Bioluminescence Measurements Research Articles

Toxin Dynamics among Populations of the Bioluminescent HAB Species Pyrodinium bahamense from the Indian River Lagoon, FL.

Dinoflagellate species that form some of the most frequent toxic blooms are also bioluminescent, yet the two traits are rarely linked when studying bloom development and persistence. P. bahamense is a toxic, bioluminescent dinoflagellate that previously bloomed in Florida with no known record of saxitoxin (STX) production. Over the past 20 years, STX was identified in P. bahamense populations. The goal of this study was to examine toxin dynamics and associated molecular mechanisms in spatially and temporally distinct P. bahamense populations from the Indian River Lagoon, FL. SxtA4 is a key gene required for toxin biosynthesis. SxtA4 genotype analysis was performed on individual cells from multiple sites. Cell abundance, toxin quota cell-1, and sxtA4 and RubisCo (rbcL) transcript abundance were also measured. There was a significant negative correlation between cell abundance and toxin quota cell-1. While the sxtA4+ genotype was dominant at all sites, its frequency varied, but it occurred at 90-100% in many samples. The underlying mechanism for toxin decrease with increased cell abundance remains unknown. However, a strong, statistically significant negative correlation was found between stxA4 transcripts and the sxtA4/rbcL ratio, suggesting cells make fewer sxtA4 transcripts as a bloom progresses. However, the influence of sxtA4- cells must also be considered. Future plans include bioluminescence measurements, normalized to a per cell basis, at sites when toxicity is measured along with concomitant quantification of sxtA4 gene and transcript copy numbers as a means to elucidate whether changes in bloom toxicity are driven more at the genetic (emergence of sxtA4- cells) or transcriptional (repression of sxtA4 in sxtA4+ cells) level. Based on the results of this study, a model is proposed that links the combined traits of toxicity and bioluminescence in P. bahamense bloom development.

Luciferase transduction and selection protocol for reliable in vivo bioluminescent measurements in cancer research

Bioluminescence imaging has become an essential non-invasive tool in cancer research for monitoring various cellular processes and tumor progression in vivo. In this article, we aimed to propose a transduction and selection protocol for reliable in vivo bioluminescent measurements in immunocompetent mouse models. Using two different heterogenous luciferase-expressing cell models, we underlined factors influencing transduction. The protocol was tested through an in vitro luciferase activity assay as well as using in vivo longitudinal monitoring of metastases formation (In Vivo Imaging System®). The data were cross validated with histological assessment. Our results demonstrated stable and proportional in vitro and in vivo bioluminescent signals correlating with actual metastatic burden. Furthermore, ex vivo analysis confirmed the accuracy of bioluminescent imaging in quantifying metastatic surface area. This protocol should ensure reliable and reproducible measurements in cancer research utilizing luciferase-positive cell lines, confirming the validity and accuracy of preclinical studies in immunocompetent models.

Bioluminescence measurement of superoxide anion in infertile men with oxidative stress

Infertility is such an important issue in society today. In some cases of male infertility, the main cause is oxidative stress and the presence of reactive oxygen species in the environment or in sperm cells. All current techniques that measure oxidative stress, including the nitroblue tetrazolium Test, DNA Fragmentation Index, Malondialdehyde, and Endz Test are qualitative and semi-quantitative. These methods do not have good sensitivity and specificity. Semen samples from 50 infertile patients and 10 normal individuals were collected. The samples were examined for laboratory routine tests according to the WHO 2010 protocol. Oxidative stress tests, including DFI, NBT, and MDA, were performed for these two groups. Bioluminescence inhibition assay was performed for detection of O2.- in semen samples by aequorin. The normal individuals showed significantly better semen parameters than the patient's group. Significantly lower O2.- levels were seen in the patient's group compared to normal individuals. The cut-off value of O2.- levels in normal individuals was determined to be 8 × 105 RLU/s with a sensitivity of 100% and a specificity of 100%. Infertile patients, despite having reduced quality of semen parameters, have high O2.- levels, and this causes the intensity of bioluminescence to be quenched in these people.

THE RESEARCH OF THE PLANT-DERIVED MOLECULES REGULATORY EFFECTS REGARDING THE BACTERIAL BIOSENSORS WITH THE DIFFERENT “QUORUM SENSING” SYSTEMS OF THE LUXI/LUXR TYPE

