High-throughput Testing Research Articles

High-throughput sgRNA testing reveals rules for Cas9 specificity and DNA repair in tomato cells.

CRISPR/Cas9 technology has the potential to significantly enhance plant breeding. To determine the specificity and the mutagenic spectrum of SpCas9 in tomato, we designed 89g(uide) RNAs targeting genes of the tomato MYB transcription factor family with varying predicted specificities. Plasmids encoding sgRNAs and Cas9 were introduced into tomato protoplasts, and target sites as well as 224 predicted off-target sites were screened for the occurrence of mutations using amplicon sequencing. Algorithms for the prediction of efficacy of the sgRNAs had little predictive power in this system. The analysis of mutations suggested predictable identity of single base insertions. Off-target mutations were found for 13 out of 89 sgRNAs and only occurred at positions with one or two mismatches (at 14 and 3 sites, respectively). We found that PAM-proximal mismatches do not preclude low frequency off-target mutations. Off-target mutations were not found at all 138 positions that had three or four mismatches. We compared off-target mutation frequencies obtained with plasmid encoding sgRNAs and Cas9 with those induced by ribonucleoprotein (RNP) transfections. The use of RNPs led to a significant decrease in relative off-target frequencies at 6 out of 17, no significant difference at 9, and an increase at 2 sites. Additionally, we show that off-target sequences with insertions or deletions relative to the sgRNA may be mutated, and should be considered during sgRNA design. Altogether, our data help sgRNA design by providing insight into the Cas9-induced double-strand break repair outcomes and the occurrence of off-target mutations.

Patient-derived MicroOrganoSpheres (MOS) and precision clinical decision-making for patients with multiple myeloma.

8047 Background: There are many multiple myeloma (MM) treatments and nearly all patients undergo continual cycles of treatment, response, and resistance. Selecting an effective therapeutic strategy is of critical importance; but there is currently a lack of patient-derived MM models that can enable functional precision medicine to help real-time clinical decision-making to guide individual patient treatment. Methods: We show a method to rapidly establish MM patient-derived MicroOrganoSheres (MOS), which are microscale, droplet-sized patient avatars that sustain the native tumor niche, including both stromal and immune compartments. We generated first-of-its-kind data showing that MM MOS predicted patient treatment outcomes and have the potential to guide clinical treatment decisions. We further developed high-throughput drug screen to demonstrate MM MOS response to FDA-approved single and combination therapies and developed MOS assays to predict patient responses to immunotherapies including bispecific and phagocytosis-inducing antibodies. Results: MM MOS established from patient bone marrow (BM) biopsies preserved CD138/CD38 tumor cells, stromal (e.g., osteoblast) cells, and all major immune cell populations (T, NK, B, macrophage, dendritic, and myeloid-derived suppressor cells) for 11 days in culture. We performed single and combo drug testing on MOS derived from 7 MM patients within 10 days of biopsy collection. Patients went on receiving treatments and their responses were assessed using International Myeloma Working Group response criteria. The MOS assay predicted responses of both treatment-naïve and refractory patient to standard-of-care regimens, including combinations of proteasome inhibitors (bortezomib, carfilzomib), immunomodulatory agents (lenalidomide) and DCEP. Moreover, the BM biopsy-derived MOS enabled high-throughput drug testing of single agents and combinations in 9-dose titrations, providing clinicians the ability to evaluate alternative treatment options. Furthermore, because MOS retain the immune compartments of the original tumor niche, we were able to evaluate patients’ response to bispecific T cell engagers and developed a phagocytosis assay to measure anti-tumor phagocytosis activity, which showed efficacy in both single-agent and combo settings. Multi-modal profiling and an AI image analysis pipeline provided robust measurements that confirmed the unique phenotypic profile of each patient sample, highlighting the critical unmet need to guide each patient to the optimal treatment. Conclusions: Based on this groundbreaking feasibility study, a subsequent clinical trial of 40 patients is current under IRB review to further validate the clinical power of MM MOS to predict patient outcome and to demonstrate utility as a viable functional precision medicine approach to inform treatment decisions in the clinic.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

3D Organoids for Regenerative Endodontics.

