Live-cell Assays Research Articles

Sandwiching Phosphorene with Iron Porphyrin Monolayer for High Stability and Its Biomimetic Sensor to Sensitively Detect Living Cell Released NO.

Instability of 2D phosphorene material is the major obstacle for its broad applications. Herein phosphorene is sandwiched with self‐assembled iron porphyrin monolayers on both sides (I‐Phene) to significantly enhance stability. Iron porphyrin has strong interaction with phosphorene through formation of P—Fe bonds. The sandwich structure offers excellent stability of phosphorene by both‐sided monolayer protections for an intact phosphorene structure more than 40 days under ambient conditions. Meanwhile, the electron transfer between iron porphyrin and phosphorene result in a high oxidation state of Fe, making I‐Phene biomimetic sensitivity toward oxidation of nitric oxide (NO) for 2.5 and 4.0 times higher than phosphorene and iron‐porphyrin alone, respectively. Moreover, I‐Phene exhibits excellent selectivity, a wide detection range, and a low detection limit at a low oxidation potential of 0.82 V, which is comparable with the reported noble metal based biomimetic sensors while ranking the best among all non‐noble biomimetic ones. I‐Phene is further used for real‐time monitoring NO released from cells. This work provides effective approach against phosphorene degrading for outstanding stability, which has universal significance for its various important applications, and holds a great promise for a highly sensitive biomimetic sensor in live‐cell assays.

Chemical Biology of Mitotic Spindle Assembly Motors.

Mitotic kinesins play essential roles during mitotic spindle assembly and in ensuring proper chromosome segregation. Chemical inhibitors of mitotic kinesins are therefore valuable tools to study kinesin function in vitro and in cells. Because cancer is a disease of unregulated cell division, inhibitors also represent potential chemotherapeutic agents. Here, we present assays that can be used to evaluate the potency and specificity of mitotic kinesin inhibitors identified from high-throughput screening. By evaluating their effects in a variety of in vitro, fixed-cell, and live cell assays, screening hits can be prioritized and optimized to produce effective, on-target inhibitors.

Single walled carbon nanotubes in high concentrations is cytotoxic to the human neuronal cell LN18

Single walled carbon nanotubes have been used in biological applications for targeted drug delivery, and as scaffolds for regeneration and diagnosis of diseases due to their excellent properties. However, their extensive application in biology is stifled by toxicity issues. The present study focuses on the effect of pristine single walled carbon nanotubes (P-SWCNT) and carboxylic functionalized single walled carbon nanotubes (C-SWCNT) on the human neuronal cell line LN18 at concentrations of 5, 10, 20, 40 µgmL−1 and time durations of 0–48 h. Cytotoxicity was evaluated by measuring the viability using Trypan Blue, MTT, and Live Dead Cell Assays, and oxidative stress was evaluated by the ROS and Lipid Peroxidation Assays. The IC70 values as measured by the cell viability assay at 12 h were found to be 20 and 10 µgmL−1 for P-SWCNT and C-SWCNT respectively. A significant amount of ROS was produced with P-SWCNT and C-SWCNT at 40 µgmL−1 and 6 h incubation onwards. At 24 and 48 h, a threefold increase in MDA concentration was observed at 40 µgmL−1 of P-SWCNT and C-SWCNT and was measured to be 0.814 ± 0.0042 nM. Analysis of the cellular outcomes revealed that the toxicity of P-SWCNT and C-SWCNT was dose-time dependent with higher toxicity observed for the latter as compared to the former. Hence, it can be concluded that P-SWCNT and C-SWCNT are toxic to LN18 cells at higher concentrations.

History-dependent dopamine release increases cAMP levels in most basal amygdala glutamatergic neurons to control learning.

