High-throughput Flow Research Articles

Label-free ghost cytometry for manufacturing of cell therapy products

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However, current QC methods, reliant on molecular staining, pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC), a machine learning-driven, multidimensional, high-content, and high-throughput flow cytometry approach, in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975), T cells and non-T cells in white blood cells (ROC-AUC = 0.969), activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990), and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers, assess viability, identify specific cell subsets or phenotypic states, and remove impurities during cell therapy manufacturing. Thus, LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency.

Deep learning classification method for boar sperm morphology analysis.

Boar semen quality emphasizes three major criteria: sperm concentration, motility, and morphology. Methods to analyze concentration and motility quickly and objectively readily exist, but few exist for analyzing morphology outside of subjective manual counting. Other vital factors for fertilization, like acrosome health, lack efficient detection methods due to limitations in detection by the human eye and costly biomarker analysis, which is rarely used in semen diagnostics. To overcome these challenges, we propose a novel approach integrating deep-learning technology with high-throughput image-based flow cytometry (IBFC) for objective and accurate analysis of both morphology and label-free acrosome health of thousands of individual spermatozoa at once, as opposed to manually counting on a microscope slide. Images of 10,000 spermatozoa were captured using an IBFC and manually annotated based on the primary morphological defect or acrosome health status for the training of the convolutional neural network (CNN). The CNN used these images to train and then applied that training to unannotated images to predict the model accuracy. Using the CNNs, high F1 scores of 96.73%, 98.55%, and 99.31% for 20x, 40x, and 60x magnifications, respectively, for morphological classification were attained. Additionally, the model demonstrates an F1 score of 99.8% in detecting subtle acrosome health variations at the 60x magnification. We have established an integrated approach to rapidly collect and classify morphological defects and acrosome health status, without the use of manual counting or biomarker labeling. Our study underscores the potential of artificial intelligence in semen diagnostics, reducing technician variability, streamlining assays, and facilitating the development of additional label-free detection methods. This innovative approach addresses the barriers hindering biomarker adoption in semen analysis, offering a promising avenue for enhancing reproductive health assessments.

High-throughput fluorescence lifetime imaging flow cytometry

Flow cytometry is a vital tool in biomedical research and laboratory medicine. However, its accuracy is often compromised by undesired fluctuations in fluorescence intensity. While fluorescence lifetime imaging microscopy (FLIM) bypasses this challenge as fluorescence lifetime remains unaffected by such fluctuations, the full integration of FLIM into flow cytometry has yet to be demonstrated due to speed limitations. Here we overcome the speed limitations in FLIM, thereby enabling high-throughput FLIM flow cytometry at a high rate of over 10,000 cells per second. This is made possible by using dual intensity-modulated continuous-wave beam arrays with complementary modulation frequency pairs for fluorophore excitation and acquiring fluorescence lifetime images of rapidly flowing cells. Moreover, our FLIM system distinguishes subpopulations in male rat glioma and captures dynamic changes in the cell nucleus induced by an anti-cancer drug. FLIM flow cytometry significantly enhances cellular analysis capabilities, providing detailed insights into cellular functions, interactions, and environments.

Analysis of cell cycle stage, replicated DNA, and chromatin-associated proteins using high-throughput flow cytometry

Abstract Flow cytometry is a versatile tool used for cell sorting, DNA content imaging, and determining various cellular characteristics. With the possibility of high-throughput analyses, it combines convenient labelling techniques to serve rapid, quantitative, and qualitative workflows. The ease of sample preparation and the broad range of applications render flow cytometry a preferred approach for many scientific questions. Yet, we lack practical adaptations to fully harness the quantitative and high-throughput capabilities of most cytometers for many organisms. Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. In proof-of-principle experiments, we show that DNA content increase in S-phase correlates with de novo DNA synthesis and can be predicted by the presence of the replicative helicase MCM2-7 on genomic DNA.

Natural products from food sources can alter the spread of antimicrobial resistance plasmids in Enterobacterales.

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum β-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum β-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Pre-existing cell subpopulations in primary prostate cancer tumors display surface fingerprints of docetaxel-resistant cells.