Apical periodontitis is the inflammation and destruction of periradicular tissues, mediated by microbial factors originating from the infected pulp space. This bacteria-mediated inflammatory disease is known to interfere with root development in immature permanent teeth. Current research on interventions in immature teeth has been dedicated to facilitating the continuation of root development as well as regenerating the dentin-pulp complex, but the fundamental knowledge on the cellular interactions and the role of periapical mediators in apical periodontitis in immature roots that govern the disease process and post-treatment healing is limited. The limitations in 2D monolayer cell culture have a substantial role in the existing limitations of understanding cell-to-cell interactions in the pulpal and periapical tissues. Three-dimensional (3D) tissue constructs with two or more different cell populations are a better physiological representation of in vivo environment. These systems allow the high-throughput testing of multi-cell interactions and can be applied to study the interactions between stem cells and immune cells, including the role of mediators/cytokines in simulated environments. Well-designed 3D models are critical for understanding cellular functions and interactions in disease and healing processes for future therapeutic optimization in regenerative endodontics. This narrative review covers the fundamentals of (1) the disease process of apical periodontitis; (2) the influence and challenges of regeneration in immature roots; (3) the introduction of and crosstalk between mesenchymal stem cells and macrophages; (4) 3D cell culture techniques and their applications for studying cellular interactions in the pulpal and periapical tissues; (5) current investigations on cellular interactions in regenerative endodontics; and, lastly, (6) the dental-pulp organoid developed for regenerative endodontics.

Development of a high-throughput minimum inhibitory concentration (HT-MIC) testing workflow.

The roots of the minimum inhibitory concentration (MIC) determination go back to the early 1900s. Since then, the test has undergone modifications and advancements in an effort to increase its dependability and accuracy. Although biological investigations use an ever-increasing number of samples, complicated processes and human error sometimes result in poor data quality, which makes it challenging to replicate scientific conclusions. Automating manual steps using protocols decipherable by machine can ease procedural difficulties. Originally relying on manual pipetting and human vision to determine the results, modern broth dilution MIC testing procedures have incorporated microplate readers to enhance sample analysis. However, current MIC testing procedures are unable to simultaneously evaluate a large number of samples efficiently. Here, we have created a proof-of-concept workflow using the Opentrons OT-2 robot to enable high-throughput MIC testing. We have further optimized the analysis by incorporating Python programming for MIC assignment to streamline the automation. In this workflow, we performed MIC tests on four different strains, three replicates per strain, and analyzed a total of 1,152 wells. Comparing our workflow to a conventional plate MIC procedure, we find that the HT-MIC method is 800% faster while simultaneously boasting a 100% accuracy. Our high-throughput MIC workflow can be adapted in both academic and clinical settings since it is faster, more efficient, and as accurate than many conventional methods.

Sulfur-fluoride exchange (SuFEx)-enabled lead discovery of AChE inhibitors by fragment linking strategies

SuFEx click chemistry has been a method for the rapid synthesis of functional molecules with desirable properties. Here, we demonstrated a workflow that allows for in situ synthesis of sulfonamide inhibitors based on SuFEx reaction for high-throughput testing of their cholinesterase activity. According to fragment-based drug discovery (FBDD), sulfonyl fluorides [R–SO2F] with moderate activity were identified as fragment hits, rapidly diversified into 102 analogs in SuFEx reactions, and the sulfonamides were directly screened to yield drug-like inhibitors with 70-fold higher potency (IC50 = 94 nM). Moreover, the improved molecule J8-A34 can ameliorate cognitive function in Aβ1−42-induced mouse model. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, this methodology can accelerate the development of robust biological probes and drug candidates.

Development and Validation of a High-Throughput Sequencing Test for Mitogenome and rDNA Assembly and Annotation, and Its Use in Support of Nematode Identification of Regulatory Concern

Nematoda is a diverse phylum, and representatives are found in most habitats, including in and on animals and plants. Nematodes are regarded as the most abundant group in terms of individuals in marine and terrestrial sediments. Plant-parasitic nematodes are globally responsible for an annual yield loss of $125 billion. Reliable species identification is essential to take appropriate phytosanitary measures. The introduction of validated Sanger sequencing of 18S, 28S, and cox1 barcode loci represented a powerful tool in support of nematode identification. However, technical challenges associated with PCR and Sanger sequencing and the need for additional loci for identification hamper the efficient use of sequence data. To overcome these challenges, we developed an automated bioinformatic pipeline for the assembly and annotation of mitochondrial genomes and ribosomal DNAs, and we defined and validated a standardized test protocol including controls for routine diagnostics (i.e., high-throughput sequencing [HTS] test). The HTS test can be performed on single nematode specimens and outperforms the Sanger-based sequencing by producing less ambiguous consensus sequences and by yielding additional sequence data offering additional diagnostic resolution when needed. Compared with Sanger sequencing, the HTS test represents a reduction in hands-on time. The HTS test is regarded as fit for the purpose of the molecular identification of single nematode specimens in support of nematode diagnostics of regulatory concern. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with thissystem allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

A compact and versatile cryogenic probe station for quantum device testing.

