Bioluminescent Biosensor Research Articles

A Novel Bioluminescent Biosensor Quantifying Intramolecular Interaction and Levels of Pyroptosis Effector GSDMD.

Gasdermin D (GSDMD) is a key executor of pyroptosis, a form of inflammation-induced programmed cell death. Recently, GSDMD has been shown to play important roles in the development of various inflammatory-related human diseases including heart failure and cancer, suggesting that it is a promising therapeutic target for these diseases. While extensive studies on GSDMD's role in pyroptosis have been reported, it is challenging to study its function due to the lack of enzymatic activity of GSDMD. In this study, we used the NanoBiT technology to develop a novel GSDMD bioluminescent biosensor (GSDMD-BS) that detects the amount of non-cleaved GSDMD. This sensor allows us to quantify GSDMD's intramolecular interactions, the amounts of uncleaved GSDMD after caspase-1 cleavage, and expression levels in living cells. In vitro experiments using purified GSDMD-BS also confirmed the sensor's accuracy in reporting GSDMD levels and cleavage. Moreover, the potential for in vivo application was demonstrated in a xenograft mouse model. In conclusion, we have developed a GSDMD biosensor that is a valuable tool for real-time monitoring of GSDMD dynamics and pyroptosis. This biosensor will significantly expedite pyroptosis research and can be used for high-throughput screening for drugs targeting GSDMD for the therapy of many inflammation-related diseases.

Abstract 4327: Development of bioluminescent biosensors to monitor the interaction betweenGSDMD and caspase-1 in living cells

Abstract Gasdermin D (GSDMD) is the key executioner of pyroptosis, an inflammatory form of programmed cell death implicated in various inflammation-driven diseases, including cancer, heart failure and strokes. The interaction between GSDMD and caspase-1 (CASP1) orchestrates the critical pathway of GSDMD-mediated pyroptosis. During pyroptosis, pyroptosis stimuli activate CASP1, which subsequently binds to and cleave GSDMD between GSDMD’s N- and C-terminal domains. The free N-terminal domains oligomerize and translocate to the cell membrane, forming pores and releasing proinflammatory cytokines and causing cellular lysis. Key interaction sites have been identified for GSDMD recognition and engagement by CASP1; therefore, blocking functionally significant residues presents a new therapeutic strategy for inhibiting CASP1-induced GSDMD cleavage and pyroptosis. Currently, there is no small molecule capable of specifically disrupting the GSDMD and CASP1 interaction. In response, the ongoing pursuit of identifying potential inhibitors is a driving force for developing a biosensor capable of monitoring this interaction and their roles in downstream pyroptotic effects. In this study, we utilized the NanoLuc Binary Technology (NanoBiT) to construct several bioluminescent biosensors comprising two NanoLuc luciferase enzyme fragments (LgBiT and SmBiT) fused to GSDMD and CASP1, respectively, in various combinations. Further validation of these biosensors by luciferase assays in living cells identified a GSDMD-CASP1 biosensor with highest luciferase activity. This newly developed biosensor paves the way for high-throughput screens to identify novel small molecule inhibitors disrupting the GSDMD-CASP1 interplay. These small molecule inhibitors will effectively impede GSDMD-mediated pyroptosis, holding promise for novel therapies of various human diseases. Citation Format: Tynan Kelly. Development of bioluminescent biosensors to monitor the interaction betweenGSDMD and caspase-1 in living cells [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 4327.

Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays.

Here we present LUCOS (Luminescent Competition Sensor), a modular and broadly applicable bioluminescent diagnostic platform enabling the detection of both small molecules and protein biomarkers. The construction of LUCOS sensors entails the covalent and site-specific coupling of a bioluminescent sensor component to an analyte-specific antibody via protein G-mediated photoconjugation. Target detection is accomplished through intramolecular competition with a tethered analyte competitor for antibody binding. We established two variants of LUCOS: an inherent ratiometric LUCOSR variant and an intensiometric LUCOSI version, which can be used for ratiometric detection upon the addition of a split calibrator luciferase. To demonstrate the versatility of the LUCOS platform, sensors were developed for the detection of the small molecule cortisol and the protein biomarker NT-proBNP. Sensors for both targets displayed analyte-dependent changes in the emission ratio and enabled detection in the micromolar concentration range (KD,app = 16-92 μM). Furthermore, we showed that the response range of the LUCOS sensor can be adjusted by attenuating the affinity of the tethered NT-proBNP competitor, which enabled detection in the nanomolar concentration range (KD,app = 317 ± 26 nM). Overall, the LUCOS platform offers a highly versatile and easy method to convert commercially available monoclonal antibodies into bioluminescent biosensors that provide a homogeneous alternative for the competitive immunoassay.

