Label-free Assay Research Articles

Profiling CCR3 target pathways for discovering novel antagonists from natural products using label-free cell phenotypic assays

CC chemokine receptor 3 (CCR3) plays important roles in atopic dermatitis (AD) and other related allergic diseases. Activation of CCR3 receptor signaling pathways regulates the recruitment of eosinophils to related tissues, releasing inflammatory mediators and causing inflammatory responses. However, none of the known CCR3 antagonists exhibit promising efficacy in clinical trials. In this work, we sought new natural CCR3 antagonists for drug development. To construct a high-throughput screening model, we established a stably transfected CHO-K1-Gα15-CCR3 cell line, and receptor expression was demonstrated by real-time quantitative PCR, confocal detection and flow cytometry analysis. Then, we applied a label-free cell phenotyping technique to profile and deconvolute CCR3 target pathways in CHO-K1-Gα15-CCR3 cells and found that activation of CCR3 triggered the Gq-PLC-Ca2+ and MAPK-P38-ERK pathways. By in vitro and in silico experiments, we discovered a novel CCR3 antagonist emodin, with an IC50 value of 27.28 ± 1.71 μM out of 266 compounds that were identified in 15 traditional Chinese medicines used in the clinical treatment of skin diseases. Molecular docking graphically presented the binding mode of emodin on CCR3. This work reports a new approach for CCR3 antagonist screening and pathway detection and identifies a new antagonist that would benefit future drug development.

Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs.

Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).

Innovative Chemical Tools to Address Analytical Challenges of Protein Phosphorylation and Glycosylation.

ConspectusProtein post-translational modifications (PTMs) modulate almost all cellular processes. Aberrant PTMs are closely bound to various human diseases, which greatly complicates the association of a specific disease with a genetic mutation and even renders genomics-driven personalized therapeutics ineffective. PTM proteomics has the potential to provide a more comprehensive understanding of disease biology and is emerging as one of the new breakthroughs of precision medicine in the postgenomics era. As two of the most prominent PTMs, phosphorylation and glycosylation have attracted broad research interest both in academia for the discovery of cancer pathogenesis, biomarkers, and therapeutic targets and in industry for the development of targeted drugs and vaccines. Despite the great rewards, current phosphorylation and glycosylation analyses are still faced with some considerable challenges due to the ultralow abundance of phosphorylation and glycosylation and the grand heterogeneity and structural complexity of glycosylation. To date, our laboratory has concentrated on protein phosphorylation and glycosylation and offered unique insights for the development of innovative separation and analytical tools. An overview of these chemical tools, together with our views on potential solutions to the challenges of protein phosphorylation and glycosylation analysis, is presented in this Account.The first part describes our efforts in phosphorylation enrichment and sensing. Given the excellent responsiveness and expandability of smart polymers, we designed a smart polymer material bearing affinity ligands that target the phosphate group and achieved efficient enrichment of multiply phosphorylated peptides through a tunable catch-and-release. Further, the combination of ligand-bearing smart polymers with solid-state nanochannels for the creation of functional nanofluidic devices is presented. The devices allow us to monitor the kinase reaction, thus providing a low-cost, label-free assay for kinase activity. In the next section, we discuss capture strategies for glycosylated species and glycan analysis methods. A Schiff base-containing material was revealed to achieve high-efficiency capture of sialylated glycopeptides through a hydrolysis reaction, demonstrating the power of dynamic covalent chemistry for enrichment. Then, a directed evolution strategy based on phage display was used to develop a lipopolysaccharide (LPS)-targeted ligand that, when incorporated into a polymer as an adsorbent, can efficiently clear the circulating LPS. Based on the distinction of simple glycan isomers with a nanofluidic device, we further explored the identification of diverse glycans with a protein nanopore through a derivatization strategy. The success of the exploration offers a solution to advance nanopore-based single-molecule glycan profiling and glycan sequencing. Although not comprehensive, this Account could provide new insight into the development of chemical tools to facilitate the analysis of phosphorylation and glycosylation as well as other PTMs.

Peroxidase-Mimicking Activity of Nanoceria for Label-Free Colorimetric Assay for Exonuclease III Activity.