Fast feedback from cryogenic electrical characterization measurements is key for the development of scalable quantum computing technology. At room temperature, high-throughput device testing is accomplished with a probe-based solution, where electrical probes are repeatedly positioned onto devices for acquiring statistical data. In this work, we present a probe station that can be operated from room temperature down to below 2K. Its small size makes it compatible with standard cryogenic measurement setups with a magnet. A large variety of electronic devices can be tested. Here, we demonstrate the performance of the prober by characterizing silicon fin field-effect transistors as a host for quantum dot spin qubits. Such a tool can massively accelerate the design-fabrication-measurement cycle and provide important feedback for process optimization toward building scalable quantum circuits.

Preview the Products on Display at Display Week 2023

Preview the Products on Display at Display Week 2023

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.

A fast and sensitive high-throughput assay to assess polysorbate-degrading hydrolytic activity in biopharmaceuticals

Hydrolysis of polysorbate in biopharmaceutical products has been ascribed to the enzymatic activity from trace levels of residual host cell proteins. In recent years, significant efforts to identify the causative enzymes typically used elaborate, material-intensive and time-consuming approaches. Therefore, the lack of fast and sensitive assays to monitor their activity remains a major bottleneck for supporting process optimization and troubleshooting activities where time and sample throughput are crucial constraints. To address this bottleneck, we developed a novel Electrochemiluminescence-based Polysorbase Activity (EPA) assay to measure hydrolytic activities in biotherapeutics throughout the drug substance manufacturing process. By combining the favorable features of an in-house designed surrogate substrate with a well-established detection platform, the method yields fast (∼36 h turnaround time) and highly sensitive readouts compatible with high-throughput testing. The assay capability for detecting substrate conversion in a precise and reliable manner was demonstrated by extensive qualification studies and by employing a number of recombinant hydrolases associated with polysorbate hydrolysis. In addition, high assay sensitivity and wide applicability were confirmed for in-process pool samples of three different antibody products by performing a head-to-head comparison between this method and an established liquid chromatography - mass spectrometry based assay for the quantification of free fatty acids. Overall, our results suggest that this new approach is well-suited to resolve differences in hydrolytic activity through all stages of purification.

Automated High-Throughput Fatigue Testing of Freestanding Thin Films.

Mechanical testing at small length scales has traditionally been resource-intensive due to difficulties with meticulous sample preparation, exacting load alignments, and precision measurements. Microscale fatigue testing can be particularly challenging due to the time-intensive, tedious repetition of single fatigue experiments. To mitigate these challenges, this work presents a new methodology for the high-throughput fatigue testing of thin films at the microscale. This methodology features a microelectromechanical systems-based Si carrier that can support the simultaneous and independent fatigue testing of an array of samples. To demonstrate this new technique, the microscale fatigue behavior of nanocrystalline Al is efficiently characterized via this Si carrier and automated fatigue testing with in situ scanning electron microscopy. This methodology reduces the total testing time by an order of magnitude, and the high-throughput fatigue results highlight the stochastic nature of the microscale fatigue response. This manuscript also discusses how this initial capability can be adapted to accommodate more samples, different materials, new geometries, and other loading modes.

Deep Mutational Scanning and High-throughput Functional Testing to Resolve Variants of Unknown Significance in Sarcoglycanopathies (N4.001)

