Downstream Assays Research Articles

Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.

The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

Gut bacteria are essential for development of an invasive bark beetle by regulating glucose transport

Insects and their gut bacteria form a tight and beneficial relationship, especially in utilization of host nutrients. The red turpentine beetle (RTB), a destructive and invasive pine pest, employs mutualistic microbes to facilitate its invasion success. However, the molecular mechanism underlying the utilization of nutrients remains unknown. In this study, we found that gut bacteria are crucial for the utilization of D-glucose, a main carbon source for RTB development. Downstream assays revealed that gut bacteria-induced gut hypoxia and the secretion of riboflavin are responsible for RTB development by regulating D-glucose transport via the activation of a hypoxia-induced transcription factor 1 (Hif-1α). Further functional investigations confirmed that Hif-1α mediates glucose transport by direct upregulation of two glucose transporters (ST10 and ST27), thereby promoting RTB development. Our findings reveal how gut bacteria regulate the development of RTB, and promote our understanding of the mutualistic relationship of animals and their gut bacteria.

Investigation of piezoelectric printing devices for oil-free and on-demand picolitre monodisperse droplet generation

Picolitre monodisperse droplet printing technology has important applications in biochemistry, such as accounting for quantitative analysis and single-cell analysis, and can be used for parallel high-throughput analysis of biomarkers and chemicals. However, commonly used droplet generation devices require complex control systems or customised microfluidic chips, making them costly and difficult for researchers to operate. Additionally, generating picolitre monodisperse droplets with microfluidic devices necessitates the introduction of an oil phase to block and separate the liquid. This requirement can reduce the throughput of the target droplets and cause cell contamination, hindering the adoption of this technology. By employing a common 1-mm-diameter capillary in the laboratory in combination with a piezoelectric transducer, we have achieved on-demand picolitre droplet printing of less than 100 pL in an oil-free environment. The device was found to be biocompatible with K562 cells. This approach is less costly, offers greater operational freedom, and is easier to integrate with other downstream assay modules or even handheld cell-printing devices. This study holds great potential for application in areas such as single-cell analysis, cell sampling, and pharmaceutical analysis.

Generation and Characterization of hiPSC-Derived Vascularized-, Perfusable Cardiac Microtissues-on-Chip.

In the heart in vivo, vasculature forms a semi-permeable endothelial barrier for selective nutrient and (immune) cell delivery to the myocardium and removal of waste products. Crosstalk between the vasculature and the heart cells regulates homeostasis in health and disease. To model heart development and disease in vitro it is important that essential features of this crosstalk are captured. Cardiac organoid and microtissue models often integrate endothelial cells (ECs) to form microvascular networks inside the 3D structure. However, in static culture without perfusion, these networks may fail to show essential functionality. Here, we describe a protocol to generate an in vitro model of human induced pluripotent stem cell (hiPSC)-derived vascularized cardiac microtissues on a microfluidic organ-on-chip platform (VMToC) in which the blood vessels are perfusable. First, prevascularized cardiac microtissues (MT) are formed by combining hiPSC-derived cardiomyocytes, ECs, and cardiac fibroblasts in a pre-defined ratio. Next, these prevascularized MTs are integrated in the chips in a fibrin hydrogel containing additional vascular cells, which self-organize into tubular structures. The MTs become vascularized through anastomosis between the pre-existing microvasculature in the MT and the external vascular network. The VMToCs are then ready for downstream structural and functional assays and basic characterization. Using this protocol, cardiac MTs can be efficiently and robustly vascularized and perfused within 7 days. In vitro vascularized organoid and MT models have the potential to transition current 3D cardiac models to more physiologically relevant organ models that allow the role of the endothelial barrier in drug and inflammatory response to be investigated. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Generation of VMToC Support Protocol 1: Functional Characterization of VMToC Support Protocol 2: Structural Characterization of VMToC.