Investigating G-protein coupled receptor signalling with light-emitting biosensors.

G protein-coupled receptors (GPCRs) are the most frequent target of currently approved drugs and play a central role in both physiological and pathophysiological processes. Beyond the canonical understanding of GPCR signal transduction, the importance of receptor conformation, beta-arrestin (β-arr) biased signalling, and signalling from intracellular locations other than the plasma membrane is becoming more apparent, along with the tight spatiotemporal compartmentalisation of downstream signals. Fluorescent and bioluminescent biosensors have played a pivotal role in elucidating GPCR signalling events in live cells. To understand the mechanisms of action of the GPCR-targeted drugs currently available, and to develop new and better GPCR-targeted therapeutics, understanding these novel aspects of GPCR signalling is critical. In this review, we present some of the tools available to interrogate each of these features of GPCR signalling, we illustrate some of the key findings which have been made possible by these tools and we discuss their limitations and possible developments.

Bioluminescence biosensing platforms for One Health: From paper sensors to thread-based analytical devices

Bioluminescence is a fascinating phenomenon in which photons are emitted as byproduct of a chemical reaction occurring in living organisms, including bacteria, fireflies, and several marine species. Here we report the development of novel bioluminescent tools and strategies which can be used to improve the analytical performance of paper-based smartphone biosensors. In particular two new applications are reported based on paper and thread-based bioluminescent biosensors.

A panel of visual bacterial biosensors for the rapid detection of genotoxic and oxidative damage: A proof of concept study.

The emergence of new compounds during the past decade requires a high-throughput screening method for toxicity assay. The stress-responsive whole-cell biosensor is a powerful tool to evaluate direct or indirect damages of biological macromolecules induced by toxic chemicals. In this proof-of-concept study, nine well-characterized stress-responsive promoters were first selected to assemble a set of blue indigoidine-based biosensors. The PuspA-based, PfabA-based, and PgrpE-based biosensors were eliminated due to their high background. A dose-dependent increase of visible blue signal was observed in PrecA-, PkatG-, and PuvrA-based biosensors, responsive to potent mutagens, including mitomycin and nalidixic acid, but not to genotoxic lead and cadmium. The PrecA, PkatG, and Ppgi gene promoters were further fused to a purple deoxyviolacein synthetic enzyme cluster. Although high basal production of deoxyviolacein is unavoidable, an enhanced visible purple signal in response to mitomycin and nalidixic acid was observed as dose-dependent, especially in PkatG-based biosensors. The study shows that a set of stress-responsive biosensors employing visible pigment as a reporter is pre-validating in detecting extensive DNA damage and intense oxidative stress. Unlike widely-used fluorescent and bioluminescent biosensors, the visual pigment-based biosensor can become a novel, low-cost, mini-equipment, and high-throughput colorimetric device for the toxicity assessment of chemicals. However, combining multiple improvements can further improve the biosensing performance in future studies.

Chitosan-Based Biosensors-A Comprehensive Review

Chitosan is a copolymer that is linearly linked to 2-amino-2-deoxy-Dglucopyronos and is synthesized by diacylation of chitin. A biosensor is an integrated and compact device that makes use of a biological component as a sensing element and is coupled to a transducer for the purpose of signal detection. This review article discusses the chitosan-based biosensors that are currently in high demand due to their advanced applications, excellent range, and detection limits in fields such as soil monitoring, water sample detection, disease detection, food quality management, and many others. These biosensors are described in this article. In this overview, we have discussed biosensors from two different points of view: the transducing and the bioluminescent perspectives. The method of transducing encompasses electrochemical, optical, and physical biosensors respectively. Additionally, a variety of electrochemical biosensors, including amperometric, voltammetric, potentimetric, and impedimetric biosensors, are discussed in detail. Other types of bioluminescent biosensors can be broken down further into categories such as those that detect enzymes, DNA/RNA, herbicides, and pesticides.