<h3>Objective:</h3> The goal of this study was to accurately predict the pathogenicity of all possible protein-coding genetic variation in genes causing sarcoglycanopathies. <h3>Background:</h3> Limb-girdle muscular dystrophies R3/R4/R5 are due to recessive mutations in <i>SGCA</i>, <i>SGCB</i> and <i>SGCG</i>. Genetic variation identified within genetic test reports leads to diagnostic uncertainty. Only 14% of missense variants in the sarcoglycan genes have been asserted as either benign or pathogenic; with the vast majority being “variants of unknown significance (VUS).” VUS in LGMD genes leads to an under recognition of these diseases that have treatments within the pre-clinical pipeline. <h3>Design/Methods:</h3> To help resolve VUSs, we performed Deep Mutational Scanning (DMS) to create lentiviral libraries of all possible coding variations in <i>SGCA</i>, <i>SGCB</i> and <i>SGCG</i>. Using these libraries we performed high throughput and orthogonal functional screens in cells followed by sequencing to determine a functional score for each unique variant. <h3>Results:</h3> We tested the effect of all possible coding variants in <i>SGCB</i> to obtain a functional score that predicts the pathogenicity of each genetic variant. The measured scores were perfectly predictive of known pathogenic and benign variants and out-performed all tested bioinformatic prediction algorithms. Validation studies of questionably pathogenic vs benign variants identified in patients support the robustness of our results. Additionally, patient acquired clinical data demonstrated that our DMS functional score was quantitatively correlated with with disease onset and severity. <h3>Conclusions:</h3> In summary, we have generated a valuable resource or “look up table” of functionally defined pathogenic variants in sarcoglycan genes. This type of approach can be utilized for other LGMD genes. In the future these mutational libraries can be used to perform high throughput analysis in the presence and absence of small molecules that may elucidate variant specific therapies. <b>Disclosure:</b> Dr. Haller has nothing to disclose. Dr. Li has nothing to disclose. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Casma therapeutics. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Abata. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Weihl has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Acceleron. Dr. Weihl has received personal compensation in the range of $5,000-$9,999 for serving as an Expert Witness for Wilson Elser Moskowitz Edelman &amp; Dicker LLP.

A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

BackgroundHigh-throughput pharmaco-toxicological testing frequently relies on the use of established liver-derived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable.MethodsWe have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions.ResultsClonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks.ConclusionOur strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality.

Abstract LB358: Efficacy of a functional precision medicine approach in relapsed/refractory pediatric cancer patients: results from a prospective clinical study

Abstract Personalized medicine often serves as the first salvage therapy strategy when standard oncology treatments fail. However, most precision oncology approaches rely on molecular profiling which, unfortunately, provides therapeutic options for less than 10% of cancer patients. Functional precision medicine (FPM) complements molecular profiling by combining it with rapid, high-throughput drug testing on live patient cells to identify promising treatment options. In this study, we investigated the efficacy of FPM in the management of pediatric patients with recurrent and/or refractory cancers. We enrolled 25 pediatric/young adult patients with refractory solid or liquid cancers in this clinical trial (number NCT03860376). Enrolled patients represented the breadth of cancer indications generally presenting in pediatric patients: acute lymphoblastic leukemia (3 patients), acute myeloid leukemia (3 patients), astrocytoma (1 patient), ependymoma (1 patient), Ewing’s sarcoma (4 patients), glioblastoma (1 patient), malignant rhabdoid tumor (1 patient), medulloblastoma (1 patient), neuroblastoma (1 patient), osteosarcoma (4 patients), rhabdomyosarcoma (4 patients) and Wilms’ tumor (1 patient). We used a functional ex vivo drug sensitivity test (DST) panel encompassing 40 formulary drugs frequently used at Nicklaus Children’s Hospital and 47 non-formulary drugs approved by FDA for cancer treatment, as well as drugs from phase III and IV clinical trials. Drug sensitivity scores (DSS) were calculated for each drug based on cancer cells’ responses. DST results were then combined with results from targeted mutation profiles to match actionable mutations with selective targeted therapies. FPM and molecular data were prospectively generated and treatment recommendations were provided to an FPM Molecular Tumor Board (MTB) of clinicians. In total, 19 of 25 patients (76%) had complete functional and molecular profiling data provided to the MTB to support clinical decision-making. Six patients had their subsequent treatment guided by FPM recommendations, of which 83% (five of six patients) demonstrated greater than 1.3x increased progression-free survival compared to their previous therapy. Interestingly, hierarchical clustering analysis of DST results shows that patients with the same subtype of cancer do not cluster together, and no cancer subtype is differentially represented in either high-level cluster, suggesting the importance of functional profiling to provide deeper insight into individual patient pharmacological response. This FPM study is the first pediatric cancer study to enroll both solid and hematologic cancers regardless of tumor type, and the first FPM study in the United States to generate prospective treatment data on pediatric oncology patients. We illustrate the feasibility and efficacy of FPM to meet the needs of cancer patients with both liquid and solid tumors, especially for high-risk populations such as pediatric cancer patients. Citation Format: Arlet Maria Acanda de la Rocha, Maggie Fader, Ebony R. Coats, Joseph Dunn, Leat Perez, Carolina Velasquez, Jeanette Galano, Cima Saghira, Ileana Sotto, Yana Vorontsova, Ziad Khatib, Haneen Abdella, Cristina M. Andrade-Feraud, Alexa Jacome, Victoria Reis, Lilliam Rimblas, Nicole Tomas, Paula S. Espinal, Noah Berlow, Tomás R. Guilarte, Jennifer McCafferty-Fernandez, Daria Salyakina, Diana J. Azzam. Efficacy of a functional precision medicine approach in relapsed/refractory pediatric cancer patients: results from a prospective clinical study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB358.