Relevance. «Quorum sensing» (QS) plays an important role in the pathogenic potential realization of many malignant bacteria, and therefore is a promising direction for the in-depth study. It could provide the basis for the antibacterial agents creation of new operating principles. The use of plant extracts and small molecules identified in them is the one option to inhibit «quorum sensing» system, but this approach has certain limitations. In addition, the use of a limited number of bacterial biosensors leaves questions related to extrapolate data on the whole system and needs more research. Aim. Comparison of the QS-inhibitory activity of plant-derived molecules against the «quorum sensing» LuxI/LuxR-type system of the violacein-producing strain Chromobacterium subtsugae 026, as well as a panel of bacterial lux-biosensors. Material and methods. The work used a library of chemically synthesized analogues of plant-derived molecules, represented by quercetin, cinnamic aldehyde, 7-hydroxycoumarin, 4-hexylresorcinol and salicylic acid. The influence on the growing and the QS-inhibitory activity of the plant-derived molecules were evaluated using the serial dilution method. The QS-inhibitory effect of the studied compounds was determined according to the optical density measurement of the Chromobacterium subtsugae 026 strain extracted pigment and the measurement of bacterial lux-biosensors bioluminescence. Results. A panel of seven bacterial biosensors was used to screen the biological activity of quercetin, cinnamic aldehyde, 7-hyd-roxycoumarin, 4-hexylresorcinol and salicylic acid against four «quorum sensing» LuxI/LuxR-type systems. The unequal nature of the action of the studied compounds on the tested «quorum sensing» systems was demonstrated. It was found that the 4-hexylresorcinol QS-inhibitory action was most pronounced regarding Chromobacterium subtsugae 026, while the trans-cinnamaldehyde was more active regarding bacterial lux-biosensors. Conclusions. The presented work demonstrates the unequal nature of the action of the plant-derived molecules under study in relation to four differ-ent QS LuxI/LuxR-type systems. It has been suggested that this circumstance may be associated with a different mechanism of action of the studied substances on the bacterial cell. The results are of practical interest and require further study.

Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power.

Bioluminescence emitted from a luciferase-catalyzed oxidation of luciferin has been broadly utilized to report on biological events, predominantly through relative changes in the light output. Recent advances in protein engineering and synthetic chemistry have yielded bioluminescent systems with markedly improved brightness and bioavailability. These developments have enabled not only the detection of biological events at far lower expression levels but also new opportunities utilizing bioluminescence to power photochemistry in cells. Regardless of the application, bioluminescence analyses have leaned heavily on the use of luminometers to measure the light output of a system. Current luminometers report the light output of a sample in relative units, limiting the ability to compare data between instruments and preventing the absolute power of a bioluminescent system from being quantified. Luminescent solution calibrants comprising luciferases and their cognate luciferins that have been characterized for absolute light output would enable calibration of any given luminometer for absolute photon counting. To this end, we have built a custom light detection apparatus and used it alongside wavelength-matched LED light sources emitting at 450 and 561 nm to characterize the absolute power of a series of NanoLuc and firefly luciferase solutions, respectively. This approach revealed that these two common luciferases produce 3.72 × 10-18 and 7.25 × 10-20 watts/molecule, respectively. Components of these luminescent solution calibrants are commercially available and produce stable bioluminescent signals over 2-5 min, enabling any luminometer to be calibrated for power measurements of bioluminescence emitted by these two luciferases in units of watts or photons per second.

SAT297 Major Immunophenotypic Abnormalities In Patients With Primary Adrenal Insufficiency Of Different Etiology

Abstract Disclosure: H.F. Nowotny: None. T. Marchant Seiter: None. J. Ju: None. A. Gottschlich: None. H. Schneider: None. S. Zopp: None. F. Vogel: None. L. Tschaidse: None. M.K. Auer: None. C. Lottspeich: None. S. Kobold: None. S. Rothenfusser: None. F. Beuschlein: None. M. Reincke: None. L. Braun: None. N. Reisch: None. Background: Increased risk of infection, adrenal crises and a higher mortality rate have been reported for patients with primary adrenal insufficiency (PAI). Such dismal outcomes have been inferred to immune cell dysregulation as a consequence of unphysiological cortisol replacement. As the immune landscape of patients with different types of PAI has not been systematically explored, we set out to immunophenotype PAI patients with different causes of glucocorticoid deficiency. Methods: The present cross-sectional single center study includes 28 patients with congenital adrenal hyperplasia (CAH), 27 patients after bilateral adrenalectomy due to Cushing’s syndrome (BADx), 21 patients with Addison’s disease (AD) and 52 healthy controls. All patients with PAI were on a stable glucocorticoid replacement regimen using a median dose of 25 mg hydrocortisone (IQR 5 mg for BADx and AD and IQR 10 mg for CAH) per day. Heparinized blood samples were processed to isolate peripheral blood mononuclear cells. All samples were processed for analysis of immune cell subsets using multicolor flow cytometry after four-hour stimulation with phorbol myristate acetate (PMA)/Ionomycin, as well as analysis of natural killer (NK-) cell cytotoxicity and clock gene expression. Results: Analysis of immune cell profiles revealed reduced percentages of IFNγ-secreting T helper (Th1) in AD (p = 0.0024) and cytotoxic (Tc1) cells in CAH (p = 0.0055) and AD patients (p = 0.0075) compared to controls. Moreover, we observed a downregulation of the percentage of IL-4+ Th2 cells in AD patients (p = 0.0157) and Tc2 cells in AD (p = 0.0154) and CAH patients (p = 0.0012) compared to controls. Th17 and Tc17 cells were also downregulated in patients with AD compared to the other patient groups and controls (p < 0.0001). NK-cell cytotoxicity (NKCC) using bioluminescence measurements was reduced in all subsets of PAI patients compared to controls with the lowest change in patients with CAH (mean specific lysis 57.5% in controls, 21.7% in CAH, -0.5% in AD and -28.7% in BADx). The percentage of activated NK-cells was upregulated in BADx (p = 0.0008) and AD patients (p = 0.0348) compared to controls, also expressed as an increase in CD107a expression as a marker of NK-cell degranulation (BADx p < 0.0001; AD p = 0.0002). While the percentage of NK-cell activating receptor expressing NK-cells did not differ from controls, expression percentage of NK-cell inhibiting receptor CD94 was upregulated in both BADx and AD patients (p < 0.0001). Conclusion: In patients with three different etiologies of PAI, distinct differences in T- and NK-cell-phenotypes became apparent despite the use of same GC preparation and dose. Our results highlight so far unsuspected differences in immune cell composition and NK-cell function in PAI patients of different causes and suggest disease-specific alterations that might necessitate disease-specific treatment. Presentation: Saturday, June 17, 2023