Docetaxel resistance is a significant obstacle in the treatment of prostate cancer (PCa), resulting in unfavorable patient prognoses. Intratumoral heterogeneity, often associated with epithelial-to-mesenchymal transition (EMT), has previously emerged as a phenomenon that facilitates adaptation to various stimuli, thus promoting cancer cell diversity and eventually resistance to chemotherapy, including docetaxel. Hence, understanding intratumoral heterogeneity is essential for better patient prognosis and the development of personalized treatment strategies. To address this, we employed a high-throughput single-cell flow cytometry approach to identify a specific surface fingerprint associated with docetaxel-resistance in PCa cells and complemented it with proteomic analysis of extracellular vesicles. We further validated selected antigens using docetaxel-resistant patient-derived xenografts in vivo and probed primary PCa specimens to interrogate of their surface fingerprint. Our approaches revealed a 6-molecule surface fingerprint linked to docetaxel resistance in primary PCa specimens. We observed consistent overexpression of CD95 (FAS/APO-1), and SSEA-4 surface antigens in both in vitro and in vivo docetaxel-resistant models, which was also observed in a cell subpopulation of primary PCa tumors exhibiting EMT features. Furthermore, CD95, along with the essential enzymes involved in SSEA-4 synthesis, ST3GAL1, and ST3GAL2, displayed a significant increase in patients with PCa undergoing docetaxel-based therapy, correlating with poor survival outcomes. In summary, we demonstrate that the identified 6-molecule surface fingerprint associated with docetaxel resistance pre-exists in a subpopulation of primary PCa tumors before docetaxel treatment. Thus, this fingerprint warrants further validation as a promising predictive tool for docetaxel resistance in PCa patients prior to therapy initiation.

Pharmacological induction of MHC-I expression in tumor cells revitalizes T cell antitumor immunity.

Antigen presentation by major histocompatibility complex class I (MHC-I) is crucial for T cell-mediated killing, and aberrant surface MHC-I expression is tightly associated with immune evasion. To address MHC-I downregulation, we conducted a high-throughput flow cytometry screen, identifying bleomycin (BLM) as a potent inducer of cell surface MHC-I expression. BLM-induced MHC-I augmentation rendered tumor cells more susceptible to T cells in coculture assays and enhanced antitumor responses in an adoptive cellular transfer mouse model. Mechanistically, BLM remodeled the tumor immune microenvironment, inducing MHC-I expression in a manner dependent on ataxia-telangiectasia mutated/ataxia telangiectasia and Rad3-related-NF-κB. Furthermore, BLM improved T cell-dependent immunotherapeutic approaches, including bispecific antibody therapy, immune checkpoint therapy, and autologous tumor-infiltrating lymphocyte therapy. Importantly, low-dose BLM treatment in mouse models amplified the antitumor effect of immunotherapy without detectable pulmonary toxicity. In summary, our findings repurpose BLM as a potential inducer of MHC-I, enhancing its expression to improve the efficacy of T cell-based immunotherapy.

A multiplexed high throughput screening assay using flow cytometry identifies glycolytic molecular probes in bloodstream form Trypanosoma brucei