Determination of the size effect on the tensile properties of miniaturized specimens

Abstract To realize high-throughput tensile testing of miniaturized specimens, the size effect was investigated. High-throughput tensile testing was used to characterize the mechanical properties of steel in the micro-zone. Miniaturized specimens with diameters ranging from 0.7 mm to 2.5 mm were cut from low-alloyed steel and tested, and the deviations between the results of miniaturized and conventional sized specimens were examined. The results showed that the size effect was negligibly small for the tensile strength. However, the upper yield strength, lower yield strength, and plastic extension strength of miniaturized specimens decreased as the specimen diameter decreased. It was found that a diameter of 1.5 mm was critical for the tensile testing of miniaturized specimens. As the specimen diameter decreased toward 1.5 mm, the strength parameters gradually decreased, and as the specimen diameter further decreased from 1.5 mm, the strength parameters increased. In contrast, as the specimen diameter decreased from 1.3 mm, the elongation after fracture decreased. However, the percentage reduction of area did not vary between miniaturized and conventional sized specimens.

Fabrication of a Low-Cost Microfluidic Device for High-Throughput Drug Testing on Static and Dynamic Cancer Spheroid Culture Models

Drug development is a complex and expensive process from new drug discovery to product approval. Most drug screening and testing rely on in vitro 2D cell culture models; however, they generally lack in vivo tissue microarchitecture and physiological functionality. Therefore, many researchers have used engineering methods, such as microfluidic devices, to culture 3D cells in dynamic conditions. In this study, a simple and low-cost microfluidic device was fabricated using Poly Methyl Methacrylate (PMMA), a widely available material, and the total cost of the completed device was USD 17.75. Dynamic and static cell culture examinations were applied to monitor the growth of 3D cells. α-MG-loaded GA liposomes were used as the drug to test cell viability in 3D cancer spheroids. Two cell culture conditions (i.e., static and dynamic) were also used in drug testing to simulate the effect of flow on drug cytotoxicity. Results from all assays showed that with the velocity of 0.005 mL/min, cell viability was significantly impaired to nearly 30% after 72 h in a dynamic culture. This device is expected to improve in vitro testing models, reduce and eliminate unsuitable compounds, and select more accurate combinations for in vivo testing.

Abstract 205: Patient-derived organoids from surgically treated, localized non-small cell lung cancer as high-throughput drug testing platforms for conventional and repurposed drugs