The effectiveness of disinfection protocols in osteopathic family medicine offices.

Healthcare-associated infections (HAIs) pose a substantial public health threat. Despite significant strides tocurb HAIs in hospital environments, outpatient settings have not received the same degree of attention. Given their emphasis on holistic, patient-centered care, osteopathic family medicine offices are pivotal in both disease prevention and comprehensive patient treatment. The importance of simple yet effective disinfection protocols, such as thorough cleaning between patient appointments, cannot be overstated in these settings because they are integral to minimizing disease transmission. This study aims to assess the effectiveness of the current disinfection protocols in osteopathic family medicine offices. A cross-sectional study evaluating disinfection practices on 18 examination tables in an osteopathic family medicine office was conducted. Two high-touch surfaces (midtorso region and table edge) were examined. Initial swab samples were collected after morning disinfection by Environmental Services, and terminal swab samples were gathered after day's-end disinfection by the medical staff. Adenosine triphosphate (ATP) bioluminescence assays were performed utilizing AccuPoint Advanced HC Reader, which quantified ATP, indicating contamination levels in the samples. The higher the ATP levels found in a sample, the greater the amount of biological contamination. All samplers were handled and tested as per manufacturer's instructions. Apreliminary trial was conducted to confirm the internal validity of ATP bioluminescence measurements. The statistical analysis involved Shapiro-Wilk and Wilcoxon signed-rank tests, with significance set at p<0.05. Cohen's d test was utilized to calculate the effect size, identifying meaningful differences in initial and terminal swab sample relative light units (RLUs). The midtorso region demonstrated an 11.1 % increase in failure rate after terminal disinfection when compared to initial disinfection. A Wilcoxon signed-rank testrevealed a median estimated pathogen level forthemidtorso region that was higher after terminal disinfection (median, 193 RLUs; range, 1-690 RLUs; n=18) compared to initial disinfection (median, 134 RLUs; range,4-946 RLUs; n=18). However, this increase was not statistically significant, p=0.9124, with a small effect size, d=0.04. The edge showed no change in failure rate after terminal disinfection, maintaining a 100 % failure rate both before and after disinfection. However, the Wilcoxon signed-rank test revealed a slight reduction in the median estimated pathogen levels after terminal disinfection (median, 2095 RLUs; range, 891-5,540 RLUs; n=18) compared to before disinfection (median, 2,257 RLUs; range, 932-5,825 RLUs; n=18). However, this reduction wasnot statistically significant, p=0.61, with a small effect size, d=0.12. The findings from this study reveal a substantial disparity in outcomes between the two sample locations, midtorso and edge. The midtorso demonstrated arelatively low failure rate in both initial and terminal swab samples, indicating successful outcomes. In contrast, the edge consistently displayed a 100 % failure rate, emphasizing the need for more care and attention when cleaning the edge of the examination to ensure better outcomes. By prioritizing adequate disinfection protocols, including thorough cleaning between patients, osteopathic family medicine offices can more effectively prevent disease transmission and promote patient safety.

A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms.

Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that Pseudomonas aeruginosa and Burkholderia cenocepacia strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.