Dopaminergic inputs to basal amygdala (BA) instruct learning of motivational salience. This learning depends on intracellular plasticity signals such as cyclic adenosine monophosphate (cAMP), which is regulated by activation of dopamine receptors. We examine the dynamics of dopamine release and downstream signaling during multiple salient events occurring within tens of seconds. We perform real-time tracking and manipulation of cAMP in BA neurons invitro and invivo. Optogenetically evoked release of dopamine drives proportional increases in cAMP in almost all BA glutamatergic neurons, suggesting widespread actions of dopamine across neurons preferring positive or negative valence. This cAMP response decreases across trials with short intertrial intervals owing to depression of dopamine release. No such depression is evident when photostimulating cAMP production directly. cAMP and protein kinase A responses to repeated appetitive or aversive stimuli also exhibit pronounced depression. Thus, history-dependent dynamics of dopamine and cAMP may regulate learning of temporally clustered, salient stimuli.

Inhibition of Neovascularization and Inflammation in a Mouse Model of Corneal Alkali Burns Using Cationic Liposomal Tacrolimus.

Eye drops account for more than 90% of commercialized ophthalmic drugs. However, eye drops have certain shortcomings, such as short precorneal retention time and weak corneal penetration. The requirement of frequent instillation of eye drops also causes poor patient compliance, which may lead to further aggravation of the disease. We aimed to develop a cationic liposome formulation to increase the bioavailability of the therapeutic agent and solve the aforementioned problems. In the present study, we prepared cationic liposomal tacrolimus (FK506) with a surface potential of approximately +30 mV, which could bind to the negatively charged mucin layer of the ocular surface. Our results showed that the content of FK506 in the cornea was increased by 93.77, 120.30, 14.24, and 20.36 times at 5, 30, 60, and 90 min, respectively, in the FK506 liposome group (0.2 mg/ml) compared with the free drug group (0.2 mg/ml). Moreover, FITC-labeled FK506 liposomes significantly prolonged the ocular surface retention time to 50 min after a single dose. In addition, the results of the Cell Counting Kit-8 assay, live and dead cell assay, sodium fluorescein staining, and hematoxylin and eosin staining all indicated that FK506 liposomes had good biological compatibility in both human corneal epithelial cells and mouse eyeballs. Compared with the free drug at the same concentration, FK506 liposomes effectively inhibited vascular endothelial growth factor-induced green fluorescent protein-transduced human umbilical vein endothelial cell migration and tube formation in vitro. In a mouse corneal neovascularization model induced by alkali burns, FK506 liposomes (0.2 mg/ml) enhanced corneal epithelial recovery, inhibited corneal neovascularization, and reduced corneal inflammation, and its therapeutic effect was better than those of the commercial FK506 eye drops (1 mg/ml) and the free drug (0.2 mg/ml). Collectively, these results indicate that cationic FK506 liposomes could increase the efficacy of FK506 in the corneal neovascularization model. Therefore, cationic FK506 liposomes can be considered as a promising ocular drug delivery system.

A novel live-cell imaging assay reveals regulation of endosome maturation.

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type-like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

Efficacy of Vaccine BNT162b2 (Pfizer-BioNTech) in Individuals with Waldenstrom's Macroglobulinemia and Follicular Lymphoma in Australia