Kinetoplastid organisms, including Trypanosoma brucei, are a significant health burden in many tropical and semitropical countries. Much of their metabolism is poorly understood. To better study kinetoplastid metabolism, chemical probes that inhibit kinetoplastid enzymes are needed. To discover chemical probes, we have developed a high-throughput flow cytometry screening assay that simultaneously measures multiple glycolysis-relevant metabolites in live T. brucei bloodstream form parasites. We transfected parasites with biosensors that measure glucose, ATP, or glycosomal pH. The glucose and ATP sensors were FRET biosensors, while the pH sensor was a GFP-based biosensor. The pH sensor exhibited a different fluorescent profile from the FRET sensors, allowing us to simultaneously measure pH and either glucose or ATP. Cell viability was measured in tandem with the biosensors using thiazole red. We pooled sensor cell lines, loaded them onto plates containing a compound library, and then analyzed them by flow cytometry. The library was analyzed twice, once with the pooled pH and glucose sensor cell lines and once with the pH and ATP sensor cell lines. Multiplexing sensors provided some internal validation of active compounds and gave potential clues for each compound's target(s). We demonstrated this using the glycolytic inhibitor 2-deoxyglucose and the alternative oxidase inhibitor salicylhydroxamic acid. Individual biosensor-based assays exhibited a Z′-factor value acceptable for high-throughput screening, including when multiplexed. We tested assay performance in a pilot screen of 14,976 compounds from the Life Chemicals Compound Library. We obtained hit rates from 0.2 to 0.4% depending on the biosensor, with many compounds impacting multiple sensors. We rescreened 44 hits, and 28 (64%) showed repeatable activity for one or more sensors. One compound exhibited EC50 values in the low micromolar range against two sensors. We expect this method will enable the discovery of glycolytic chemical probes to improve metabolic studies in kinetoplastid parasites.

Deciphering the Relationship between Cell Growth and Cell Cycle in Individual Escherichia coli Cells by Flow Cytometry.

Accurate coordination of chromosome replication and cell division is essential for cellular processes, yet the regulatory mechanisms governing the bacterial cell cycle remain contentious. The lack of quantitative data connecting key cell cycle players at the single-cell level across large samples hinders consensus. Employing high-throughput flow cytometry, we quantitatively correlated the expression levels of key cell cycle proteins (FtsZ, MreB, and DnaA) with DNA content in individual bacteria. Our findings reveal distinct correlations depending on the chromosome number (CN), specifically whether CN ≤2 or ≥4, unveiling a mixed regulatory scenario in populations where CN of 2 or 4 coexist. We observed function-dependent regulations for these key proteins across nonoverlapping division cycles and various nutrient conditions. Notably, a logarithmic relationship between total protein content and replication origin number across nutrient conditions suggests a unified mechanism governing cell cycle progression, confirming the applicability of Schaechter's growth law to cells with CN ≥4. For the first time, we established a proportional relationship between the synthesis rates of key cell cycle proteins and chromosome dynamics in cells with CN ≥4. Drug experiments highlighted CN 2 and 4 as pivotal turning points influencing cellular resource allocation. This high-throughput, single-cell analysis provides interconnected quantitative insights into key molecular events, facilitating a predictive understanding of the relationship between cell growth and cell cycle.

ATRT-19. CELL SURFACE PROTEOME ANALYSIS OF ATRT IDENTIFIES CD44V6 AS A CAR TARGET

Abstract Atypical teratoid rhabdoid tumor (ATRT) is an uncommon, aggressive tumor affecting the brain and spinal cord primarily in children. Despite significant strides in understanding cancer biology through DNA and RNA sequencing, nearly 90% of ATRTs exhibit a crucial deletion that triggers rampant tumor growth. The absence of easily targetable genetic mutations in ATRT complicates the development of effective treatments, necessitating innovative strategies for therapy. Evading immune system detection is a key feature of cancer cells. In our study, we conducted an exhaustive, unbiased examination of 350 surface receptors through high-throughput multicolor flow cytometry on samples from ATRT surgeries, cell lines, and xenograft models derived from patients. By using a combination of antibodies targeting CD31, CD45, CD11b, CCR2, Cx3CR1, CD4, and CD8, we could identify immune cell populations within the tumors. Our findings revealed a heightened presence of CD44, CD146, CD59, CD151, and CD276 on the surface. Using qPCR we identified CD44V6 a variant isoform of the CD44 molecule, which is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. CD44 has multiple isoforms generated through alternative splicing, with CD44V6 being one of these variants. This specific isoform includes an additional variable exon (exon v6) in its extracellular domain. CD44v6-directed CAR-T cells effectively controlled tumor growth in multiple myeloma and solid tumors such as HNSCC, lung and ovarian adenocarcinomas. We developed CD44V6-directed CAR-T cells and investigated the capacity of Anti-CD44v6 CAR-T cells to execute cytotoxicity against ATRT tumor cells. We see significant cell killing by Anti-CD44v6 CAR-T cells in vitro and a significant increase in the survival of ATRT tumor-bearing mice.