Analytical and bioluminescence-based non-invasive quality assessment of differentially grown strawberry (Fragaria x ananassa Duch. ‘Asia’) during household refrigeration storage

Strawberry is a typical spring fruit, for which consumer demand is particularly high; however, information is scarce on the comparison of quality traits of strawberries of local and imported origin during household refrigeration storage. That is why we sought to answer the question of to what extent the quality of strawberries changes during 5 days of household refrigeration storage. The choice of methods was focused on fast and at the same time informative analytical methods: pH, total antioxidant capacity, and lipid oxidation. Furthermore, a non-invasive imaging technique: ultra-weak bioluminescence measurement was carried out along with the determination of fruit morphological (weight, height, width, and color) and analytical (antioxidant capacity and lipid oxidation) traits. The data indicate that the longer strawberries are cultivated in the same plantation, the smaller their fruit weight and the lower their fruit quality become.In addition, the results cast light on a controversial fact: despite the fact that the sample from the store had the finest appearance, as determined, its antioxidant capacity was the lowest for the duration of home refrigeration storage, indicating the lowest nutritional value.This was validated by the lipid oxidation levels, which were defined by the amount of malondialdehyde as well as by the rate of ultra-weak bioluminescence. The research underlines the exceptional value of local fruits over importand provides valuable information to customers, encouraging them to purchase strawberries from local farms in order to not only support the local economy but also to adopt a more health-conscious attitude. In addition, ultra-weak bioluminescence testing offers a non-invasive method for assessing fruit quality.

Impairment of endothelial Pannexin1-TRPV4 signaling and flow-induced vasodilation in obesity-induced pulmonary hypertension

Obesity is a highly prevalent and debilitating disease that significantly contributes to the development of other pathologies, such as pulmonary hypertension (PH). However, the mechanisms triggering obesity-induced PH (OiPH) are currently unknown. Endothelial cell (EC) Ca 2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca 2+ signaling mechanisms is a crucial contributor to elevated PAP in PH. Although PAs are a “high-flow” vascular bed, flow-induced endothelial Ca 2+ signaling mechanisms have not been investigated in OiPH. In this regard, we recently showed that Ca 2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4 EC ) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress (F/SS) activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1 EC ) in PAs. Panx1 EC -effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2R EC ) to increase TRPV4 EC channel activity (Daneva et al., eLife, 2021). Therefore, I hypothesized that impaired Panx1 EC -P2Y2R EC -TRPV4 EC signaling axis reduces flow-induced dilation of PAs and elevates PAP in OiPH. Right ventricular systolic pressure, an indicator of PAP, was significantly higher in mice exposed to a high-fat diet (HFD, 60%, 24 weeks) compared to normal chow-fed mice, confirming OiPH in these mice. F/SS (4–15 dynes/cm 2 )-induced dilation was drastically reduced in small PAs (~ 50 μm), cannulated at both ends and pressurized to 15 mm Hg, from HFD mice, possibly indicating impaired flow-induced endothelial signaling in OiPH. Confocal Ca 2+ imaging studies in fluo-4-loaded PAs showed that the baseline and agonist (GSK1016790A)-induced activity of TRPV4 EC sparklets (unitary Ca 2+ influx signals through TRPV4 EC channels) is reduced in HFD mice compared to normal mice, confirming a decrease in TRPV4 EC channel activity in OiPH. F/SS (4–15 dynes/cm 2 ) resulted in increased activity of TRPV4 EC sparklets in PAs from normal mice, but not in PAs from HFD mice, suggesting that flow-induced TRPV4 EC activity is reduced in OiPH. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from HFD mice than normal mice. Importantly, F/SS (4 – 15 dynes/cm 2 ) increased ATP efflux in PAs from normal mice, but not in PAs from HFD mice, indicating that flow-Panx1 EC signaling is also impaired in OiPH. Together, these data support the idea that flow-induced Panx1 EC -TRPV4 EC signaling is impaired in OiPH. Targeting the individual elements of this pathway may provide novel therapies in OiPH. Future studies will assess the signaling linkages of the flow-Panx1 EC -P2Y2R EC -TRPV4 EC signaling axis in OiPH. R01 HL142808, R01 HL146914, R01 HL157407 to Dr. S.K. Sonkusare; APS Postdoctoral Fellowship Award to Dr. Z. Daneva This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1789: Pre-clinical studies to advance anti-ROR1 CAR-T cell therapy for metastatic prostate cancer