We present a novel label-free colorimetric method for detecting exonuclease III (Exo III) activity using the peroxidase-mimicking activity of cerium oxide nanoparticles (nanoceria). Exo III, an enzyme that specifically catalyzes the stepwise removal of mononucleotides from the 3'-OH termini of double-stranded DNA, plays a significant role in various cellular and physiological processes, including DNA proofreading and repair. Malfunctions of Exo III have been associated with increased cancer risks. To assay the activity of Exo III, we applied the previous reports in that the peroxidase-mimicking activity of nanoceria is inhibited due to the aggregation induced by the electrostatic attraction between DNA and nanoceria. In the presence of Exo III, the substrate DNA (subDNA), which inhibits nanoceria's activity, is degraded, thereby restoring the peroxidase-mimicking activity of nanoceria. Consequently, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate is oxidized, leading to a color change from colorless to blue, along with an increase in the absorbance intensity. This approach enabled us to reliably detect Exo III at a limit of detection (LOD) of 0.263 units/mL across a broad dynamic range from 3.1 to 400 units/mL, respectively, with an outstanding specificity. Since this approach does not require radiolabels, complex DNA design, or sophisticated experimental techniques, it provides a simpler and more feasible alternative to standard methods.

Construction of a Dendritic Nanoassembly-Based Fluorescent Biosensor for Electrostatic Interaction-Independent and Label-Free Measurement of Human Poly(ADP-ribose) Polymerase 1 in Lung Tissues.

Poly(ADP-ribose) polymerase 1 (PARP-1) is responsible for catalyzing the creation of poly(ADP-ribose) polymer and involved in DNA replication and repair. Sensitive measurement of PARP-1 is critical for clinical diagnosis. However, the conventional electrostatic attraction-based PAPR-1 assays usually involve laborious procedures, poor sensitivity, and false positives. Herein, we demonstrate the construction of a dendritic nanoassembly-based fluorescent biosensor for electrostatic interaction-independent and label-free measurement of human PARP-1 in lung tumor tissues. When PARP-1 is present, the specific double-stranded DNA (dsDNA)-activated PARP-1 transfers the ADP-ribosyl group from nicotinamide adenine dinucleotide (NAD+)/biotinylated NAD+ to the PARP-1 itself, resulting in the formation of biotinylated dsDNA-PARP-1-PAR polymer bioconjugates that can be captured by magnetic beads. Upon the addition of TdT, APE1, and NH2-modified T-rich probe, the captured dsDNAs with dual 3'-OH termini initiate TdT-activated APE1-mediated hyperbranched amplification to produce abundant dendritic DNA nanoassemblies that can be stained by SYBR Green I to generate a high fluorescence signal. This biosensor is characterized by a template-free, electrostatic interaction-independent, high sensitivity, and label-free assay. It enables rapid (less than 3 h) measurement of PARP-1 with a limit of detection of 4.37 × 10-8 U/μL and accurate measurement of cellular PARP-1 activity with single-cell sensitivity. Moreover, it is capable of screening potential inhibitors and discriminating the PARP-1 level in normal person tissues and lung cancer patient tissues, with great potential in PARP-1-related clinical diagnosis and drug discovery.

Fluorometric Assay of Tyrosinase and Atrazine Based on the Use of Carbon Dots and the Inhibition of Tyrosinase Activity.

Sensitive and convenient strategy of tyrosinase (TYR) and its inhibitor atrazine is in pressing demand for essential research as well as pragmatic application. In this work, an exquisite label-free fluorometric assay with high sensitivity, convenience and efficiency was described for detecting TYR and the herbicide atrazine on the basis of fluorescent nitrogen-doped carbon dots (CDs). The CDs were prepared via one-pot hydrothermal reaction starting from citric acid and diethylenetriamine. TYR catalyzed the oxidation of dopamine to dopaquinone derivative which could quench the fluorescence of CDs through a fluorescence resonance energy transfer (FRET) process. Thus, a sensitive and selective quantitative evaluation of TYR can be constructed on the basis of the relationship between the fluorescence of CDs and TYR activity. Atrazine, a typical inhibitor of TYR, inhibited the catalytic activity of TYR, leading to the reduced dopaquinone and the fluorescence was retained. The strategy covered a broad linear range of 0.1-150 U/mL and 4.0-80.0 nM for TYR and atrazine respectively with a low detection limit of 0.02 U/mL and 2.4 nM/mL. It is also demonstrated that the assay can be applied to detect TYR and atrazine in spiked complex real samples, which provides infinite potential in application of disease monitoring along with environmental analysis.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

