studies are expected to advance understanding of the role of neutrophils in H. pylori pathogenesis.

INSIDIA 2.0 High-Throughput Analysis of 3D Cancer Models: Multiparametric Quantification of Graphene Quantum Dots Photothermal Therapy for Glioblastoma and Pancreatic Cancer.

Cancer spheroids are in vitro 3D models that became crucial in nanomaterials science thanks to the possibility of performing high throughput screening of nanoparticles and combined nanoparticle-drug therapies on in vitro models. However, most of the current spheroid analysis methods involve manual steps. This is a time-consuming process and is extremely liable to the variability of individual operators. For this reason, rapid, user-friendly, ready-to-use, high-throughput image analysis software is necessary. In this work, we report the INSIDIA 2.0 macro, which offers researchers high-throughput and high content quantitative analysis of in vitro 3D cancer cell spheroids and allows advanced parametrization of the expanding and invading cancer cellular mass. INSIDIA has been implemented to provide in-depth morphologic analysis and has been used for the analysis of the effect of graphene quantum dots photothermal therapy on glioblastoma (U87) and pancreatic cancer (PANC-1) spheroids. Thanks to INSIDIA 2.0 analysis, two types of effects have been observed: In U87 spheroids, death is accompanied by a decrease in area of the entire spheroid, with a decrease in entropy due to the generation of a high uniform density spheroid core. On the other hand, PANC-1 spheroids’ death caused by nanoparticle photothermal disruption is accompanied with an overall increase in area and entropy due to the progressive loss of integrity and increase in variability of spheroid texture. We have summarized these effects in a quantitative parameter of spheroid disruption demonstrating that INSIDIA 2.0 multiparametric analysis can be used to quantify cell death in a non-invasive, fast, and high-throughput fashion.

The pro-apoptotic properties of a phytonutrient rich infusion of A. cherimola leaf extract on AML cells

Annonaceae family has broad uses in herbal medicine for treatment of several diseases, whether through seeds’ or leaves’ extracts. The present study investigates the antiproliferative and antitumor activity of Annona cherimola aqueous leaf (AAL) extract/infusion in acute myeloid leukemia (AML) cell lines in vitro. High-resolution LC-MS was first used to analyze the composition of the aqueous extract. Cell proliferation assay, Annexin V staining, cell cycle analysis, dual Annexin V/PI staining, cell death quantification by ELISA, ROS level detection and Western Blotting were then performed to elucidate the therapeutic effects of AAL extract. The results obtained revealed a potent antioxidant activity of AAL extract. Moreover, the extract exhibited dose- and time-dependent antiproliferative effects on AML cell lines by decreasing cell viability with an IC50 of 5.03% (v/v) at 24 h of treatment of KG-1 cells. This decrease in viability was accompanied with a significant increase in apoptotic cell death with cell cycle arrest and flipping of the phosphatidylserine from the inner to the outer leaflet of the cell membrane. The respective overexpression and downregulation of proapoptotic proteins like cleaved caspase-8, cleaved PARP-1 and Bax and antiapoptotic proteins like Bcl-2 further validated the apoptotic pathway induced by AAL on AML cells. Finally, LC-MS revealed the presence of several compounds like fatty acids, terpenes, phenolics, cinnamic acids and flavonoids that could contribute to the antioxidant and anti-cancer effects of this herbal infusion. In addition to the generally known nutritional effects of the Annona cherimola fruit and leaves, the presented data validates the antioxidant and anti-cancerous effects of the leaf infusion on AML cell lines, proposing its potential therapeutic use against acute myeloid leukemia with future in vivo and clinical trials.

Quantifying cell death induced by doxorubicin, hyperthermia or HIFU ablation with flow cytometry

Triggered release and targeted drug delivery of potent anti-cancer agents using hyperthermia-mediated focused-ultrasound (FUS) is gaining momentum in the clinical setting. In early phase studies, tissue biopsy samples may be harvested to assess drug delivery efficacy and demonstrate lack of instantaneous cell death due to FUS exposure. We present an optimised tissue cell recovery method and a cell viability assay, compatible with intra-cellular doxorubicin. Flow cytometry was used to determine levels of cell death with suspensions comprised of: (i) HT29 cell line exposed to hyperthermia (30 min at 47 °C) and/or doxorubicin, or ex-vivo bovine liver tissue exposed to (ii) hyperthermia (up to 2 h at 45 °C), or (iii) ablative high intensity FUS (HIFU). Flow cytometric analysis revealed maximal cell death in HT29 receiving both heat and doxorubicin insults and increases in both cell granularity (p < 0.01) and cell death (p < 0.01) in cells recovered from ex-vivo liver tissue exposed to hyperthermia and high pressures of HIFU (8.2 MPa peak-to-peak free-field at 1 MHz) relative to controls. Ex-vivo results were validated with microscopy using pan-cytokeratin stain. This rapid, sensitive and highly quantitative cell-viability method is applicable to the small masses of liver tissue typically recovered from a standard core biopsy (5–20 mg) and may be applied to tissues of other histological origins including immunostaining.