Abstract Background: Alterations in WNT signaling are frequently associated with tumorigenesis and metastasis in many cancers including prostate cancer (PCa). The Wnt ligand, WNT5A, is required for normal prostate gland development and is increased in bone metastatic, castration resistant PCa (CRPC) patients. WNT5A signaling is mediated in part through ROR1, a non-canonical Wnt receptor and fetal oncoprotein for which the therapeutic inhibitory antibody, Zilovertamab, has been developed. Its safety has been clinically proven in trials for chronic lymphocytic leukemia (CLL) and metastatic breast cancer. We sought to investigate Zilovertamab-based anti-ROR1 therapies for metastatic prostate cancer.Our Hypothesis is that WNT5A may activate a stem-cell-like program via ROR1 which leads to therapy resistance in metastatic prostate cancer. The anti-ROR1 biologic, Zilovertamab, may inhibit this mechanism of resistance. The expression of ROR1 on CRPC and NEPC tumors and its lack of expression on normal adult tissues makes it a promising CART cell target. Methods: We used the patient-derived xenograft (PDX), PCSD13, small cell bone metastatic prostate cancer model and the neuroendocrine PCa cell lines, PC3 and DU145, to test the effect of Zilovertamab and Zilovertamab-CART cells in vitro using the real time cell viability, proliferation, and cell cycle tracking assays in an Incucyte S3. We used in vivo bioluminescence and tumor caliper measurements to monitor effects in vivo. Results: Studies using RNASeq, qRT-PCR, FACS and Westerns showed high expression of ROR1 in PC3, DU145, and in PCSD13. CRISPR-Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased inhibition of proliferation at lower docetaxel concentrations. Treatment of PCSD13 PDX in vivo with Zilovertamab increased docetaxel-mediated tumor growth inhibition. Mice bearing PC3 xenografts injected intravenously with Zilovertamab anti-ROR1 CAR-T cells showed durable, tumor ablation in 67% of mice compared to 22% of mice injected with activated T cells from the same donor and 0% of untreated mice. Survival of mice at Day 70 injected with CART was 78% compared to 0% of mice injected with control donor T cells and 0% of untreated mice. Zilovertamab antibody could inhibit Zilovertamab CART cell tumor cell killing. Conclusions: ROR1 was expressed at high levels on castration resistant small cell PCa and neuroendocrine PCa cell lines and PDX models. Zilovertamab synergized with docetaxel to inhibit tumor growth in patient derived xenograft in vivo and in organoid cultures. Zilovertamab-based CART cells durably eradicated ROR1+ prostate cancer xenograft tumors. These studies supported the recently launched Phase 1b clinical trial of Zilovertamab plus docetaxel in metastatic CRPC patients. Clinical development of GMP Zilovertamab CART cells for a clinical trial in CLL is in progress and may lead to rapid progression to a clinical trial for metastatic CRPC and NEPC. Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle Muldong, Evodie Koutouan, JOngwook Kim, Niloofar Etemadfard, Hae Soo Choo, Navyaa Sinha, Sanghee Lee, Christina Wu, Gabriel Pineda, Kathleen Lennon, Karl Willert, Catriona H. Jamieson, Terry Gaasterland, Rana Mckay, Christopher J. Kane, Thomas J. Kipps, Anna A. Kulidjian, Nicholas A. Cacalano, Charles Prussak. Pre-clinical studies to advance anti-ROR1 CAR-T cell therapy for metastatic prostate cancer [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 1789.

Abstract 33: Humanized MOLM-13 AML model as a versatile tool to study immune-activating agents

Abstract Acute myeloid leukemia (AML) is the most common acute leukemia and second most common form of blood cancer in children and adolescents. Despite progress in understanding the biology of AML, therapeutic intervention plans have not changed nor outcomes significantly improved over the last years. Current AML treatment consists of intensive chemotherapy followed by stem cell transplantation. While initially many patients respond well to chemotherapy, relapse rates are high and still associated with poor prognosis. Evidence suggests that chemo-resistant leukemic stem cells (LSCs) have the ability to reinitiate and sustain the disease. Therefore, novel therapies are urgently needed to improve AML treatment and well-characterized in vivo models modelling this disease are a prerequisite for improving the current state. At Reaction Biology, we have used NOG and NOD-SCID mice to establish a humanized MOLM-13 AML model concomitantly engrafted with human T cells. Luciferase positive MOLM-13_Luc AML cells were injected both in naïve mice and mice pre-treated with Cyclophosphamide. Thereafter, tumor growth was monitored by bioluminescence measurements to track disseminated tumor burden and isolated T cells were applied upon tumor growth detection (generally observed on day 8 or 15 post-tumor cell inoculation for NOG or NOD-SCID mice, respectively). For each of the inoculations, fresh T cells were obtained by negative selection from buffy coat derived PBMCs with a purity of &amp;gt;95 %. Following administration of human T cells, animals were monitored daily, including the collection of animal weights (3x/w), and tumor burden (2x/w) via in vivo bioluminescence imaging technique. MOLM-13_Luc tumor cells grew rapidly in all NOG mice with termination of the last animals as early as day 17. Neither Cyclophosphamide pre-treatment, nor the addition of T cells influenced tumor growth or prolonged survival. In Cyclophosphamide pre-treated NOD-SCID mice, onset of tumor growth was observed to initiate homogenously at day 13 post inoculation (with applied T cells being ineffective in reducing tumor burden), whereas tumor growth in non-pre-treated animals was heterogenous and delayed by more than 7d. The presence of T cells affected tumor growth even further. Beside T cell addition, also the application of NK cell therapies were successfully investigated (data not shown). Taken together, the MOLM-13_Luc AML tumor model is found to be suitable for the investigation of bi-specific engagers using isolated T or NK cells. Citation Format: Carla Castro, Philipp Metzger, Cynthia Obodozie, Holger Weber. Humanized MOLM-13 AML model as a versatile tool to study immune-activating agents [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 33.