IntroductionLymphoid malignancies are a risk factor for severe COVID-19. Vaccination with BNT162b2 protects the general population from severe disease, but recent studies have shown limited seroconversion after vaccination in patients with lymphoid malignancy. This reduced response is likely related to disease and treatment factors altering both humoral and cellular immunity. Assessing response in patients with the indolent lymphomas, Waldenström's Macroglobulinaemia (WM) and Follicular Lymphoma (FL), including cohorts on differing treatment regimens, may help elucidate some of these factors. Australia has had low prevalence of SARS-CoV-2 infection to date, affording a unique opportunity to assess efficacy to vaccination without the confounding impact of endemic infection.MethodsPatients with WM and FL and controls were enrolled in a prospective study of immune response after two doses of BNT162b2 administered 21 days apart. The study had Human Research Ethics Committee approval and all patients gave informed consent prior to participation. Recruitment was targeted to obtain comparable proportions of controls to treatment cohorts. PBMC and sera were collected from participants immediately prior to the first dose (T1), at day 21 immediately prior to the second dose (T2), and day 49 (+/- 7d) (T3). Immune response was measured by: flow cytometric detection of anti-SARS-CoV-2 spike antibodies (ASAb), performed using our recently validated flow cytometric live cell assay (Tea et.al. PLoS Medicine 2021) with increased sensitivity compared to currently available commercial ELISAs; live virus neutralisation to a panel of SARS-CoV-2 variants of concern; and CD4+and CD8+ antigen-specific T cell responses. Statistical analysis of medians between cohorts were compared by the Mann-Whitney non-parametric test using Graphpad Prism. Initial ASAb IgG data for T1 and T2 is presented here. Complete immune response data at all time points will be available for the ASH meeting.ResultsEighty-five participants received their first dose of BNT162b2 from 18 May 2021 to 7 June 2021 with a second dose 21 days later: 72 lymphoma patients (WM and FL) and 13 age-matched healthy volunteers (controls). Of 37 with WM [19 (51.3%) female, median 71 years (IQR 63-74)] 9 were treatment naïve (WMN), 15 had received rituximab-chemotherapy (WMT), and 13 were currently treated with a Bruton Tyrosine Kinase inhibitor (BTKi): 5 ibrutinib, 8 zanubrutinib (WMB). Of 35 patients with FL [16 (45.7%) female, median 65 years (IQR 54-71)], 11 were treatment naïve (FLN), and 24 had received rituximab-chemotherapy (FLT). Of the 13 controls 8 (61.5%) were female, median age 72 years (IQR 57-74)].No participants had detectable ASAb at T1, confirming no prior SARS-CoV-2 exposure. Figure 1 shows ASAb results at T2. The median mean fluorescence intensity (MFI) of healthy controls: 60802 (IQR 17565 -78443), is higher than all WM: 0 (IQR 0 - 15010) p<0.0001, and all FL patients: 1687 (IQR 0 - 25421) p=0.002, Fig 1A. The median MFI of controls was higher than WMN (p=0.036), but not higher than FLN (p=0.28). The median MFI of WMN: 20074 (IQR 5421 - 35695), is higher than WMB: 0 (IQR 0 - 4217) p=0.018, but not significantly higher than WMT: 0 (IQR 0 - 14356) p=0.13, Fig 1B. Median MFI of FLN: 31476 (IQR 19351 - 51317), is higher than FLT: 0 (IQR 0 - 32849) p=0.01, Fig 1B.ConclusionThese early serological data show measurable ASAb in all healthy controls 21 days post first dose of BNT162b2 vaccination. Treatment naïve patients had a better response than treated patients, and this did not differ significantly to healthy controls in the FL cohort. In WM, patients on BTKi had a significantly reduced response compared to treatment-naïve patients. This same reduction was not observed in the chemotherapy-rituximab cohort, but the characteristics of early responders versus non-responders, including time since last therapy, is being analysed. FL patients treated with chemotherapy-rituximab had a significantly reduced response compared to the treatment-naïve cohort.Time constraints before the abstract submission deadline prevented reporting of all mature vaccination response data. Measurement of ASAb one month after second vaccination, live virus neutralisation to a panel of SARS-CoV-2 variants of concern, and CD4+ and CD8+ antigen-specific T cell responses at T1, T2 and T3, to fully characterise the immune response to BNT162b2, will be reported at the ASH meeting. [Display omitted] DisclosuresRankin: Novo Nordisk Pharmaceuticals: Other: Travel reimbursement to attend haemophilia academy. Kelleher: CSL/Sequiris: Honoraria; Aegros and CSL: Consultancy; Merck: Speakers Bureau; Aegros, CSL, Merek: Membership on an entity's Board of Directors or advisory committees. Brilot: Novartis: Research Funding; Biogen, Merck: Honoraria. Trotman: beigene: Research Funding; roche: Research Funding; PCYC: Research Funding; BMS: Research Funding; TAKEDA: Research Funding; JANSSEN: Research Funding.

The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction.

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of Drosophila melanogaster KNL1 (Spc105) has never been shown to bind MTs or to recruit PP1. Furthermore, the phosphoregulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell-based assays. A phosphoregulatory circuit that utilizes Aurora B kinase promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag and deletion/chimera mutants are used to define the interplay of MT and PP1 binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction.