Immobilization of Firefly Bioluminescent System: Development and Application of Reagents.

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL-1. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP-0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination.

A Luciferase Mutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing.

The availability of new bioluminescent proteins with tuned properties, both in terms of emission wavelength, kinetics and protein stability, is highly valuable in the bioanalytical field, with the potential to improve the sensitivity and analytical performance of the currently used methods for ATP detection, whole-cell biosensors, and viability assays among others. We present a new luciferase mutant, called BgLuc, suitable for developing whole-cell biosensors and in vitro biosensors characterized by a bioluminescence maximum of 548 nm, narrow emission bandwidth, favorable kinetic properties, and excellent pH- and thermo-stabilities at 37 and 45 °C and pH from 5.0 to 8.0. We assessed the suitability of this new luciferase for whole-cell biosensing with a cell-based bioreporter assay for Nuclear Factor-kappa B (NF-kB) signal transduction pathway using 2D and 3D human embryonic kidney (HEK293T) cells, and for ATP detection with the purified enzyme. In both cases the luciferase showed suitable for sensitive detection of the target analytes, with better or similar performance than the commercial counterparts.

Antibacterial and Oxidative Stress-Protective Effects of Five Monoterpenes from Softwood.

Volatile organic compounds (VOC) affect the quality of indoor air. Terpenes and especially monoterpenes are the main molecules emitted from softwood material (coniferous species), which is widely used in construction. The corneal epithelium is one of the first human membranes to encounter VOCs in the air. Moreover, the industrial use of pleasant-scented monoterpenes in cosmetics, food, and detergents exposes people to monoterpenes in their daily lives. In the present study, the health effective properties of five monoterpenes from softwood were tested; cytotoxicity and oxidative stress-protective effects of α- and β-pinenes, R- and S-limonene, and 3-carene were tested in a human corneal epithelial (HCE) cell model system and with two additional in vitro antioxidant tests: oxygen radical absorbance capacity (ORAC) and hydrogen peroxide (H2O2) scavenging. Antibacterial efficacies were tested with two bioluminescent bacterial biosensor strains (Escherichia coli K12+pcGLS11 and Staphylococcus aureus RN4220+pAT19) and with minimum inhibitory concentration (MIC) test against Escherichia coli. Only very high concentrations of monoterpenes (0.3–0.5 mg/mL) demonstrated cytotoxicity against HCE cells. Contrary to the original hypothesis, monoterpenes did not exhibit strong antioxidant properties in tested concentrations. However, biosensors and MIC tests indicated clear antibacterial activities for all tested monoterpenes.