EXTH-57. INVESTIGATION OF THE TUMORICIDAL EFFECTS OF SONODYNAMIC THERAPY IN MALIGNANT GLIOBLASTOMA BRAIN TUMORS

Abstract OBJECTIVE Glioblastoma is the most common primary brain tumor; survival is typically 12–18 months after diagnosis. We sought to study the effects of sonodynamic therapy (SDT) using 5-Aminolevulinic acid hydrochloride (5-ALA) and high frequency focused ultrasound (FUS) on 2 glioblastoma cell lines. PROCEDURE Rat C6 and human U87 glioblastoma cells were studied under the following conditions: 1mM 5-ALA (5-ALA); Focused ultrasound (FUS); 5-ALA and focused ultrasound (SDT); control. Studied responses included cell viability using an MTT assay, microscopic changes using phase contract microscopy, apoptotic induction through a caspase-3 assay, and apoptosis staining to quantify cell death. RESULTS SDT led to a marked decrease in cell extension and reduction in cell size. For C6, the MTT assay showed reductions in cell viability for 5-ALA, FUS, and SDT groups of 5%, 16%, and 47%, respectively compared to control (p&amp;lt; 0.05). Caspase 3 induction in C6 cells relative to control showed increases of 109%, 110%, and 278% for 5-ALA, FUS, and SDT groups, respectively (p&amp;lt; 0.05). For the C6 cells, caspase 3 staining positivity was 2.1%, 6.7%, 11.2%, and 39.8% for control, 5-ALA, FUS, and SDT groups, respectively. C6 Parp-1 staining positivity was 1.9%, 6.5%, 9.0%, and 37.8% for control, 5-ALA, FUS, and SDT groups, respectively. U87 cells showed similar responses to the treatments. CONCLUSIONS Sonodynamic therapy resulted in appreciable glioblastoma cell death as compared to 5-ALA or FUS alone. The approach couples two already FDA approved techniques in a novel way to treat the most aggressive and malignant of brain tumors. Further study of this promising technique is planned using glioma and also brain metastasis models.

Targeting 1q21 Amplification with APG2575 and Lenalidomide to Sensitize BCL-2 Inhibition with the Decrease of MCL-1 Protein in High Risk MM Models

Background Multiple myeloma (MM) is a heterogenous plasma cell malignancy. About 30-40% of newly diagnosed and 20-60% of relapsed/refractory MM patients carry 1q21 amplification worldwide, a high-risk indicator for poor prognosis in RRMM. Different BCL-2 family anti-death proteins play important roles in MM survival and drug resistance. High expression of BCL-2 due to t (11;14) renders cell vulnerability to BCL-2 antagonist, however, patients carrying other genetic abnormality including 1q21 amplification have limited response to the newly emerged treatment choice. With MCL-1 upregulation accompanied with 1q21 amplification, we tested whether BCL-2 antagonist combined with IMiDs (immunomodulatory imide drugs), improves cell killing in high-risk MM patient models, including those resistant to bortezomib and lenalidomide. For that aim, we studied APG-2575, a novel and potent BCL-2 inhibitor developed by Ascentage Pharma Group and it is currently in clinical trials for hematologic malignances, including MM. Methods 1. Cells were treated with APG-2575 single agent or in combination with lenalidomide; 2. Cell line viability was assessed by CellTiter-Glo (CTG) assay; 3. Flow cytometry analysis was used to detect CD138+ cell surface marker in primary cells derived from MM patients; 4. Western blot analysis for BCL-2 family and IMiD signaling proteins. Results We first determined the cell sensitivity of APG-2575 as a single agent in a panel of MM cell lines, and as expected, those carrying t (11;14) were very sensitive to APG-2575, with low IC50 values ranging 7-23 nM. The IC50 values for cells carrying other genetic markers were greater than 5-10 μM. We then evaluated cell-death inducing activity of APG-2575 in MM patient-derived primary cells ex vivo, which were freshly prepared from patients' bone marrow aspirates. Primary cells were treated with APG-2575 or ABT-199 (venetoclax) for 18-24 hours, and the loss of CD138 surface marker was used to quantify cell death. As shown in Figure 1a, APG-2575 induced cell death (CD138+ loss) in a dose-dependent manner, and significant cell death was observed at 0.37-3.3 μM of APG-2575, indicating primary cells more sensitive than MM cell lines. Interestingly, the sensitivity to APG-2575 is similar in primary MM cells with or without 1q21 amplification. Since moderate cell death inducing activity was observed in MM cells when APG-2575 used as a single agent, we combined APG-2575 with lenalidomide. Cell death was increased in the combination groups compared with single agent, and it was dose-dependent (Figure 1b).Similar enhanced antiproliferative activity was confirmed in RPMI 8226 cell line, which carries 1q21 amplification (Figure 1c). We furthered to understand the mechanism of action (MoA) of this combination. In Figure 1d, Western blot analysis of RPMI 8226 cell line revealed that IKZF1 and IKZF3 proteins from the NF-KB pathway were downregulated by lenalidomide. And the decrease of MCL-1 protein and the strong induction of pro-death protein BAK were evident in the combination group of APG-2575 and lenalidomide, which helps to illustrate MoA underlying the synergistic effect of APG-2575 and lenalidomide in MM models. Conclusions In both cell lines and primary samples derived from MM patients, APG-2575 demonstrates cell death inducing activity as a single agent, and enhanced/synergistic effects when it combines with lenalidomide in RRMM resistant to lenalidomide and bortezomib. The combination decreases IKZF1, IKZF3 and MCL-1 proteins, and upregulates pro-death protein BAK, thus providing a strong rationale to combine BCL-2 inhibitor and lenalidomide to treat high-risk patient populations carrying 1q21 amplification. Disclosures FU: Ascentage Pharma (SuZhou) Co., Ltd: Other: research agreement contract . Yin:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Mao:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Deng:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Fang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment. Yang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests. Zhai:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests.