Nanoscale coordination polymers with live-cell imaging property

The reaction of semi flexible ligand, 9,10-bis((4-carboxylatopyridinium-1-methylene)anthracene dihyrdrobromide ([H2cpma]Br2) with transition metal ions [Cu(II), Zn(II) and Cd(II)] in solvothermal condition resulted in the formation of three new coordination polymers i.e., Cu3(cpma)7/2(O)2(H2O)3Cl](NO3)2(H2O)2(S)} (Cu-cpma), {[Zn(cpma)Cl2] (S)} (Zn-cpma) and {[Cd(cpma)(Br)(NO3)]2(S)} (Cd-cpma) where cpma denotes 9,10-bis((4-carboxylatopyridinium-1-methylene)anthracene and S denotes the squeezed solvent molecules. These coordination polymers have been characterized by single crystal X-ray, powder XRD, TGA, IR, elemental analysis and fluorescence emission spectroscopy techniques. Single crystal X-ray studies revealed that both Cu-cpma and Zn-cpma are one dimensional chain whereas Cd-cpma has zero dimensional structure. The appropriate particle size of all CPs was calculated by the PXRD pattern using Debye-Scherrer equation and found that all the CPs is of nano range. These nano coordination polymers (NCPs) were evaluated in live-cell imaging assays using HCT-15 as a model cell-line. The results showed that Zn-cpma has highest fluorescence value.

Massively parallel quantification of phenotypic heterogeneity in single-cell drug responses.

Single-cell analysis tools have made substantial advances in characterizing genomic heterogeneity; however, tools for measuring phenotypic heterogeneity have lagged due to the increased difficulty of handling live biology. Here, we report a single-cell phenotyping tool capable of measuring image-based clonal properties at scales approaching 100,000 clones per experiment. These advances are achieved by exploiting a previously unidentified flow regime in ladder microfluidic networks that, under appropriate conditions, yield a mathematically perfect cell trap. Machine learning and computer vision tools are used to control the imaging hardware and analyze the cellular phenotypic parameters within these images. Using this platform, we quantified the responses of tens of thousands of single cell–derived acute myeloid leukemia (AML) clones to targeted therapy, identifying rare resistance and morphological phenotypes at frequencies down to 0.05%. This approach can be extended to higher-level cellular architectures such as cell pairs and organoids and on-chip live-cell fluorescence assays.

Hyperlipidemia-induced metabolic changes in regulatory T cells result in altered function.

Regulatory T cells (Tregs) play a critical role in maintaining self-tolerance and controlling inflammation. However, physiologically relevant conditions that alter Treg function and drive disease pathogenesis are poorly understood and few have been defined. We have previously shown that induction of hyperlipidemia in mice results in changes in Tregs that reduce their function. Here, we set out to examine mechanisms by which hyperlipidemia alters Tregs. Using live-cell metabolic assays, we observed that induction of hyperlipidemia increases metabolism in Tregs but not conventional T cells. Increased metabolism resulted from preferential activation of the serine/threonine kinase Akt2 (PKB-β). Expression of a constitutively activated form of Akt2 in CD4 T cells was sufficient to increase glycolysis in Tregs and drive changes in Treg subsets. Induction of hyperlipidemia did not alter Treg metabolism in mice lacking Akt2. Activation of Akt2 was sufficient to drive the production of inflammatory cytokines by Tregs. We suggest that hyperlipidemia alters Treg function through effects on metabolism via Akt2 activation thereby promoting plasticity and decreased function of FoxP3+ T cells.

Porous tantalum-composited gelatin nanoparticles hydrogel integrated with mesenchymal stem cell-derived endothelial cells to construct vascularized tissue in vivo.