Measurement of Patient-Derived Glioblastoma Cell Response to Temozolomide Using Fluorescence Lifetime Imaging of NAD(P)H.

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α2 associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response-cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients.

CETSA and thermal proteome profiling strategies for target identification and drug discovery of natural products

BackgroundMonitoring target engagement at various stages of drug development is essential for natural product (NP)-based drug discovery and development. The cellular thermal shift assay (CETSA) developed in 2013 is a novel, broadly applicable, label-free biophysical assay based on the principle of ligand-induced thermal stabilization of target proteins, which enables direct assessment of drug-target engagement in physiologically relevant contexts, including intact cells, cell lysates and tissues. This review aims to provide an overview of the work principles of CETSA and its derivative strategies and their recent progress in protein target validation, target identification and drug lead discovery of NPs. MethodsA literature-based survey was conducted using the Web of Science and PubMed databases. The required information was reviewed and discussed to highlight the important role of CETSA-derived strategies in NP studies. ResultsAfter nearly ten years of upgrading and evolution, CETSA has been mainly developed into three formats: classic Western blotting (WB)-CETSA for target validation, thermal proteome profiling (TPP, also known as MS-CETSA) for unbiased proteome-wide target identification, and high-throughput (HT)-CETSA for drug hit discovery and lead optimization. Importantly, the application possibilities of a variety of TPP approaches for the target discovery of bioactive NPs are highlighted and discussed, including TPP-temperature range (TPP-TR), TPP-compound concentration range (TPP-CCR), two-dimensional TPP (2D-TPP), cell surface-TPP (CS-TPP), simplified TPP (STPP), thermal stability shift-based fluorescence difference in 2D gel electrophoresis (TS-FITGE) and precipitate supported TPP (PSTPP). In addition, the key advantages, limitations and future outlook of CETSA strategies for NP studies are discussed. ConclusionThe accumulation of CETSA-based data can significantly accelerate the elucidation of the mechanism of action and drug lead discovery of NPs, and provide strong evidence for NP treatment against certain diseases. The CETSA strategy will certainly bring a great return far beyond the initial investment and open up more possibilities for future NP-based drug research and development.

Dimorphism of Candida tropicalis and its effect on nitrogen and phosphorus removal and sludge settleability

Candida tropicalis PNY, a novel dimorphic strain with the capacity of simultaneous carbon, nitrogen and phosphorus removal in anaerobic and aerobic conditions, was isolated from activated sludge. Dimorphism of C. tropicalis PNY had effect on removing nitrogen and phosphorous and slightly affected COD removal under aerobic condition. Sample with high hypha formation rate (40 ± 5%) had more removal efficiencies of NH4+-N (50 mg/L) and PO43−-P (10 mg/L), which could achieve 82.19% and 97.53%, respectively. High hypha cells dosage exhibited good settleability and filamentous overgrowth was not observed. According to label-free quantitative proteomics assays. Up-regulated proteins involved in the mitogen-activated protein kinase (MAPK) pathway indicated the active growth and metabolism process of sample with high hypha formation rate (40 ± 5%). And proteins concerning about glutamate synthetase and SPX domain-contain protein explain for the nutrient removal mechanism including assimilation of ammonia and polyphosphates synthesis.