Abstract 858: Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor

Abstract Understanding features common to all forms of cancer is a vital component of treatment and research into malignant disease. All solid tumours share the ability to overcome contact inhibition of proliferation (CIP), the process by which intercellular contacts engage signalling to stop proliferation. Merlin, a tumour suppressor protein that is inactivated in a wide variety of cancers, plays a crucial role in CIP; merlin-deficient cells lose the ability to be contact-inhibited and subsequently form tumours. Although the role of merlin in cancer has been investigated extensively over the past 28 years, this protein has been notoriously difficult to study. To address this issue, we develop and utilize 2 split-luciferase biosensor systems that enable accurate quantification of merlin activity in real time. Merlin undergoes a conformational change that is functionally important in tumour suppression. Phosphorylation at a key C-terminal residue promotes transition of the protein from an open, active conformation to a closed, N-to-C terminal autoinhibited conformation. Therefore, monitoring this conformation change in real time could provide unique insights into merlin function. To do this, we apply NanoBiT split-luciferase technology to develop an intramolecular merlin biosensor (intra-Mer-BS). In brief, 2 split-luciferase components, LgBiT and SmBiT, are fused to the N- and C-terminus of merlin, respectively. Upon open-to-closed conformation change of merlin, LgBiT and SmBiT complement to reconstitute a functional luciferase and emit light. This enables accurate quantification of merlin’s functionally relevant conformation changes in real time. Importantly, cotransfection of the intra-Mer-BS alongside PAK1, an upstream merlin regulator that promotes transition to the closed conformation, significantly increases luminescent activity of the intra-Mer-BS, indicating that the biosensor faithfully reports on merlin’s conformation. Moreover, merlin has been shown to exert its tumour suppressive function through activation of LATS, the central mediator of the Hippo signalling pathway. In addition to the intra-Mer-BS, we develop and validate a NanoBiT biosensor to monitor the interaction between merlin and LATS (Mer-LATS-BS). This Mer-LATS-BS is used to quantify the effect of merlin activators and inhibitors on merlin/LATS tumour suppressive activity in cancer cells. In summary, we develop and validate 2 novel bioluminescent biosensors to monitor merlin’s conformation changes and activity in cancer cells. The intra-Mer-BS and Mer-LATS-BS provide real time information with high sensitivity and excellent reproducibility. Ultimately, these biosensors enable high throughput screening to discover novel upstream regulators of merlin in cancer and provide mechanistic insight into how contact inhibitive signalling is propagated through merlin and the Hippo pathway. Citation Format: Alexander J. Pipchuk, Xiaolong Yang. Development of ultrasensitive split luciferase biosensors monitoring activity of the merlin tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 858.

The state-of-the-art in bioluminescent whole-cell biosensor technology for detecting various organic compounds in oil and grease content in wastewater: From the lab to the field.

Oil and grease content in wastewater is used as an environmental monitoring parameter in the oil and gas industry to prevent serious pollution. Conventional oil and grease laboratory testing is time-consuming and necessitates the use of a hazardous chemical solvent, resulting in non-real-time test data and unnecessary chemical waste. On-site or real-time analysis can enable monitoring of oil and grease in wastewater before discharge to the environment from an operating plant, allowing immediate action to be taken to mitigate environmental impact before contamination spirals out of control. Bioluminescent whole-cell biosensors have been reported to have high sensitivity and selectivity in environmental samples, but only for a few traces of organic compounds such as polycyclic aromatics and naphthalene, allowing for faster analysis times. However, no evaluation of biosensor application for oil and grease (a mixture of hydrocarbons) detection in wastewater, which is critical in the oil and gas industry, has been published to date. Herein, the advantages, disadvantages, challenges, and limitations of using a whole-cell bioluminescent biosensor technology to measure oil and grease content in wastewater are carefully reviewed. This review attempts to bridge the knowledge gap between conventional laboratory methods and biosensor technology in terms of analytical challenges, identifying areas for improvement as well as real-world applications for oil and grease content detection in wastewater.

Modeling and characterization of an engineered microbial biosensor for high-throughput screening of arsenic in rural water

Current arsenic analysis methods in groundwater samples rely on expensive apparatus, complicated procedures, and dangerous chemical reagents. Also, delays in detecting arsenic harm to public health, the environment, agriculture and food sectors. Therefore, in this study, a bioluminescent biosensor has been optimized and used to detect and measure arsenic concentration in groundwater. Optimum conditions for the appropriate performance of E. coli DH5α (pJAMA-arsR) were determined and the luminescent calibration curve was drawn. The optimization results showed that maximum luminescent light output could occur at the end of the logarithmic phase or the beginning of the stationary phase, the temperature of 37 °C, and pH between 5.5 and 7 upon adding 10 μl n-decanal (18 mM). Increasing the duration of bacterial induction by arsenic leads to elevation of biosensor luminescent light yield. Functional stability of the biosensor with 20 % glycerol (V/V) at −20 °C was verified for at least six months. Luminescence reaction of the bacterial biosensor cells to arsenic concentration in the range of 0–90 ppb was promising (R2 = 0.948 by linear regression), but higher arsenic concentration had poisonous effect on biosensor cells. The modified Gompertz model derived here could successfully predict the bacterial biosensor growth under the optimum condition compared with experimental data. In this study critical challenges, such as technical and appropriate performance, are defined to interpret the bacterial biosensor’s true perspective to promote its broad adoption and usage. The work is concluded with closing remarks and potential perspectives to emphasize the importance of the bacterial biosensor, which could detect arsenic from a wide scope in real-time, quickly, and environmentally friendly signaling tool with high sensitivity and selectivity.