Detecting Necroptosis in Virus-Infected Cells.

Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer's and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic membrane allowing a rapid influx of water leading to a loss of cellular integrity. As its role in human disease becomes apparent, methods identifying necroptosis will need to be further developed and optimized. Here we describe identification of necroptosis through quantifying cell death with pathway inhibitors and using western blots to identify end points of MLKL activation and protein-protein interactions leading to it.

Memantine, Simvastatin, and Epicatechin Inhibit 7-Ketocholesterol-induced Apoptosis in Retinal Pigment Epithelial Cells But Not Neurosensory Retinal Cells In Vitro.

Purpose7-ketocholesterol (7kCh), a natural byproduct of oxidation in lipoprotein deposits is implicated in the pathogenesis of diabetic retinopathy and age-related macular degeneration (AMD). This study was performed to investigate whether several clinical drugs can inhibit 7kCh-induced caspase activation and mitigate its apoptotic effects on retinal cells in vitro.MethodsTwo populations of retinal cells, human retinal pigment epithelial cells (ARPE-19) and rat neuroretinal cells (R28) were exposed to 7kCh in the presence of the following inhibitors: Z-VAD-FMK (pan-caspase inhibitor), simvastatin, memantine, epicatechin, and Z-IETD-FMK (caspase-8 inhibitor) or Z-ATAD-FMK (caspase-12 inhibitor). Caspase-3/7, -8, and -12 activity levels were measured by fluorochrome caspase assays to quantify cell death. IncuCyte live-cell microscopic images were obtained to quantify cell counts.ResultsExposure to 7kCh for 24 hours significantly increased caspase activities for both ARPE-19 and R28 cells (P 0.05). In ARPE cells, pretreatment with various drugs had significantly lower caspase-3/7, -8, and -12 activities, reported in % change in mean signal intensity (msi): Z-VAD-FMK (48% decrease, P 0.01), memantine (decreased 47.8% at 1 µM, P = 0.0039 and 81.9% at 1 mM, P 0.001), simvastatin (decreased 85.3% at 0.01 µM, P 0.001 and 84.8% at 0.05 µM, P 0.001) or epicatechin (83.6% decrease, P 0.05), Z-IETD-FMK (68.1% decrease, P 0.01), and Z-ATAD-FMK (47.7% decrease, P = 0.0017). In contrast, R28 cells exposed to 7kCh continued to have elevated caspase-3/7, -8, and -12 activities (between 25.7% decrease and 17.5% increase in msi, P 0.05) regardless of the pretreatment.ConclusionSeveral current drugs protect ARPE-19 cells but not R28 cells from 7kCh-induced apoptosis, suggesting that a multiple-drug approach is needed to protect both cells types in various retinal diseases.