PD04-10 PRE-CLINICAL STUDIES TO ADVANCE ZILOVERTAMAB-BASED ANTI-ROR1 DUAL SPECIFICITY CAR T-CELL THERAPY FOR METASTATIC PROSTATE CANCER.

PD04-10 PRE-CLINICAL STUDIES TO ADVANCE ZILOVERTAMAB-BASED ANTI-ROR1 DUAL SPECIFICITY CAR T-CELL THERAPY FOR METASTATIC PROSTATE CANCER.

A Bioluminescent Protein-Graphene Oxide Donor-Quencher Pair in DNA Hybridization Assays.

Despite fluorescent quenching with graphene oxide (GO) having shown great success in various applications - bioluminescent quenching has not yet been demonstrated using GO as a quencher. To explore the ability of GO to quench bioluminescence, we used Gaussia luciferase (Gluc) as a donor and GO as a quencher and demonstrated its application in sensing of two target analytes, HIV-1 DNA and IFN-γ. We demonstrated that the incubation of Gluc conjugated HIV-1 and IFN-γ oligonucleotide probes with GO provided for monitoring of probe-target interactions based on bioluminescence measurement in a solution phase sensing system. The limits of detection obtained for IFN-γ and HIV-1 DNA detection were 17 nM and 7.59 nM, respectively. Both sensing systems showed selectivity toward the target analyte. The detection of IFN-γ in saliva matrix was demonstrated. The use of GO as a quencher provides for high sensitivity while maintaining the selectivity of designed probes to their respective targets. The use of GO as a quencher provides for an easy assay design and low cost, environmentally friendly reporter.

Relationship between ATP Bioluminescence Measurements and Microbial Assessments in Studies Conducted in Food Establishments: A Systematic Literature Review and Meta-Analysis

Adherence to proper environmental cleaning practices is critical in food establishments. To validate cleanliness, cleaning practices should be routinely monitored, preferably by a rapid, reliable, and cost-effective method. The aim of this study was to determine whether a correlation exists between ATP bioluminescence measurements and selected microbial assessments in studies conducted in food establishments. A systematic literature review and meta-analysis was conducted using the principles of preferred reporting items for systematic reviews and meta-analyses. Twelve online databases and search engines were selected for the review. Peer-reviewed articles published in English between January 2000 and July 2020 were included in the search. From a total of 19 eligible studies, 3 that included Pearson correlation coefficients (r) between ATP bioluminescence measurements and microbial assessments were used for the meta-analysis calculations. Only the fixed-effect model produced a strong correlation because one value dominated the estimates: r = 0.9339 (0.9278, 0.9399). In contrast, both the random effects model, 0.2978 (0.24, 0.3471), and the mixed effects model, r = 0.3162 (-0.0387, 0.6711), indicated a weak relationship between ATP bioluminescence and microbial assessments, with no evidence of a strong correlation. The meta-analysis results indicated no sufficient evidence of a strong correlation between ATP bioluminescence measurements and microbial assessments when applied within food establishments. This lack of evidence for a strong correlation between the results of these two monitoring tools suggests that food establishments cannot depend on only one method. Yet, with immediate feedback and quantification of organic soiling, ATP bioluminescence could be an effective monitoring tool to use in food establishments.

Investigating the Relationship between Primary Bone Marrow Adipocytes and Myeloma Cells with Three Cell Culture Systems