The challenges and breakthroughs in the development of diagnostic monoclonal antibodies

AbstractOver the past century, the field of antibody discovery has undergone significant evolution, excluding the current exploration stage of artificial intelligence‐based antibody generation and the often overlooked non‐animal sourced antibody discovery, which typically requires mature in vitro affinity and the selection of high‐quality antigen formulations. This journey has traversed various stages, from methods involving serum‐based antibody acquisition, the isolation of B cells capable of perpetual antibody production through hybridoma technology, to the in‐depth exploration of genetic material using the phage display system, and the current stage involving diverse single B cell screening techniques. Additionally, the emergence of machine learning has brought impressive scientific and technological breakthroughs across research domains, proving to be a powerful application in the field of antibody discovery. However, each technique comes with its limitations, such as variability and control challenges in serum‐based acquisition, lengthy and difficult hybridoma‐derived antibody development, potential limitations in sequence and epitope diversity due to immunization biases in phage display techniques, and costly single B cell screening. Protein mass spectrometry sequencing, with shorter acquisition time and lower costs, is seen as a shortcut by diagnostic companies, impacting traditional antibody development. In diagnostic antibody development, methodological differences in downstream assays and the impact of constant regions outside the Fv core are often neglected. This paper deeply analyzes challenges, proposing innovative strategies for the next generation of diagnostic antibody development. Aimed at moving closer to the gold standard of antibody discovery, these strategies enhance the competitiveness of diagnostic reagent products.

Isolation of Mouse Pancreatic Endothelial Cells.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

DIPG-08. PROBING THE BLOOD-BRAIN-TUMOR-BARRIER WITH A MICROVASCULAR MODEL OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Pediatric diffuse midline glioma (DMG) is a uniformly fatal brain tumor in children, with most patients surviving less than 12 months. Current treatment for DMG is limited, and outcomes for this tumor have not improved. The blood-brain-barrier (BBB) remains a key challenge for DMG therapeutics, preventing most drugs from entering brain tissue. Functional characterization of the blood-brain-tumor-barrier (BBTB) – the interaction between tumors and BBB vasculature – remains largely unexplored for DMG. There is a need for a preclinical model of DMG that can probe the functional, physiological, and biological properties of the BBTB in vitro as a platform to evaluate therapeutic efficacy that is representative of human physiology. METHODS To generate an in vitro model of DMG with human microvasculature (DMG-BBTB model), we co-cultured DMG neurospheres, human brain microvascular endothelial cells, human brain vascular pericytes, and human astrocytes. Cells are simultaneously seeded into microfluidic devices at varied concentrations within a fibrin matrix. Self-assembled microvascular networks are formed within 7-8 days and used for downstream functional and phenotypic assays. RESULTS Vascular morphology and solute permeability in microvascular networks are influenced by cell identity, growth pattern, and relative concentration. We show that DMG neurospheres exhibit different patterns of growth when co-cultured as uniform spheroids versus single cells in suspension. Devices representing each growth pattern are further characterized for paracellular diffusion and permeability compared to devices without DMG cells through perfusion of high molecular weight dextran. Permeability values are correlated to biologic features such as the abundance and localization of junctional proteins and transporters. CONCLUSIONS We developed a platform to evaluate the microvasculature of the DMG BBTB as a tool to develop new therapies informed by vascular biology. We probed the functional, physiological, and biological properties of the human DMG BBTB in vitro, and in ongoing work are assessing approved and experimental therapeutics.

Collection of biospecimens from the inspiration4 mission establishes the standards for the space omics and medical atlas (SOMA)

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R + 1, R + 45, R + 82, R + 194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations.As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can aid future human spaceflight and space biology experiments.

Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens

Effective pathogen detection is essential for plant disease control. However, plant sample preparation for downstream assays, such as quantitative polymerase chain reaction (qPCR), is challenging to perform outside of a laboratory. This paper reports two sample preparation methods featuring chemical and mechanical lysis and nucleic acid extraction using a micro-homogenizer, followed by serial dilution or nucleic acid purification with a paper disk before assay. Five minutes of lysis and extraction resulted in DNA and RNA yields of up to 76.5% and 63.3%, respectively, compared to mortar and pestle controls. Crude lysates were unsuitable for direct use in qPCR assays; however, serial dilution or quick wash using chromatography paper rendered samples ready for such assays. Additionally, the nucleic acids stored on paper disks under various storage conditions remained stable for one month. These methods can facilitate the in-field preparation of citrus samples and allow for both onsite and mail-in diagnostics for growers.

Picosecond laser capture microdissection based on edge catapulting combined with dielectrophoretic force.