Development of a liquid tumor potency assay for evaluation of immune cell-mediated cytotoxicity in vitro

Abstract The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells—to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, with each representing an important aspect driving clinical efficacy of the cellular product. Previous work has shown that impedance-based potency assays provide a real-time, label-free measure of effector cell-mediated cytotoxicity for adherent target cells. Here, we describe an in vitro potency assay that quantifies effector cell-mediated cytotoxicity of liquid tumor target cells. First, the wells were coated with antibody to tether the liquid tumor target cells (Raji = anti-CD40, K562 = anti-CD71, Daudi = anti-CD40). Tethering the liquid tumor target cells significantly increased the impedance measurement, as compared to untethered controls. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of effector to target ratios. Cytolysis of the target cells was calculated by comparing the impedance in treated wells to no treatment control wells. As expected, the CD19-specific CAR T cells produced significantly higher cytolysis in the Daudi (54.3%) and Raji (70.4%) cells, which express CD19, as compared to the K562 cells (30.5%). These results support a real-time, label-free potency assay with liquid tumor target cells.

Rapid Testing of Δ9-Tetrahydrocannabinol and Its Metabolite On-Site Using a Label-Free Ratiometric Fluorescence Assay on a Smartphone.

Excessive consumption of Δ9-tetrahydrocannabinol (THC) severely endangers human health and has raised public safety concerns. However, its quantification by readily rapid tools with simplicity and low cost is still challenging. Herein, we found that a G-rich THC aptamer (THC1.2) can tightly bind to thioflavin T (ThT) with strong fluorescence, which would be specifically quenched in the presence of THC. Based on that, a label-free ratiometric fluorescent sensor for the sensing of THC and its metabolite (THC-COOH) based on THC1.2/ThT as a color emitter and red CdTe quantum dots as reference fluorescence was constructed. Notably, a transition of the fluorescent color of the ratiometric probe from green to red can be instantly observed upon the increased concentration of THC and THC-COOH. Furthermore, a portable smartphone-based fluorescence device integrated with a self-programmed Python program was fabricated and used to accomplish on-site monitoring of THC and THC-COOH within 5 min. Under optimized conditions, this ratiometric fluorescent sensor allowed for an instant response toward THC and its metabolite with considerable limits of detection of 97 and 254 nM, respectively. The established sensor has been successfully applied to urine and saliva samples and exhibited satisfactory recoveries (88-116%). This ratiometric fluorescent sensor can be used for the simultaneous detection of THC and THC-COOH with the advantages of rapidness, low cost, ease of operation, and portability, providing a promising strategy for on-site detection and facilitating law enforcement regulation and roadside control of THC.

Monitoring the Reversibility of GPCR Signaling by Combining Photochromic Ligands with Label-free Impedance Analysis.

G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.

Highly sensitive quantitative phase microscopy and deep learning aided with whole genome sequencing for rapid detection of infection and antimicrobial resistance.

Current state-of-the-art infection and antimicrobial resistance (AMR) diagnostics are based on culture-based methods with a detection time of 48-96 h. Therefore, it is essential to develop novel methods that can do real-time diagnoses. Here, we demonstrate that the complimentary use of label-free optical assay with whole-genome sequencing (WGS) can enable rapid diagnosis of infection and AMR. Our assay is based on microscopy methods exploiting label-free, highly sensitive quantitative phase microscopy (QPM) followed by deep convolutional neural networks-based classification. The workflow was benchmarked on 21 clinical isolates from four WHO priority pathogens that were antibiotic susceptibility tested, and their AMR profile was determined by WGS. The proposed optical assay was in good agreement with the WGS characterization. Accurate classification based on the gram staining (100% recall for gram-negative and 83.4% for gram-positive), species (98.6%), and resistant/susceptible type (96.4%), as well as at the individual strain level (100% sensitivity in predicting 19 out of the 21 strains, with an overall accuracy of 95.45%). The results from this initial proof-of-concept study demonstrate the potential of the QPM assay as a rapid and first-stage tool for species, strain-level classification, and the presence or absence of AMR, which WGS can follow up for confirmation. Overall, a combined workflow with QPM and WGS complemented with deep learning data analyses could, in the future, be transformative for detecting and identifying pathogens and characterization of the AMR profile and antibiotic susceptibility.