Introduction & Aims of the Study: The unique properties of the bone marrow allow for migration and proliferation of multiple myeloma (MM) cells, while also providing the ideal environment for development of quiescent, drug-resistant MM cell clones. Recent studies suggest that bone marrow adipocytes (BMAds) can contribute to myeloma-induced bone disease, and reciprocally, that BMAds support MM cells in vitro. Importantly, most data investigating BMAds have been generated using adipocytes differentiated from bone marrow-derived mesenchymal stromal cells (MSCs) in vitro due to the technical challenges associated with isolating and culturing primary adipocytes. To that end, we investigated the MM-BMAd relationship ex vivo utilizing primary adipocytes (pBMAds), which overcome many of the drawbacks associated with using MSC-derived adipocytes. Methods: pBMAds were isolated from the top of human bone marrow biopsies after centrifugation and seeded onto one of three in vitro systems. To develop an in vitro model with a tissue-like structure to mimic the bone marrow microenvironment, we developed the first 3D, tissue engineered model utilizing pBMAds derived from human bone marrow. Following adherence of pBMAds, MM cells were added to investigate bidirectional tumor-host signaling. Viable pBMAds were measured utilizing CellTiterGlo and RealTime Glo (bioluminescence), alone and in culture with MM cells (MM.1Sgfp+luc+ and RPMI-8226). MM.1Sgfp+luc+ cell number was determined by luciferin spike-in and measurement of bioluminescence. To investigate the effects of myeloma cells on pBMAds, we used fluorescence and confocal microscopy to image cell morphology, and mass spectrometry proteomics of conditioned media samples to specifically interrogate soluble signals. Gene expression in both cell types was investigated by qPCR. Results: Utilizing adipocytes isolated from 12 different human donors, we found that pBMAds, which are extremely fragile, can be isolated and stably cultured in 2D for 10 days and in 3D for short-terms (~2 weeks) or long-terms (1 month) in vitro (Figure 1A). We monitored stable pBMAd presence in 2D transwells via Cell TiterGlo and on 3D scaffolds via RealTime Glo up to 9 days post-seeding, and by calcein staining after 10 days in culture. Using bioluminescence in pBMAs (Cell TiterGlo) and MM.1S (luciferin spike-in) we were able to detect viability of both cell types in co-culture. Utilizing mass spectrometry proteomics, in pBMAds alone, we detected secretion of CXCL12 (SDF1; p<0.01), as well as TGFB1 (p<0.01) which has implications for bone remodeling. Interestingly, in conditioned media collected from the co-culture condition, we found increased secretion of the WNT inhibitor DKK3 (p<0.05) and detection of proteins known to be associated with extracellular exosomes including: CNTN1, ACP1, OMD, RPS18, RPL24, PGAM1, LAMA4, CTSB, PLOD2 and ARHGDIA (Figure 1B), suggesting that pBMAds and MM cells likely interact via exosomes. Conclusions: In sum, we developed three in vitro cell culture systems to study primary bone marrow adipocytes and myeloma cells, which could be adapted to investigate many diseases and biological processes involving the bone marrow, including other bone-homing tumor types. Our use of these models to examine the interaction between MM cells and pBMAds has revealed new roles for pBMAds in the MM bone marrow microenvironment, with implications for MM-induced bone disease and pBMAd-MM bidirectional signaling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A flexible electronic strain sensor for the real-time monitoring of tumor regression.

Assessing the efficacy of cancer therapeutics in mouse models is a critical step in treatment development. However, low-resolution measurement tools and small sample sizes make determining drug efficacy in vivo a difficult and time-intensive task. Here, we present a commercially scalable wearable electronic strain sensor that automates the in vivo testing of cancer therapeutics by continuously monitoring the micrometer-scale progression or regression of subcutaneously implanted tumors at the minute time scale. In two in vivo cancer mouse models, our sensor discerned differences in tumor volume dynamics between drug- and vehicle-treated tumors within 5 hours following therapy initiation. These short-term regression measurements were validated through histology, and caliper and bioluminescence measurements taken over weeklong treatment periods demonstrated the correlation with longer-term treatment response. We anticipate that real-time tumor regression datasets could help expedite and automate the process of screening cancer therapies in vivo.

Comparative Analysis of Cell Metabolic Activity Sensing by Escherichia coli rrnB P1-lux and Cd Responsive-Lux Biosensors: Time-Resolved Experiments and Mechanistic Modelling

Whole-cell bacterial sensors are used in medical/environmental applications to detect chemicals, and to assess medium toxicity or stress. Non-specific constitutive biosensors generally serve the latter purpose, whereas chemical detection is performed with biosensors involving a specific chemical-inducible promoter. Herein, we show that functioning principles of specific and non-specific whole-cell biosensors are not exclusive as both can probe modulations of cell metabolic activity under stressing conditions. The demonstration is based on (i) time-resolved measurements of bioluminescence produced by constitutive rrnB P1-luxCDABE Escherichia coli biosensor in media differing with respect to carbon source, (ii) theoretical reconstruction of the measured signals using a here-reported theory for bioluminescence generated by constitutive cells, (iii) comparison between time-dependent cell photoactivity (reflecting metabolic activity) retrieved by theory with that we reported recently for cadmium-inducible PzntA-luxCDABE E. coli in media of similar compositions. Whereas signals of constitutive and non-constitutive biosensors differ in terms of shape, amplitude and peak number depending on nutritional medium conditions, analysis highlights the features shared by their respective cell photoactivity patterns mediated by the interplay between stringent response and catabolite repressions. The work advocates for the benefits of a theoretical interpretation for the time-dependent response of biosensors to unravel metabolic and physicochemical contributions to the bioluminescence signal.