High-speed optical imaging with sCMOS pixel reassignment

Fluorescence microscopy has undergone rapid advancements, offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However, the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here, we introduce sHAPR, a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings, enabling acquisition at the maximum camera readout rate, typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems, including high-throughput flow cytometry, cardiomyocyte contraction, and neuronal calcium waves, using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities, opening up new avenues for investigating high-speed biological phenomena.

Arctic phytoplankton microdiversity across the marginal ice zone: Subspecies vulnerability to sea-ice loss

Seasonal phytoplankton blooms are important Arctic phenomena, contributing to global primary production and biogeochemical cycling. The decline in sea-ice extent and thickness favors a longer open-water period with impacts on phytoplankton dynamics. Arctic net productivity is influenced by microalgae living associated with sea ice, with distinct species thought to be favored by ice-covered and ice-free waters. In this study, we investigated the phytoplankton community structure in Baffin Bay, a semi-enclosed sea where Arctic and North Atlantic water masses interact. We compared communities from the ice-free Atlantic-influenced eastern, the marginal ice zone, and the ice-covered Arctic-influenced western Baffin Bay. The community was characterized using 18S rRNA high-throughput amplicon sequencing and flow cytometry cell counting, and compared to environmental data collected during the Green Edge campaign. We sampled 16 stations grouped by sectors according to sea-ice cover. In the sectors associated with sea ice, phytoplankton formed a highly diverse community of smaller taxa, which contrasted with a low-diversity community in ice-free sectors, dominated by larger centric diatoms and Phaeocystis pouchetii adapted to high light/low nutrient conditions. Several phytoplankton species were flagged as indicators for the under-ice and marginal ice zone sectors, including ice-associated taxa such as the diatoms Melosira arctica and Pseudo-nitzschia seriata, but also subspecies representatives of the early-blooming alga Micromonas polaris and the cryptophyte Baffinella frigidus. The strong association of certain taxa with under-ice and marginal ice zone sectors, including Pterosperma sp., Chrysochromulina sp., Micromonas polaris, and B. frigidus, suggest that they might be indicators of diversity loss due to ongoing sea-ice changes in Baffin Bay. We report new intra-species variability of Micromonas polaris suggesting that seasonal specialists could wax and wane over the bloom and non-bloom periods, highlighting the need for detailed year-long studies and the importance of microdiversity when assessing the diversity and distribution of polar phytoplankton.

Flow cytometry as a tool for the rapid enumeration of 1-μm microplastics spiked in wastewater and activated sludge after coagulation-flocculation-sedimentation

Considering the limited literature and the difficulty of quantifying 1-μm micro-nanoplastics (1-μm MNP) in complex aqueous matrices such as wastewater and sludge, the removal rate of these very small particles in wastewater treatment plants (WWTP) represents a major challenge. In this study, coagulation-flocculation-sedimentation (CFS) with aluminum salts was investigated to evaluate the removal of 1-μm MNPs spiked in tap water, raw wastewater, pre-settled wastewater, and activated sludge. Quantification of 1-μm MNP was performed using the high-throughput flow cytometry (FCM) analysis which takes only a few minutes and produces results with high accuracy and reproducibly.The results indicated that the 1-μm MNPs were highly stable in pure water and unable to settle rapidly. In raw wastewater, sedimentation without coagulants removed less than 4% of 1-μm MNP. Conversely, CFS treatment showed a significant improvement in the removal of 1-μm MNP from wastewater. At dosages of 0.3–3 mg Al3+/L, the removal of MNPs in wastewater reached 30% and no flocs were observed, while floc formation was visible with increased dosages of 3–12 mg Al3+/L, obtaining MNP removal greater than 90%. CFS in activated sludge with a solids content of 5800 mg MLSS/L registered the highest removal efficiency (95–99%) even for dosages of 0.3–60 mg Al3+/L and pH dropping to 5. However, activated sludge showed extremely high removal efficiency of MNPs (97.3 ± 0.9%) even without coagulants. The large, dense flocs that constitute activated sludge appear particularly efficient in capturing 1-μm MNPs during the sedimentation process even in the absence of coagulants.