Target-induced site-specific cleavage of phosphorothioate-modified hairpin DNA for amplified and label-free sensitive electrochemical myeloperoxidase assay

Abnormal myeloperoxidase (MPO) expression is associated with inflammation and neurodegenerative diseases. By using a new phosphorothioate (PT)-modified DNA hairpin probe, we fabricate a label-free and highly sensitive electrochemical sensor for monitoring MPO via integration of DNAzyme-assisted cleavage recycling with DNA polymerization reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). The target MPO mediates the specific cleavage of PT-modified hairpin probes with assistance of hydrogen peroxide and chloride ion to initiate DNAzyme-based cyclic cleavage of substrate hairpin probes for the release of numerous trigger strands. Subsequent hybridizations of the trigger strands with the thiolated-hairpin probes on the electrode surface expose the 3′-OH termini of hairpin probes for triggering TdT–catalyzed in situ DNA polymerization formation of long ssDNAs containing multiple G-quadruplex repeats. Hemin is confined on the electrode surface via the formation of G-quadruplex/hemin complexes to produce highly enhanced current signals for the determination of MPO in a sensitive and label-free way. The proposed sensor exhibits a linear range of 0.1–100 ng mL−1 and achieves a low detection limit of 0.046 ng mL−1 for MPO. Such sensor also shows high selectivity against other interferents and shows the capability to detect MPO in diluted human serum samples.

Abstract 3907: Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor

Abstract Resistance to targeted therapies is a major challenge in oncology. Disease progression is caused by multiple resistance mechanisms. Besides pre-existing and acquired genetic alternations, adaptive non-mutational reprogramming as well as modulation of phenotypic plasticity have emerged as drivers of disease progression. For example, in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) patients who were treated with osimertinib, ~50% of progressive disease could not be attributed to genetic mutations (Leonetti et al., BJC 2019). Similarly, in ~ 40% of patients suffering from KRAS G12C-mutated NSCLC and colorectal cancer (CRC), disease progressed under sotorasib treatment without identifiable acquired mutations (Zhao et al., Nature 2021). Thus, targeting non-genetic adaptive resistance mechanism such as drug-induced transcriptional reprogramming might be of great therapeutic benefit. Here we identified small molecules, that interfere with cancer drug-induced transcriptional escape mechanisms using a phenotypic screen based on an SRY-Box Transcription Factor 2 (SOX2) reporter system. The screen led to the development of TT125-802, a highly specific and potent, orally available small molecule inhibitor of the bromodomain of the transcriptional and epigenetic regulator CBP [cyclic adenosine monophosphate response element binding protein (CREB) binding protein] and its paralogue p300. TT125-802 dose-dependently prevented osimertinib resistance development in EGFR-mutated NSCLC cell lines HCC827 and HCC4006, as well as sotorasib resistance development in KRAS G12C-mutated NCI-H358 (NSCLC), SW837 and SNU-1411 (CRCs) as assessed by label-free long-term live microscopy assays. Data generated in mouse xenograft studies confirmed the ability of TT125-802 increasing response rates and prolonging the duration of response to osimertinib and sotorasib in vivo. In cells and tumours which were already resistant to osimertinib or sotorasib, TT125-802 could still delay cell or tumor growth. Complementary and longitudinal analysis of transcriptional changes using RNA sequencing in vitro and in vivo identified several early adaptive and late acquired resistance signatures that were reversed by TT125-802. A first-in-human study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Thomas Bohnacker, Dorothea Gruber, Sara Laudato, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3907.

Abstract 3694: Metabolic response in human cervical cancer cells to modifications in bioavailable ATP