The ideal scaffold material of angiogenesis should have mechanical strength and provide appropriate physiological microporous structures to mimic the extracellular matrix environment. In this study, we constructed an integrated three-dimensional scaffold material using porous tantalum (pTa), gelatin nanoparticles (GNPs) hydrogel, and seeded with bone marrow mesenchymal stem cells (BMSCs)-derived endothelial cells (ECs) for vascular tissue engineering. The characteristics and biocompatibility of pTa and GNPs hydrogel were evaluated by mechanical testing, scanning electron microscopy, cell counting kit, and live-cell assay. The BMSCs-derived ECs were identified by flow cytometry and angiogenesis assay. BMSCs-derived ECs were seeded on the pTa-GNPs hydrogel scaffold and implanted subcutaneously in nude mice. Four weeks after the operation, the scaffold material was evaluated by histomorphology. The superior biocompatible ability of pTa-GNPs hydrogel scaffold was observed. Our in vivo results suggested that 28 days after implantation, the formation of the stable capillary-like network in scaffold material could be promoted significantly. The novel, integrated pTa-GNPs hydrogel scaffold is biocompatible with the host, and exhibits biomechanical and angiogenic properties. Moreover, combined with BMSCs-derived ECs, it could construct vascular engineered tissue in vivo. This study may provide a basis for applying pTa in bone regeneration and autologous BMSCs in tissue-engineered vascular grafts.

The human cytomegalovirus protein pUL13 targets mitochondrial cristae architecture to increase cellular respiration during infection

Viruses modulate mitochondrial processes during infection to increase biosynthetic precursors and energy output, fueling virus replication. In a surprising fashion, although it triggers mitochondrial fragmentation, the prevalent pathogen human cytomegalovirus (HCMV) increases mitochondrial metabolism through a yet-unknown mechanism. Here, we integrate molecular virology, metabolic assays, quantitative proteomics, and superresolution confocal microscopy to define this mechanism. We establish that the previously uncharacterized viral protein pUL13 is required for productive HCMV replication, targets the mitochondria, and functions to increase oxidative phosphorylation during infection. We demonstrate that pUL13 forms temporally tuned interactions with the mitochondrial contact site and cristae organizing system (MICOS) complex, a critical regulator of cristae architecture and electron transport chain (ETC) function. Stimulated emission depletion superresolution microscopy shows that expression of pUL13 alters cristae architecture. Indeed, using live-cell Seahorse assays, we establish that pUL13 alone is sufficient to increase cellular respiration, not requiring the presence of other viral proteins. Our findings address the outstanding question of how HCMV targets mitochondria to increase bioenergetic output and expands the knowledge of the intricate connection between mitochondrial architecture and ETC function.

Natural Compounds Isolated from Stachybotrys chartarum Are Potent Inhibitors of Human Protein Kinase CK2

A large number of secondary metabolites have been isolated from the filamentous fungus Stachybotrys chartarum and have been described before. Fourteen of these natural compounds were evaluated in vitro in the present study for their inhibitory activity towards the cancer target CK2. Among these compounds, stachybotrychromene C, stachybotrydial acetate and acetoxystachybotrydial acetate turned out to be potent inhibitors with IC50 values of 0.32 µM, 0.69 µM and 1.86 µM, respectively. The effects of these three compounds on cell proliferation, growth and viability of MCF7 cells, representing human breast adenocarcinoma as well as A427 (human lung carcinoma) and A431 (human epidermoid carcinoma) cells, were tested using EdU assay, IncuCyte® live-cell imaging and MTT assay. The most active compound in inhibiting MCF7 cell proliferation was acetoxystachybotrydial acetate with an EC50 value of 0.39 µM. In addition, acetoxystachybotrydial acetate turned out to inhibit the growth of all three cell lines completely at a concentration of 1 µM. In contrast, cell viability was impaired only moderately, to 37%, 14% and 23% in MCF7, A427 and A431 cells, respectively.

Development of a specific live-cell assay for native autophagic flux

Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.

Phagocytic Activities of Reactive Microglia and Astrocytes Associated with Prion Diseases Are Dysregulated in Opposite Directions.