Effect of AR42 in Primary Vestibular Schwannoma Cells and a Xenograft Model of Vestibular Schwannoma.

AR42, a histone deacetylase (HDAC) inhibitor, reduces viability of primary vestibular schwannoma (VS) cells and delays tumor progression and hearing loss (HL) in a xenograft model of VS. The impact of HDAC expression on AR42 response in primary VS cells is unknown, as well as the effects of AR42 on VS-associated HL and imbalance. Primary human VS cells (n = 7) were treated with AR42 (0-3.0 μM), and viability assays were conducted. Immunohistochemistry and western blotting for phosphorylated-HDAC2 (pHDAC2) were performed on tumor chunks. Pharmacokinetic studies were conducted in Fischer rats using mass spectrometry. Merlin-deficient Schwann cells were grafted onto cochleovestibular nerves of immunodeficient rats and treated with vehicle (n=7) or AR42 (25 mg/kg/day for 4weeks; n=12). Tumor bioluminescence imaging, auditory brainstem response (ABR), and rotarod tests were conducted to 6weeks. Final tumor weight and toxicities were measured. AR42 caused dose-dependent reductions in viability of VS cells. Tumors with higher pHDAC2:HDAC2 ratios had greater reductions in viability with AR42. On pharmacokinetic studies, AR42 reached peak levels in nerve ~24 hours after oral administration. Although AR42-treated rats demonstrated mean ABR threshold shifts ~10 to 20 dB lower than controls, this did not persist nor reach significance. When compared to controls, AR42 did not affect tumor bioluminescence, tumor weight, and rotarod measurements. Response of primary VS cells to AR42 may be influenced by pHDAC2 expression in tumor. Although AR42 may delay HL in our xenograft model, it did not halt tumor growth or vestibular dysfunction. Further investigations are warranted to evaluate the AR42 effectiveness in NF2-associated VS.

A next generation inducible armored CAR to overcome the immunosuppressive tumor microenvironment and enhances cytotoxicity of CAR-T and TILs.

e14517 Background: The full potential of CAR-T cells in clinic is limited by the immunosuppressive tumour microenvironment (TME), which induces expression of exhaustion markers and limits the activity of tumour infiltrating lymphocytes (TILs) including CAR-T cells. We developed a next generation inducible Armored CAR platform (aCAR) that releases an anti-PD-1 checkpoint inhibitor upon CAR-T cell activation, limiting payload release exclusively to the TME, thereby reducing the risk of systemic exposure. This differentiated strategy facilitates activation of CAR-T cells and TILs within the TME and has the potential for lower toxicity. Methods: A ROR1-targeting second-generation CAR containing a 41BB-CD3ζ intracellular domain was cloned into a lentivirus transfer vector alongside a prototypic anti-PD1 antibody. The CAR was under the control of a constitutively active promoter, and the PD1 targeting payload was controlled by an inducible promoter that was activated upon engagement of CAR with ROR1 on tumour cells. CAR-T cells were subject to in vitro co-culture assays with target ROR1+ tumour cells and their cytotoxic responses evaluated by flow cytometry. Levels and binding functionality of released payload were analyzed by ELISA and flow cytometry. In vivo xenograft models were performed in NSG mice with tumor growth assessed by bioluminescent imaging (BLI) and caliper measurement of the tumour volume. Results: Non-Armored CAR cells displayed potent and specific cytotoxic responses directed towards ROR1+ TNBC and NSCLC cells lines at different effector to target (E:T) ratios. The results were equivalent or superior to CARs generated with comparator anti-ROR1 antibodies, which may be due to the ROR1 CAR targeting a membrane proximal epitope within the ROR1 frizzled domain. The aCAR cells released measurable levels of anti-PD1 payload within 5 hours of binding to ROR1 on tumors and enhanced the cytotoxic effects at challenging 1:10 E:T ratios. Established PDL1+ TNBC xenograft models using the aCAR cells and comparing with non-Armored CAR cells displayed a qualitative abrogation in tumor growth by BLI, which was confirmed and shown to be significant by caliper measurement of the tumor volume. Continuing the experiment out to 3 months showed a significant survival advantage for the animals receiving aCAR. All other cohorts were terminated by day 70, however 20% of the aCAR cohort survived at day 95. Conclusions: Our next generation inducible aCAR platform enables the release of an immune stimulating payload only in the presence of target tumor cells, enhancing the therapeutic activity of the CAR-T cells and limiting payload exposure to the site of action. This technology provided a significant survival advantage in challenging in vivo xenograft models. This coupled with its potential safety attributes merits further clinical evaluation of this approach.