Abstract Introduction: Cancer cells utilize a markedly reduced oxidative phosphorylation (OXPHOS) when experiencing conditions of high oxygen stress (Warburg Effect/aerobic glycolysis), a phenomenon first studied by Otto Warburg. While there have been many discoveries associated with metabolic differences across cancer cells, one of the most common alterations is the primary means of generating ATP. The importance of deciphering these cellular decisions will enable an understanding of the multifaceted role ATP has in cancer proliferation, senescence, and chemotherapeutic resistance. The use of synthetic protein DX, a man-made ATP chelator with high specificity and affinity, is a proposed method of investigating the metabolic response in cancer cells. Hypothesis: Following DX expression, HeLa cells respond to energy stress (reduced intracellular ATP) via metabolic adaptations, specifically inducing the spare respiratory capacity. Methods: The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX expression. Additionally, the relative contribution of glycolysis and OXPHOS to the total ATP production rate was measured using the label-free XF Real Time ATP Rate Assay and XF Cell Mito Stress Test (XFe96 Seahorse, Agilent Technologies) over time post DX expression. To determine the impact of ATP stress on mitochondrial and nuclear content, both were quantified following DX-expression. Results: In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. In response to DX over time, the total ATP production rates in HeLa cells significantly decreased. Importantly, this reduction in the rate of ATP production was associated with a significant downregulation in both OXPHOS and glycolysis. Conclusion: Advances in synthetic biology have allowed for the intentional design of artificial proteins that maintain function in vivo. These proteins can be developed into powerful investigative tools to study biological questions, such as cellular decisions involved in cancer cell metabolism. In response to DX-potentiated ATP stress, the significant reduction in both glycolysis and oxidative phosphorylation suggests metabolic downregulation is required to maintain HeLa cell viability. Citation Format: Parth K. Jayaswal, Taha Muhammad, Ashley Brown, Jeffrey Norris, Shaleen B. Korch. Metabolic response in human cervical cancer cells to modifications in bioavailable ATP. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3694.

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.

The demethylase of fat mass and obesity related protein (FTO) is critical to regulate the dynamic N6-methyladenosine (m6A) modification of eukaryotic mRNAs, and its overexpression has found to be closely related to the initiation of several cancers. On the basis of a target-promoted specific activation of DNAzyme strategy coupled with self-primer exponential amplification reaction (SPEXPAR) cycles and DNA supersandwich assemblies, the highly sensitive and label-free electrochemical FTO assay approach is established. The modification of the catalytic core nucleobase of the DNAzyme probe by m6A can inhibit its cleavage activity. The presence of target FTO catalyzes the elimination of the methyl group to restore the DNAzyme activity, which cleaves the hairpin substrates to trigger the SPEXPAR for yielding many ssDNAs. The capture of these DNAs on the sensor electrode leads to the initiation of supersandwich assembly formation of long dsDNAs. Tremendous electrochemical signal probe of [Ru(NH3)6]Cl3 are then absorbed on these dsDNAs to produce highly amplified catalytic currents with the assistance of K3[Fe(CN)6] for detecting trace FTO with 63.1fM detection limit. Furthermore, the sensor can be employed for selective assay of FTO in cell lysates, revealing the great potential of this sensing strategy for biomedical and biological study applications.

Plasmonic Dual-Gap Nanodumbbells for Label-Free On-Particle Raman DNA Assays.

Metal nanostructures with a tunable plasmonic gap are useful for photonics, surface-enhanced spectroscopy, biosensing, and bioimaging applications. The use of these structures as chemical and biological sensing/imaging probes typically requires an ultra-precise synthesis of the targeted nanostructure in a high yield,with Raman dye-labeling and complex assay components and procedures. Here, a plasmonic nanostructure with tunable dual nanogaps, Au dual-gap nanodumbbells (AuDGNs), is designed and synthesizedvia the anisotropic adsorption of polyethyleneimine on Au nanorods to facilitate tip-selective Au growths on nanorod tips for forming mushroom-shaped dumbbell-head structures at both tips and results in dual gaps (intra-head and inter-head gaps) within a single particle. AuDGNs are synthesized in a high yield (>90%) while controlling the inter-head gap size, and the average surface-enhanced Raman scattering (SERS) enhancement factor (EF) value is 7.5×108 with a very narrow EF distribution from 1.5×108 to 1.5×109 for >90% of analyzed particles. Importantly, AuDGNs enable label-free on-particle SERS detection assays through the diffusion of target molecules into the intraparticle gap for different DNA sequences with varying ATGC combinations in a highly specific and sensitive manner without a need for Raman dyes.

Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products

Background aimsChimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells. MethodsWe used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency. ResultsResults indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells. ConclusionsThese studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.