A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

Design of bioluminescent biosensors for assessing contamination of complex matrices

The presence of potentially toxic xenobiotics in complex matrices has become rather the rule than the exception. Therefore, there is a need for highly sensitive inexpensive techniques for analyzing environmental and food matrices for toxicants. Enzymes are selectively sensitive to various toxic compounds, and, thus, they can be used as the basis for detection of contaminants in complex matrices. There are, however, a number of difficulties associated with the analysis of complex matrices using enzyme assays, including the necessity to take into account properties and effects of the natural components of the test media for accurate interpretation of results. The present study describes the six-stage procedure for designing new enzyme sensors intended for assessing the quality of complex matrices. This procedure should be followed both to achieve the highest possible sensitivity of the biosensor to potentially toxic substances and to minimize the effect of the uncontaminated components of complex mixtures on the activity of the biosensor. The proposed strategy has been tested in designing a bioluminescent biosensor for integrated rapid assessment of the safety of fruits and vegetables. The biosensor is based on the coupled enzyme system NAD(P)H:FMN-oxidoreductase and luciferase as the biorecognition element. The study describes methods and techniques for attaining the desired result in each stage. The proposed six-stage procedure for designing bioluminescent enzyme biosensors can be used to design the enzymatic biosensors based on other enzymes.

Effects of Linker Flexibility and Conformational Changes of IP3 Receptor on Split Luciferase Complementation Assay.

Background: IP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.Objectives: Based on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1–604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and Methods: In the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.Results: As expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.Conclusions: It seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Abstracts of the Molecular Epidemiology Group (MEG-UK) Annual Conference on “Prediction & Causal Pathways Towards Disease – What Can Molecular Markers Tell Us?”, 12th March 2019, Bristol, United Kingdom

Resistance to therapy remains one of the greatest challenges in the management of the blood and bone marrow (BM) disorder, acute myeloid leukaemia (AML). This study aimed to investigate how leukaemic and BM stromal cells contribute to altered chemo-sensitivity; with particular interest in genotoxicity protection of leukaemic cells and sensitisation of the supportive BM. AML cells (HL-60 and K562) were mono-cultured or cocultured with a stromal (HS-5) cell line using trans-well inserts, prior to treatment with a physiological dose of cytarabine (25 µM); standard AML induction therapy. Toxicity protection and sensitisation were determined by genotoxicity (micronucleus/alkaline comet) assays, viability (dye exclusion), chemo-sensitivity (bacterial bioluminescent biosensor) and proliferation (CFSE fluorescence). Leukaemic-stromal co-culture altered both cell types in favour of disease progression. HS-5 significantly reduced micronuclei formation (4.72/3.45%; p

A biosensor for measuring NAD+ levels at the point of care

The cellular level of nicotinamide adenine dinucleotide (NAD+), through its different functions, affects cellular metabolism and signalling1-3. A decrease in the NAD+ content has been associated with various pathologies and physiological aging4,5, while strategies to boost cellular NAD+ levels have been shown to be effective against age-related diseases in many animal models6. The link between decreased NAD+ levels and numerous pathologies and physiological aging has triggered the need for a simple quantification method for NAD+, ideally applicable at the point of care. Here, we introduce a bioluminescent biosensor for the rapid quantification of NAD+ levels in biological samples, which can be used either in laboratories or at the point of care. The biosensor is a semisynthetic, light-emitting sensor protein that changes the colour of emitted light from blue to red on binding of NAD+. This NAD+-dependent colour change enables the use of the biosensor in paper-based assays in which NAD+ is quantified by measuring the colour of the emitted light by using either a simple digital camera or a plate reader. We used the approach to quantify NAD+ levels in cell culture, tissue and blood samples, yielding results that agreed with those from standard testing methods. The same biosensor furthermore allows the quantification of NAD+-dependent enzymatic activities in blood samples, thus expanding its utility as a tool for point-of-care diagnostics.