Phagocytosis is one of the most important physiological functions of the glia directed at maintaining a healthy, homeostatic environment in the brain. Under a homeostatic environment, the phagocytic activities of astrocytes and microglia are tightly coordinated in time and space. In neurodegenerative diseases, both microglia and astrocytes contribute to neuroinflammation and disease pathogenesis, however, whether their phagocytic activities are up- or downregulated in reactive states is not known. To address this question, this current study isolated microglia and astrocytes from C57BL/6J mice infected with prions and tested their phagocytic activities in live-cell imaging assays that used synaptosomes and myelin debris as substrates. The phagocytic uptake by the reactive microglia was found to be significantly upregulated, whereas that of the reactive astrocytes was strongly downregulated. The up- and downregulation of phagocytosis by the two cell types were observed irrespective of whether disease-associated synaptosomes, normal synaptosomes, or myelin debris were used in the assays, indicating that dysregulations are dictated by cell reactive states, not substrates. Analysis of gene expression confirmed dysregulation of phagocytic functions in both cell types. Immunostaining of animal brains infected with prions revealed that at the terminal stage of disease, neuronal cell bodies were subject to engulfment by reactive microglia. This study suggests that imbalance in the phagocytic activities of the reactive microglia and astrocytes, which are dysregulated in opposite directions, is likely to lead to excessive microglia-mediated neuronal death on the one hand, and the inability of astrocytes to clear cell debris on the other hand, contributing to the neurotoxic effects of glia as a whole.

Abstract 2472: Effective ER antagonists resist conformational restrictions imposed by somatic mutation but do not correlate with effects on receptor stability in live cells

Abstract Approximately 50% of luminal breast cancer patients become resistant to first-line endocrine therapies after an average of 5 years. Acquired activating mutations to the ESR1 ligand binding domain enable hormone therapy resistance in approximately 25-40% of these patients. Competitive estrogen antagonists function by maintaining the receptor in a transcriptionally inactive state by forcing estrogen receptor alpha ligand binding domain (ERα LBD) helix 12 into the AF-2 cleft to prevent the docking of LXXLL-containing transcriptional coactivators. Multiple next-generation ERα antagonists that function as molecular degraders of the receptor have been described and shown activity in breast cancers harboring the activating ESR1 mutations. To understand whether ERα antagonism correlates with receptor stability, we examined a comprehensive panel of structurally diverse antagonists in live breast cancer cell assays using WT, Y537S, and D538G-mutant receptors. We found that potent antagonists are able to both stabilize and degrade the receptor and further demonstrate that the D538G mutation reduces turnover with ICI due to specific defects in receptor ubiquitination and SUMOylation. High resolution x-ray crystal structures of WT and Y537S ERα LBD in complex with stabilizing and degrading antiestrogens suggest that modulators that enforce the classical antagonist-like helix 12 conformation in the mutant receptor achieve the greatest activity in transcriptional assays, regardless of their effects on receptor stability Citation Format: David Hosfield, Nan-Sheng Li, Ross Han, Muriel Laine, Sandra Weber, Madaline Sauvage, Sylvie Madar, Geoffrey Greene, Sean Fanning. Effective ER antagonists resist conformational restrictions imposed by somatic mutation but do not correlate with effects on receptor stability in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2472.

Linking Biophysical and Transcriptional Profiles of In Vivo-Treated Human Leukemias on a Single-Cell Level Uniquely Resolves Subpopulations of Response