Abstract Background: Non-metastatic NSCLC treated with curative surgery has a five-year survival of ~50%, mostly due to development of recurrences. Despite being considered as potential drug screening platforms, patient-derived xenograft (PDX) models are inefficient due to low tumor engraftment rates and complex animal care. Patient-derived organoid (PDO) models overcome these limitations as potential clinically applicable drug testing platforms. Our objective was to develop PDO models from non-metastatic NSCLC patients to study epithelial cell type heterogeneity and drug sensitivities for precision medicine. Hypothesis: PDO models reliably capitulate the patient primary tumor and serve as versatile platforms for high-throughput screening of standard-of-care and repurposed drugs. Methods: Single cell suspensions prepared from resected lung tumor tissues from ten NSCLC patients (both adenocarcinoma and squamous cell carcinoma) were mixed with Matrigel (growth factor-reduced) and cultured in organoid growth medium. Organoids and matched primary tumors were compared by histopathology [H&amp;E staining and immunohistochemistry for cytokeratin (CK) 5/6, CK7, Napsin A, Thyroid transcription factor-1 (TTF-1) and p40] and by bulk RNA sequencing. Upon passaging, PDOs were seeded in triplicates, treated with carboplatin/paclitaxel doublet chemotherapy, and drug responses were determined using bright-field 3D imaging (z-stack method). Tumor growth (%) was determined on day 3 and 6 of treatments. RNA sequencing analyses identified a potential drug targets and repurposed drug (aldoketoreductase inhibitor Epalrestat) was tested to overcome chemoresistance in PDOs. Results: NSCLC PDO growth was established from 11/12 (91.7%) primary tumors with a median time of 11 days (range 4-18 days) to reach volume of 100 μm3. PDOs retained histopathological features and biomarker expression of the matched tumors. As determined by growth differences (p&amp;lt;0.05), 5/9 (55.6%) PDOs were chemosensitive and 4/9 (44.4%) PDOs were chemoresistant against carboplatin/paclitaxel. Whole transcriptome analysis confirmed conservation of the epithelial cell composition in all PDOs in comparison to the matched primary tumors. Potentially druggable genes associated with chemotherapy resistance were identified and the aldoketoreductase inhibitor Epalrestat was repurposed and shown to be effective in overcoming chemoresistance towards carboplatin/paclitaxel. Conclusions: PDOs can be established from resectable NSCLC patients with high success rates, while preserving histopathological, cellular, and molecular characteristics of the matched primary tumors. Considering the time frames of growth and drug testing, PDOs can serve as clinically applicable drug response testing platforms to traditional and repurposed drugs. Citation Format: Yariswamy Manjunath, Suvilesh Kanve Nagaraj, Yulia I. Nussbaum, Mohamed Gadelkarim, Kevin F. Staveley-O’Carroll, Eric T. Kimchi, Guangfu Li, Wesley Warren, Chi-Ren Shyu, Matthew Ciorba, Jonathan B. Mitchem, Jussuf T. Kaifi. Patient-derived organoids from surgically treated, localized non-small cell lung cancer as high-throughput drug testing platforms for conventional and repurposed drugs [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 205.

Abstract 2912: High-throughput screening and single-cell transcriptomics identify synergies between oncology drugs and natural killer cell immunotherapy

Abstract Background &amp; Objective: Natural killer (NK) cell-based immunotherapies are emerging therapeutic approaches that aim to target malignant cells resistant to conventional treatments and T cell-based immunotherapies. Existing oncology drugs may enhance the anti-cancer function of NK cells, although large-scale high-throughput testing of NK cell-drug combinations has not been previously attempted. Our objective was to evaluate the potential of approved and investigational oncology drugs to synergize with NK cell-based immunotherapy in acute myeloid leukemia (AML), with the aim to discover both potentiating and inhibiting compounds. Methods: A high-throughput drug sensitivity and resistance screen including 528 oncology drugs was combined with expanded NK cells to evaluate their cytotoxicity against luciferase-expressing AML cells (MOLM-14). The most prominent drugs were investigated further with single-cell RNA sequencing to reveal underlying biological mechanisms. Results: Overall, 191 drugs had an inhibitory effect on cytotoxicity, 294 had no effect, and 43 had an enhancing effect. The most notable NK cell cytotoxicity-potentiating drugs were pevonedistat (NEDD8 inhibitor), daporinad (NAMPT inhibitor), and JAK inhibitors. These drugs also induced distinct changes in NK and target cell transcriptional profiles. When combined with NK cells, pevonedistat and daporinad induced an upregulation of HLA-E and CXCL10 in target cells. In addition, MYC and FLT3 proto-oncogenes were downregulated in target cells when combining NK cells with pevonedistat. Moreover, in NK cells, daporinad induced dysregulated expression of genes related to NK cell effector functions such as TYROBP, TXNIP and KLRC1. Treatment with JAK inhibitors downregulated MHC I expression on target cells, providing a putative mechanism for increased NK cell activity. Validation of key hits is ongoing in additional AML cell lines. Conclusion: We discovered novel drugs with both potentiating and inhibiting effects on NK cell cytotoxicity against AML cells in vitro. Our results demonstrate the potential of using a high-throughput framework for studying NK cell-drug interactions against malignant cells, while aiming to improve treatment for AML and other malignancies through combination immunotherapies. Citation Format: Jonas O. Bouhlal, Petra Nygren, Essi Laajala, Aleksandr Ianevski, Jay Klievink, Hanna Lähteenmäki, Khalid Saeed, Dean Lee, Tero Aittokallio, Olli Dufva, Satu Mustjoki. High-throughput screening and single-cell transcriptomics identify synergies between oncology drugs and natural killer cell immunotherapy [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 2912.