The spatial omics information analysis of heterogeneous cells or cell populations is of great importance for biomedical research. Herein, we proposed a picosecond laser capture microdissection boosted by edge catapulting combined with dielectrophoretic force (ps-LMED) that enables fast and non-invasive acquisition of uncontaminated cells and cell populations for downstream molecular assays. The target cells were positioned under a microscope and separated by a focused picosecond pulsed laser. The system employed the plasma expansion force during cutting to lift the target and captured it under dielectrophoretic force from the charged collection cap eventually. The principle of our system has been validated by both theoretical analysis and practical experiments. The results indicated that our system can collect samples ranging from a single cell with a diameter of a few microns to large tissues with a volume of 532,500 µm3 at the moment finishing the cutting, without further operations. The cutting experiments of living cells and ribonucleic acid (RNA) and protein omics analysis results of collected targets demonstrated the advantage of non-destructiveness to the samples and feasibility in omics applications.

Glycoprotein-glycoprotein Receptor Binding Detection Using Bioluminescence Resonance Energy Transfer.

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.

Abstract 5702: BTX-9341, a bifunctional degrader of CDK4 and CDK6 for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with limited treatment options. GBM has a high frequency of dysregulation of the CDKN2A-cyclin D-CDK4/CDK6 signaling node and should subsequently be sensitive to CDK4/6 inhibition. Despite this, the CDK4/6 inhibitors that are approved for HR+/HER2- breast cancer have shown limited efficacy in GBM due to poor blood brain barrier (BBB) penetration. BTX-9341 is a Cereblon (CRBN) mediated CDK4/6 bifunctional degrader that we have developed for HR+/HER2- breast cancer. This degrader shows good exposure in brain tissues with a high brain to plasma ratio. Given the exposure in the brain, we explored the in vitro and in vivo efficacy of BTX-9341 in GBM cell line and xenograft models. GBM cell lines treated with BTX-9341 showed up to 86% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. We examined RB phosphorylation by immunoblot and cell cycle progression by propidium iodide staining followed by flow cytometry. BTX-9341 was potent in all downstream assays, with phospho-RB IC50s <10nM, and G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation by cell cycle arrest. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 13-100nM in GBM cell lines. BTX-9341 displays excellent pharmacokinetic properties with good oral bioavailability and high brain to plasma ratios which allowed for oral dosing in both subcutaneous and orthotopic xenograft studies. We examined tumor growth inhibition efficacy in two GBM xenograft models: an intracranial U87 model and a subcutaneous U118 model and saw robust tumor growth inhibition in both models with BTX-9341 treatment. The U118 cells have partially unmethylated MGMT, which lead to a loss of efficacy of temozolomide over time, while BTX-9341 maintained inhibition of tumor growth. This indicates that BTX-9341 may be effective in patient populations for which temozolomide is ineffective. The U87 intracranial xenograft model also showed dose-dependent tumor growth inhibition upon treatment with BTX-9341, indicating that BTX-9341 can cross the BBB and reach concentrations high enough to execute cell proliferation inhibition effects. BTX-9341 treated mice also had much better survival than abemaciclib treated mice, the only CDK4/6i which has BBB penetration. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo in GBM models. The xenograft data indicates that BTX-9341 can inhibit growth of MGMT methylated and unmethylated GBM cell lines, and that BTX-9341 can inhibit tumor growth in the brain more effectively than BBB penetrant CDK4/6i. Together this data shows that BTX-9341 may be a promising candidate for treating GBM. Citation Format: Hannah Majeski, Kirti Chahal, Akinori Okano, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. BTX-9341, a bifunctional degrader of CDK4 and CDK6 for glioblastoma multiforme [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 5702.