KMT2D regulates activation, localization, and integrin expression by T-cells.

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D's influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8+ single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4+ recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling.

The determination of mycotoxins using isotope-coded labeling and dispersive liquid–liquid microextraction by high throughput flow injection analysis coupled with tandem mass spectrometry

A novel and rapid method for the determination of ochratoxin A (OTA), funmonisin B1 (FB1) and zearalenone (ZEN) in legumes samples by high throughput flow injection analysis-tandem mass spectrometry (FIA-MS/MS) in conjunction with isotope-coded labeling (ICD) coupled with dispersive liquid–liquid microextraction (DLLME) was developed. The samples were extracted by a known QuEChERS method (Quick, Easy, Cheap, Effective, Rugged, Safe). The extracted components and mycotoxin standards were, respectively, labeled by d0-10-methyl-acridone-2-sulfonyl chloride and its deuterated counter part (d3-MASC). Subsequently, the mixed light-/heavy-labeled solutions were enriched and purified by DLLME method and corresponding extracts were directly used for FIA-MS/MS analysis. The FIA-MS/MS method coupled with ICD-coded labeling technique for the determination of mycotoxins in legume samples could be achieved within 2 min, which was much suitable for high throughput screening analysis. Meanwhile, the labeled mycotoxins with d3-MASC as internal standards could effectively eliminate matrix interference and ensure the accuracy and precision of mass spectrometry analysis. The limits of detection (LODs) and the limits of quantification (LOQs) ranged from 0.29 to 0.42 μg/L and 0.85 to 1.30 μg/L, respectively. The recoveries were 66.9 %–85.2 %, and the daily and inter day RSDs were within the range of 2.0 %–4.9 % and 2.4 %–5.0 %, respectively. The established method could be feasibility applied to the determination of mycotoxins in legume samples with satisfactory results.

Harnessing Microbial Effectors for Macrophage-Mediated Drug Delivery.

Macrophage-based drug delivery systems are promising, but their development is still in its infancy, with many limitations remaining to be addressed. Our aim was to design a system harnessing microbial effectors to facilitate controlled drug cargo expulsion from macrophages to enable the use of more toxic drugs without adding to the risk of off-target detrimental effects. The pore forming and actin polymerizing Listeria monocytogenes effectors listeriolysin-O (LLO) and actin assembly-inducing protein (ActA) were synthesized using a novel green fluorescent protein (GFP)-linked heterologous expression system. These effectors were coated onto polystyrene beads to generate "synthetic cargo" before loading into primary M1 macrophages. Bead uptake and release from macrophages were evaluated by using high-throughput quantitative imaging flow cytometry and confocal microscopy. In vitro results confirmed appropriate activity of synthesized effectors. Coating of these effector proteins onto polystyrene beads (simulated drug cargo) resulted in changes in cellular morphology, bead content, and intracellular bead localization, which may support an interpretation of the induced release of these beads from the cells. This forms the basis for further investigation to fully elucidate any potential release mechanisms. Bacterial effectors ActA and LLO successfully effectuated actin polarization and protrusions from cell membranes similar to those seen in cells infected with Listeria spp., illustrating the potential of using these effectors and production methods for the development of an endogenous drug delivery system capable of low-risk, targeted release of high potency drugs.