Introduction: Assays are needed to define the susceptibility of heterogeneous primary human tumor cells. We previously showed that single-cell mass accumulation rate (MAR) - conceptually similar to growth rate - can accurately define drug sensitivity ex vivo (Stevens et al. Nature Biotechnology, 2016). This live-cell assay is performed using a suspended microchannel resonator (SMR), which maintains cell viability and permits the downstream application of single-cell whole transcriptome sequencing (scRNA-seq) to identify molecular correlates of drug response. By comparing leukemias exposed to antineoplastic therapy or vehicle in vivo , we sought to identify unique transcriptional signatures that, when coupled with single-cell biophysical properties, provide a more nuanced view of therapeutic susceptibility.Methods: We transplanted a patient-derived xenograft of BCR-ABL-rearranged, B-cell leukemia into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ recipients. This model was derived from a patient whose leukemia relapsed following combination chemotherapy and multiple tyrosine kinase inhibitors. Clinical sequencing revealed an ABL1 Y253H resistance mutation that was confirmed in the PDX. Upon engraftment, animals initiated therapy with ponatinib 40 mg/kg/day or vehicle via oral gavage. Splenocytes were harvested on treatment day 5, and immunomagnetically enriched leukemia cells were individually profiled for mass and MAR using the SMR, from which they were sequentially released directly into micro PCR tubes. Smart-Seq2 whole transcriptome amplification, library preparation, and 30-bp paired-end sequencing was then performed on these individual cells.Results: Leukemia cells treated with ponatinib in vivo had significantly lower mass and normalized MAR (MAR/mass) than vehicle-treated controls (p<2e-16 by Welch two-sample t-test, Figure 1A). Single-cell RNA-seq libraries generated from both treatment conditions were of appropriate quality and complexity, with 5,540 ± 81 and 3,826 ± 92 genes mapped per cell for vehicle- and ponatinib-treated cells, respectively. These figures are in line with our published experience (Kimmerling et al. Nature Communications , 2016). Library quality was temporally stable, with consistent average single-cell complexities maintained over the full course of collection experiments.Principal components analysis segregated cells by treatment status according to PC1 (positive enrichment for protein translation genes) and PC2 (negative enrichment for B-cell activation and B-cell receptor signaling genes) (Figure 1B). While single-cell measurements of biophysical properties or gene expression independently resolved treated and untreated cells at the population level (p<2e-16 by Welch two-sample t-test for both normalized MAR as well as PC1), layering single-cell mass and MAR on top of the transcriptomic data enabled us to identify and characterize subpopulations within treatment groups. For instance, biophysical measurements defined a subset of ponatinib-treated cells that maintained high mass and MAR despite continued therapeutic pressure. No subset of cells corresponding to this physically distinct subpopulation was identified by analysis of single-cell transcriptional profiles alone. Critically, linking biophysical and gene expression measurements from the same cells revealed gene sets with functional enrichment for cell cycle and translation - distinct from the gene sets identified from principal components analysis - that correlated with single-cell mass and mass-normalized MAR, respectively.Conclusions: Integrating the biophysical and molecular profiles of human leukemias on a single-cell level using the SMR platform coupled with scRNA-seq permits assessment of in vivo therapeutic susceptibility within hours of tissue sampling, with the potential to resolve and characterize intratumoral heterogeneity with greater precision. We are now focused on defining the clinical significance of such physical-molecular tumor subpopulations and interrogating mechanisms of therapeutic resistance illuminated by these methods in the context of phase II-like in vivo treatment studies. [Display omitted] DisclosuresStevens:Travera: Equity Ownership, Other: Co-founder. Kimmerling:Travera: Equity Ownership, Other: Co-founder. Manalis:Affinity Bio: Equity Ownership, Other: Co-founder; Travera: Consultancy, Equity Ownership, Other: Co-founder.

Live Cell Assays for the Assessment of Antioxidant Activities of Plant Extracts.

Plant extracts and pharmacopoeias represent an exceptional breeding ground for the discovery of new antioxidants. Until recently, the antioxidant activity was only measured by chemical hydrogen atom transfer (HAT) and single-electron transfer (SET) cell-free assays that do not inform about the actual effect of antioxidants in living systems. By providing information about the mode of action of antioxidants at the subcellular level, recently developed live cell assays are now changing the game. The idea of this review is to present the different cell-based approaches allowing a quantitative measurement of antioxidant effects of plant extracts. Up to date, only four different approaches have reached a certain degree of standardization: (1) the catalase-like assay using H2O2 as a stressor, (2) the cell antioxidant assay (CAA) using AAPH as a stressor and DCFH-DA as a readout, (3) the AOP1 assay which uses photoinduction to monitor and control cell ROS production, and (4) the Nrf2/ARE gene reporter system. The molecular aspects of these assays are presented in detail along with their features, drawbacks, and benefits. The Nrf2/ARE gene reporter system dedicated to indirect antioxidant effect measurement currently represents the most standardized approach with high-throughput applications. AOP1, the first technology linking a fine-tuning of cell ROS production with a quantitative signal, appears to be the most promising tool for the assessment of direct cellular ROS-scavenging effects at an industrial scale.