Abstract 2311: Precise assessment of cancer cell growth and survival by artificial intelligence

Abstract Introduction: Cancer research requires accurate methods for measuring analytical parameters like cell culture confluence, cell count, colony numbers, viability, and motility. These methods must be unbiased and user-independent for reproducible data. Cell analytics involves manual processes (e.g., manual cell counting) or reagent-based approaches (e.g., viability kits). In recent years, semi-automated systems have been introduced that can either count cells, measure cell growth by density tracking, and/or determine cell viability. However, these methods are often time-consuming, require reagents and labeling, and may involve costly instrumentation. Artificial Intelligence (AI) has made strides in clinical and laboratory research, holding promise for swift integration into cancer research. Here, we present the development and validation of SnapCyte™, an AI performing accurate, unbiased, label- and reagent-free cell analytics from basic cell culture images, independent of specialized instrumentation. Materials and Methods: Cell images were generated using diverse cell lines (MCF7, PC3, Hela, Raji⋯) cultured in various vessels with and without treatments (Taxane, Cisplatin, Heatshock⋯). For the cell count dataset, adherent cells were detached using trypsin, stained with trypan blue and loaded into a standard hemocytometer. Images were acquired using the SnapCyte™ adaptor and diverse microscopes (Leica, Hund, Zeiss, Nikon) and cell phones. Multiple datasets of 500 annotated images each were created, with images masked for confluency or for singular live and dead cells by experienced scientists. Object segmentation utilized the UNet architecture for localizing cells in cell culture images and iterative training was applied to achieve the required accuracy. Results: After multiple training iterations, SnapCyte™ AI detection models achieved 99% precision for confluency and >95% precision and recall for cell count. SnapCyte™ surpassed standard methods (Crystal Violet, WST1, MTT, Presto blue, CyQuant, Incucyte®, and Bio-Rad TC20 cell counter), displaying high accuracy and smaller standard error variation than reagent-based assays. Compared to IncuCyte® Bio-Rad TC20, SnapCyte™ demonstrated similar accuracy and greater user-independent results. Furthermore, SnapCyte™ acquired data in under 10 minutes, with non-invasive measurements, allowing direct use of cells in downstream assays. Conclusion: We have developed and validated an AI model for advanced cell analytics. Our data show that the SnapCyte ™ AI is at par or better than existing reagent and instrument-based solutions in assessing cell confluency, number, and viability. This technology offers a fast, accurate, and unbiased cell analytics platform that is resistant to user variations, and independent of reagents and costly equipment. Citation Format: Cheung Pang Wong, Nasrin Khazamipour, Soroush Alibagi, Joya Saade, Daria Golanarian, Negin Farivar, Mads Daugaard, Nader Al Nakouzi. Precise assessment of cancer cell growth and survival by artificial intelligence [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 2311.

Abstract 6068: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of the cyclin D-CDK4/6 signaling node. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate. Breast cancer cell lines treated with BTX-9341 showed up to 85% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. We examined RB phosphorylation by in-cell western, E2F target gene expression by qPCR and cell cycle progression by propidium iodide staining followed by flow cytometry. BTX-9341 was potent in all downstream assays, with phospho-RB IC50s <30nM, E2F target gene downregulation and G0/G1 cell cycle arrest at concentrations as low as 10nM. These downstream effects were sustained throughout 72 hours with BTX-9341 treatment but recovered more rapidly with Palbociclib treatment. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation by cell cycle arrest. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 20-50nM while CDK4/6i had CFA IC50s of 50-1000nM. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell line models BTX-9341 maintained a low CFA IC50 (<150nM) while CDK4/6i displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties which allowed for oral dosing in xenograft studies. In several breast cancer xenograft models, BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher dose levels, and was effective with multiple alternate dosing regimens. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [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 6068.