Leveraging Temperature-Dependent (Electro)Chemical Kinetics for High-Throughput Flow Battery Characterization

The library of redox-active organics that are potential candidates for electrochemical energy storage in flow batteries is exceedingly vast, necessitating high-throughput characterization of molecular lifetimes. Demonstrated extremely stable chemistries require accurate yet rapid cell cycling tests, a demand often frustrated by time-denominated capacity fade mechanisms. We have developed a high-throughput setup for elevated temperature cycling of redox flow batteries, providing a new dimension in characterization parameter space to explore. We utilize it to evaluate capacity fade rates of aqueous redox-active organic molecules, as functions of temperature. We demonstrate Arrhenius-like behavior in the temporal capacity fade rates of multiple flow battery electrolytes, permitting extrapolation to lower operating temperatures. Collectively, these results highlight the importance of accelerated decomposition protocols to expedite the screening process of candidate molecules for long lifetime flow batteries.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP

2’-Fucosyllactose (2’-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2’-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2′-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Abstract 5798: Preclinical safety assessment for specificity of biotherapeutics using the membrane proteome array

Abstract Rigorous specificity analysis is critical for the development of antibody-based therapies, as even minimal off-target binding can lead to toxicity and clinical failures. Long believed to be exquisitely specific, recent preclinical data and our own work indicate that monoclonal antibodies (MAbs) frequently (~25%) display cross-reactivity. In many cases, off-target interactions occur with unrelated proteins that cannot be predicted by protein sequence homology. Cross-reactivity can lead to serious or even life-threatening consequences especially when MAbs are configured as bispecifics, antibody-drug conjugates (ADC) or CAR-T cell therapies, and IND applications for biotherapeutics require cross-reactivity assessment to prevent adverse events. Tissue cross-reactivity (TCR) studies have traditionally been used to screen for off-target binding, however, with poor predictive value for in vivo safety and toxicity. We developed the Membrane Proteome Array (MPA) platform to de-risk MAb-based therapeutics by testing for specificity across 6,000 human membrane proteins expressed in unfixed cells. The MPA assesses binding interactions by high-throughput flow cytometry allowing for high sensitivity detection and rapid analysis. All targets identified on the MPA screen are validated by secondary titration analysis. In contrast to TCR studies, proteins in the MPA exist in their native conformations and are not altered by fixation. Recently, we used the MPA to select a lead antibody candidate targeting the oncotherapeutic target Claudin 6 (CLDN6). Through MPA screening, we successfully identified a lead candidate devoid of cross-reactivity against other CLDN family members or any other membrane protein across the human genome. The MPA has also been used to rapidly evaluate the specificity of novel CAR-T cell therapies for cancer and autoimmunity. The MPA has been used for lead selection and data from the MPA has been accepted by the FDA as a part of IND applications for antibody-based therapies. The MPA is currently under consideration for being developed as an FDA-qualified Drug Development Tool. Citation Format: Kristopher S. Raghavan, Jonathan T. Sullivan, Carmen Navia, Rachel H. Fong, Benjamin J. Doranz. Preclinical safety assessment for specificity of biotherapeutics using the membrane proteome array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5798.

Astragaloside IV Alleviates Acute Liver Failure Induced by D-GalN/LPS by Upregulating Autophagy and Reducing Inflammation

Abstract Acute liver failure (ALF) is a life-threatening condition that manifests in an extremely serious manner and progresses rapidly. The following study investigated the protective effect of astragaloside IV (AS-IV), a traditional Chinese drug, on ALF, and its underlying mechanisms, focusing on autophagy and inflammation regulation. Mice were randomly divided into a saline group, a D-galactosamine and lipopolysaccharide (D-GalN/LPS) group and an AS-IV group. Biochemical analysis, immunohistochemistry, cytometric bead array, high-throughput quantitative PCR, flow cytometry and Western analysis were used to assess inflammation and liver damage 5 hours after D-GalN/LPS exposure. Astragaloside IV treatment reduced mortality by alleviating D-GalN/LPS–induced hepatic damage and decreasing inflammation (decreasing Ly6c+ monocyte levels, reducing inflammatory cytokines and increasing anti-inflammatory factors) as well as upregulating autophagy. Furthermore, PCR array was used to detect expression of autophagy-related genes, which demonstrated a Log2 fold change in gene expression between the AS-IV and D-GalN/LPS groups ranging from 1.19 to −3.53, with Tnfsf10 showing the largest alteration between the two groups. These data suggest that AS-IV may alleviate ALF by upregulating autophagy and reducing inflammation, and it may therefore be an interesting drug for alleviating ALF.