Influence of Bone Marrow Stromal and Leukemic Cells on Cytarabine Chemo-Toxicity in Acute Myeloid Leukemia (AML)

Mesenchymal stromal cells (MSC) are known to protect leukemic cells from drug-induced toxicity within the bone marrow (BM) niche, however, less is known about leukemic impact on supportive MSC. The nucleoside-analogue, cytarabine (Ara-C), is a front-line therapy for acute myeloid leukemia (AML), entering cells via the human equilibrative nucleoside transporter (hENT1). Over a third of AML patients do not show continued response to Ara-C-based regimens, with chemo-resistance linked with repressed hENT1 availability in some patients, while other mechanisms remain unknown. In addition to chemo-resistance, DNA damage caused by chemotherapeutics such as Ara-C can persist in BM-MSC, which remain of host origin following allogeneic stem cell transplantation. This genotoxicity hinders cellular functionality, and may be implicated in long-term hematopoietic failure and secondary malignancies. This study aimed to further elucidate chemo-resistance mechanisms, with particular focus on the contribution of leukemic cells to stromal cell toxicity; aiming to uncover potentially targetable features of resistant AML and reduce treatment burden on the BM. Primary MSC were isolated from BM aspirates from patients both at diagnosis and post-treatment; following ethical approval and informed consent. MSC cultures were confirmed by immunophenotype (flow cytometry) and differentiation capacity (cytological staining) and used in a similar manner to that of cell lines. AML (HL-60, K562) and stromal (HS-5) cell lines were mono- or co-cultured using trans-well inserts for 24h, prior to 1-24h treatment with 25µM Ara-C (equivalent to in vivo standard dose; 100-200mg/m2). Cytotoxicity was monitored by viability and proliferation (CFSE tracing) assays, and chemo-sensitivity assessed with a drug-efficacy screening tool (bacterial bioluminescent biosensor). Genotoxicity was determined by micronucleus and alkaline comet assays, assessing division abnormalities and DNA fragmentation respectively. Differential cytokine secretion utilised an array, with quantification by ELISA. In co-culture, stromal cells were sensitised to drug-induced cytotoxicity, while leukemic cells were themselves protected from treatment. Genotoxicity was also significantly increased in stromal cells (p=0.0397), while being significantly decreased in leukemic cells when co-cultured (p=0.0089), conferring with cytotoxicity findings. Similarly, BM-MSC from previously treated patients had significantly higher genotoxicity than patients at diagnosis (p=0.0138). While stromal cell proliferation remained unchanged regardless of intervention, data suggest increased proliferation in co-cultured leukemic cells compared to cells cultured alone. Chemo-sensitivity also increased in stromal cells in co-culture, while the opposite was seen for leukemic cells. Seven of 32 cytokines were differentially secreted by cell lines in co-culture compared to combined values from mono-cultured cells; CCL2, CXCL1, G-CSF, GM-CSF, IL-6, MIF and Serpin E1. Of these, the inflammatory cytokine MIF, macrophage migration inhibitory factor, was decreased in co-culture (p<0.0001), and has been implicated in the progression of other malignancies. Separation of cells following co-culture and treatment uncovered opposing MIF secretion profiles in cells with high (HL-60) and low (K562) sensitivity to Ara-C. Despite differential secretion, neither MIF, nor the MIF inhibitor ISO-1, had significant effects on chemo-sensitivity when cells were cultured in each condition for 24 hours. Chemo-resistance is evidently a network of complex, interlinking mechanisms which are not easily identified in vitro. MIF remains a likely candidate for studies into AML chemo-resistance, with research ongoing. This study shows for the first time that the co-culture of AML and MSC alters the genotoxic effect of chemotherapy. Future research utilising larger patient cohorts is required to fully understand how cells in the BM micro-environment can be targeted. This could potentially improve not only the overall outcome for AML patients, but reduce the cytotoxic and long-term genotoxic complications of current therapies. Disclosures No relevant conflicts of interest to declare.

Bacterial Bioluminescent Biosensors: Principle and Applications

Bacterial Bioluminescent Biosensors: Principle and Applications