Abstract 7248: Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy

Abstract Introduction Viral vectors, such as adeno-associated vector (AAV), play a crucial role in delivering transgenes for gene therapy research. The success of vector transduction is vital for efficacy, which is measured through vector copy number (VCN) and transgene expression. In preclinical studies, both VCN and transgene expression are commonly tested from the same sample to ensure accuracy. We have reported a method for simultaneous high-quality genomic DNA and RNA extraction without splitting the sample, utilizing SPRI bead-based technology that has consistent performance across diverse tissue types and brain regions. Methods AAV was administered to mouse and non-human primate (NHP) models. At the conclusion of each experiment, tissue samples from the adrenal gland, brain, gonads, heart, kidney, liver, lung, skeletal muscle, spinal cord, and spleen were harvested. All tissue samples that demonstrated around 15 mm3 were snap-frozen in liquid nitrogen before nucleic acid extraction. The extraction process was carried out manually or on an automated liquid handler. The quality and quantity of DNA and RNA were then assessed from all tested tissues before downstream assay. Results Our study found that all mouse tissue types (n=72/tissue type) and NHP (n=11/tissue type) had an average gDNA and RNA yield that exceeded the minimum yield cutoff of 25 ng/μL for DNA and 6.25 ng/μL for RNA. We also analyzed DNA from tissues treated with two different AAV serotype-treated tissues in the mouse model, which showed that transduction across all the tissues was highly efficient for both AAV serotypes. Furthermore, we observed variations in transgene expression levels (copies/1 μg RNA) among different tissue types. Similar patterns of VCN and transgene expression were also observed in NHP models. Conclusions Efficient gene therapy depends on a safe and effective viral vector. The simultaneous extraction of gDNA and RNA simplifies the process, saves time, ensures quality, and facilitates integration into existing lab automation. This leads to consistent and error-resistant performance in analyzing vector copy and transgene levels. Citation Format: Han Wei. Simultaneous isolation and parallel analysis of genomic DNA and RNA for gene therapy [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 7248.

Abstract 4459: Metabolic liabilities of iron-dependent ferroptosis mediated through NRF2/GPX4 axis in osteosarcoma

Abstract Osteosarcoma is an aggressive bone malignancy that predominantly affects adolescents and young adults. Despite recent advancements in treatments, the challenges of recurrence and chemoresistance persist. Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation, and emerging evidence suggests significant roles for NRF2 and GPX4 in iron metabolism, regulating tumor cell functions. In osteosarcoma, however, the molecular mechanisms of the NRF2/GPX4 axis and its therapeutic potential remain unknown. In this study, we first determined GPX4 and xCT expression levels and cellular responses to RSL3, a GPX4 inhibitor, and Erastin, an xCT inhibitor in osteosarcoma cells. A total of seven osteosarcoma cell lines, consisting of five human cell lines (HOS, 143B, Saos-2, MG-63, U2OS) and two canine cell lines (COS31 and DOUG), were used. Our results revealed that the IC50 values ranged from 0.02 uM to 1.01 uM in cells treated with RSL3 and from 0.35 uM to 1.41 uM in cells treated with Erastin, respectively. These values were proportional to GPX4 and xCT levels across the cell lines. Then, U2OS, as the most sensitive, and Saos-2, the least sensitive based on their IC50 values, were selected to examine the iron regulatory machinery. In U2OS cells, RSL3 treatment significantly decreased NRF2 and GPX4, while increasing ferroportin-1 and ferritin. GPX4 knockdown also led to comparable changes in NRF2, ferroportin-1, and ferritin expression. NRF2 appears to mediate iron metabolism, ferroportin-1 acts as an iron exporter, and ferritin stores intracellular iron. Therefore, our findings suggest that GPX4-mediated ferroptosis is regulated by iron metabolic pathways, potentially to prevent ferroptotic cell death. We also found that Ferrostatin-1, a lipid peroxidation inhibitor showed no changes in ferroportin-1, ferritin, and NRF2. In Saos-2, RSL3 induced a remarkable reduction in GPX4 expression, akin to U2OS. However, no significant change was found in NRF2, ferroportin-1, and ferritin. Instead, Ferrostatin-1 restored the decreased GPX4 and increased NRF2, ferroportin-1, and ferritin levels. In addition, Z-VAD (a pan-caspase inhibitor), Necrosatin-1s (a RIPK1 inhibitor), and Ferrostatin-1 were capable of preventing Saos-2, COS31, and DOUG cells from RSL3-induced cell death at lethal doses. Such protection was undetected in U2OS. These data suggest that metabolic pathways controlling intracellular iron are crucial for NRF2/GPX4-mediated ferroptosis in osteosarcoma cells. Furthermore, we generated RNA-seq and LC-MS/MS data to identify novel molecular targets governing the metabolic pathways of ferroptosis in osteosarcoma. Our ongoing work includes downstream molecular and functional assays following gene modifications in osteosarcoma cells (overexpression and knockout) and assessing metabolic vulnerabilities by targeting iron-mediated ferroptosis in xenografts. Citation Format: Md Abdullah, Donghee Lee, Rong Li, Jong Hyuk Kim. Metabolic liabilities of iron-dependent ferroptosis mediated through NRF2/GPX4 axis in osteosarcoma [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 4459.

Abstract 324: Comparison of xGen Exome Hyb Panel v2 (IDT) kit and Twist Exome 2.0 (TWIST) kit for whole exome sequencing optimization from low input and degraded FFPE samples

Abstract FFPE is the most common method used to store precious clinical samples at room temperature for extended periods (sometimes>15 years). FFPE samples are therefore one of the most commonly available samples that can be used for discovery and diagnosis. Whole Exome Sequencing (WES) is one of the most used NGS assays that is used for profiling somatic and tumor mutations. With future clinical trials moving towards less invasive collection methodology, core needle biopsy samples are becoming a method of choice. The FFPE fixation process leads to the degradation of nucleic acids due to cross-linkage and the availability of sufficient nucleic acid from CNB fixed samples for downstream NGS assays is becoming limited. Here we report a comparison of two Whole Exome capture kits from several degraded FFPE samples, NA12878, OncoSpan gDNA HD827 and SeraSeq Breast CNV mix gDNA samples using the xGen Exome Hyb Panel v2 (IDT) Kit and the Twist Exome 2.0 (TWIST) Kit. Libraries were prepared in triplicate at 50ng, and 10ng using the Ultra™ II DNA Library Prep (NEB) kit. Pre-capture libraries were evaluated to determine the total DNA library available for capture using the KAPA Library Quantification Kit (Roche) kit. The recommendations for input amount vary between the kits, with IDT recommending 500ng, and TWIST recommending 200ng per library. KAPA Library Quantification Kit kit was used to assess the nanomolar amounts of ligated libraries. Final libraries were pooled equimolar and then sequenced as 2 × 100bp Paired-end sequencing on the NovaSeq 6000 instrument to achieve a target of 150-300X overall coverage. The performance of each capture kit was evaluated on the number of libraries eligible for capture, percent duplication rate, median insert size, and mean target coverage. IDT capture had 49% of libraries eligible at recommended input of 500ng, with 34% of libraries generating between 100-499ng input and the remaining 17% failing to generate 100ng input for hybridization. TWIST capture had 83% of libraries eligible at recommended input of 200ng, with 4% of libraries generating between 50-199ng input and the remaining 13% failing to generate 50ng input for hybridization. Most notably, 96% of libraries at 50ng DNA input were eligible for TWIST capture, while only 46% of libraries at 50 ng DNA input were eligible for IDT capture. The average Q30 for IDT and TWIST capture were 93-94% respectively. Both IDT and TWIST capture kits produced similar duplication rates and average mean target coverage was comparable. Taken together, our testing shows that TWIST and IDT kits generated comparable sequencing metrics. However, the TWIST Kit outperforms the IDT Kit in percent eligible libraries qualifying for hybridization capture. TWIST Kit is a great qualifier for highly degraded FFPE samples, especially those with limited input DNA material. Citation Format: Rachel Rock, Nripesh Prasad, Melanie Robinson, Dinnen Wildman, Olivia Montoya, Michael Sykes, Boris Umylyn, Thomas Halsey. Comparison of xGen Exome Hyb Panel v2 (IDT) kit and Twist Exome 2.0 (TWIST) kit for whole exome sequencing optimization from low input and degraded FFPE samples [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 324.

Abstract 3523: Self-supervised foundation model captures high-dimensional morphology data from single cell brightfield images

Abstract Our knowledge of cancer biology has advanced with the characterization of distinct cell types and cell states within heterogeneous tumor environments. Many methods for imaging and sorting tumor cells require biomarker labels that alter cell characteristics and create a selection bias. The use of label-free single-cell methods should further improve cancer studies involving viable, unperturbed cells for downstream assays such as RNA-Seq, cell culture expansion, and functional assays. To eliminate biomarkers and enable broader assessment of cells, we developed REM-I, a platform that characterizes and sorts unlabeled single cells based on high-dimensional morphology. Cells are captured with brightfield imaging and processed in real-time by self-supervised deep learning models to generate quantitative AI embeddings representative of cell morphology. A significant technical challenge in building REM-I was developing an AI model based on extracted features from cell images without prior knowledge of cell types, cell preparation, or other application-specific knowledge. Accordingly, we developed the Human Foundation Model (HFM), a hybrid architecture that combines self-supervised learning (SSL) and morphometrics (computer vision) to extract 115 dimensional embeddings representing cell morphology from high-resolution REM-I cell images. SSL produces a foundation model with high generalization capacity that enables hypothesis-free sample exploration and efficient generation of application-specific models. Meanwhile, computer vision extracts features that represent measurable and interpretable concepts (e.g., cell size, shape, texture, intensity). The training process for the HFM self-supervised backbone model utilizes the discriminatory power of supervised tasks. Using synthetic cells and three cancer cell lines, we trained and then validated the reproducibility and generalization capabilities of the resulting model. Results show combining deep learning and morphometrics improve interpretability of data and enable rapid characterization and classification of tumor cells with high accuracy. We also report on the Deepcell® Axon data suite, a tool to analyze data and customize reusable workflows. This includes the ability to store and manage data, visualize high dimensional data as low-dimensional projections, and train classifiers to identify and sort cell populations. To enable compatibility with user-preferred downstream analysis pipelines, Axon provides data export options for images, plots, and embeddings. Our approach allows users of all skill levels to access and interpret AI-enabled morphological profiling. Applications of REM-I include hypothesis-free evaluation of heterogeneous tumor samples, label-free cancer cell enrichment, characterization of distinct cell states, and multi-omic integration. Citation Format: Kiran Saini, Senzeyu Zhang, Ryan Carelli, Kevin B. Jacobs, Amy Wong-Thai, Cris Luengo, Vivian Lu, Anastasia Mavropoulos, Andreja Jovic, Jeanette Mei, Thomas Vollbrecht, Stephane C. Boutet, Mahyar Salek, Maddison Masaeli. Self-supervised foundation model captures high-dimensional morphology data from single cell brightfield images [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 3523.

Abstract 2047: Scale up and scale down approaches for screening of 3D patient-derived cell models

Abstract 3D cell culture is becoming more prevalent as scientists work to better model human physiology using in vitro systems. However, differences in handling 3D cell cultures compared to traditional 2D cell lines have limited adoption of 3D techniques in compound and cell therapy screening experiments. To facilitate use of patient-derived 3D tumoroid models, we developed protocols for both scale up and scale down of these models in Gibco™ OncoPro™ Tumoroid Culture Medium. OncoPro Tumoroid Culture Medium is a serum-free, conditioned medium-free culture medium designed to promote the growth of patient-derived 3D cancer models (tumoroids, cancer organoids). OncoPro medium is compatible with a suspension culture approach for easier handling compared to traditional embedded culture formats. We studied the growth of tumoroids in different flask sizes during suspension culture to optimize scale up for downstream assays. Tumoroid growth rates and morphologies were consistent across non-tissue culture treated flask sizes, and viable cell yield increased with surface area during scale up. In some cases, as many as 100 × 106 dissociated tumoroid cells can be recovered from a single Nunc™ TripleFlask™ after a week of growth. After scaling up, tumoroids were dissociated and seeded in multiwell plates to test multiple screening conditions (compound identity, compound concentration) in parallel. Dissociated cells were seeded in 96-well plates using both manual and automated liquid handling (Tecan Fluent 780). Due to the tendency of 3D cell models to aggregate and fall out of solution, initial seeding in a 96-well flat bottom plate resulted in high well-to-well variability. Optimization of cell seeding protocols led to standard deviations in tumoroid metabolic activity, number, and size that were comparable to manual seeding, with a coefficient of variation of less than 3% between wells for multiple tumoroid lines when automation was implemented. Generally, variability in cell seeding was lower when using the optimized, automated cell seeding protocol compared to manual methods. After seeding, cells were grown for three days to form tumoroids prior to the addition of compounds. At this time point, there was low well-to-well variability in tumoroid metabolic activity (CV<2%). Compounds and reagents to analyze cell health could also be added using automated liquid handling prior to analysis on plate readers or high-content imaging platforms. Citation Format: Colin Paul, Anthony Chatman, Amber Bullock, Xiaoyu Jenny Yang, Garrett Wong, Brittany Balhouse, Chris Yankaskas, Erik Willems, Matt Dallas, David Kuninger. Scale up and scale down approaches for screening of 3D patient-derived cell models [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 2047.