High-content Screening Platform Research Articles

Arvanil induces ferroptosis of hepatocellular carcinoma by binding to MICU1.

Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate that requires research and improved treatment strategies. Chemotherapy is still one of the main methods of HCC treatment, but it may lead to drug resistance and damage to normal organs. Capsaicin, a naturally occurring active ingredient in chili peppers, has demonstrated anticancer properties in a variety of malignant tumor cell lines. However, the anti-cancer mechanism of capsaicin needs to be further explored in HCC. In this study, we utilized Arvanil, a non-stimulating synthetic capsaicin analog, in place of capsaicin. We found that Arvanil induced high mitochondrial calcium flow, which contributed to a decrease in mitochondrial membrane permeability transition pore (mPTP) opening and oxidative phosphorylation levels, ultimately triggering cellular ferroptosis by live cells in real time with a high content screening (HCS) platform and confocal microscopy. It was further confirmed by vina molecular docking and point mutation experiments that Arvanil directly binds to two amino acid sites of mitochondrial calcium uptake protein 1 (MICU1), namely Ser47 and Phe128, to trigger this process, which in turn inhibits the growth of HCC cells. In addition, it was confirmed that Arvanil enhances cisplatin chemosensitivity by inducing HCC cellular ferroptosis in vivo. In conclusion, our study suggests that Arvanil induces ferroptosis in HCC cells and is a candidate drug for the treatment of HCC.

Invasion-Block and S-MARVEL: A high-content screening and image analysis platform identifies ATM kinase as a modulator of melanoma invasion and metastasis.

Robust high-throughput assays are crucial for the effective functioning of a drug discovery pipeline. Herein, we report the development of Invasion-Block, an automated high-content screening platform for measuring invadopodia-mediated matrix degradation as a readout for the invasive capacity of cancer cells. Combined with Smoothen-Mask and Reveal, a custom-designed, automated image analysis pipeline, this platform allowed us to evaluate melanoma cell invasion capacity posttreatment with two libraries of compounds comprising 3840 U.S. Food and Drug Administration (FDA)-approved drugs with well-characterized safety and bioavailability profiles in humans as well as a kinase inhibitor library comprising 210 biologically active compounds. We found that Abl/Src, PKC, PI3K, and Ataxia-telangiectasia mutated (ATM) kinase inhibitors significantly reduced melanoma cell invadopodia formation and cell invasion. Abrogation of ATM expression in melanoma cells via CRISPR-mediated gene knockout reduced 3D invasion in vitro as well as spontaneous lymph node metastasis in vivo. Together, this study established a rapid screening assay coupled with a customized image-analysis pipeline for the identification of antimetastatic drugs. Our study implicates that ATM may serve as a potent therapeutic target for the treatment of melanoma cell spread in patients.

#6189 PRECLINICAL VALIDATION OF REPURPOSABLE CANDIDATE DRUG COMPOUNDS FOR CYSTIC NEPHROPATHIES

Abstract Background and Aims Early-onset cystic kidney disease is a clinically and genetically heterogeneous and overlapping group of rare and often severe disorders with limited, mostly symptomatic treatment options. We developed and utilized inherited polycystic cystic kidney disease (PKD) models to search for pharmacological agents that can rescue or mitigate the disease phenotype. Method A high-content screening platform for identification of chemical modifiers of cyst formation and progression in a zebrafish genetic cystic nephropathy model (IFT172 Morpholino-based knockdown in Tg(wt1b:eGFP)), using robotic microscopy with automated image classification and phenotype quantification was used to screen 1280 compounds from the Prestwick library for their cyst-inhibiting activity. Compounds with cyst-inhibitory efficacy and low toxicity in the Zebrafish and oral and trans-placental availability were selected for further study in two rodent PKD models, i.e. PCK and PKD/Mhm (cy/+) rats. Groups of 3-week-old homozygous PCK and heterozygous PKD/Mhm rats were administered orally for 10 weeks three candidate compounds, which included an antihistamine, an anthelmintic and an antifungal agent, or regular ROD16 chow (sick controls). In experimental week 5, blood was taken, and the kidneys were imaged sonographically under anaesthesia. In the last experimental week, glomerular filtration rate was determined by transcutaneous FITC-sinistrin injection. Masson trichrome staining was used to evaluate cyst formation in kidneys. Results 60 compounds with cyst preventive or cyst size reducing activity were identified in the zebrafish screening model. While one third of these compounds caused severe general toxicity, 12 of them showed medium and 4 strong cyst-inhibitory activity with no or mild off-target effects. These substances were from anthelminthic, antihistamine, antifungal and proton pump inhibitor drug classes. Preliminary results in the rodent models suggest that anthelminthic treatment reduced the number of cysts in female (3.3±1.3 vs. untreated control, 5.4±0.9) but not male PCK rats (3.7±0.6 vs. 3.9±0.7) after 10 weeks of treatment. These changes were accompanied by improved glomerular filtration rate (GFR) in treated females (t½: 0.22±0.04 min vs. 0.3±0 min). An improvement in GFR was also observed in female PKD/Mhm rats after 10 weeks of treatment with the anthelmintic compound compared to untreated control animals (t½: 0.24±0.06 vs. 0.51±0.16 min, p = 0.02). Conclusion Candidate cyst-inhibitory drugs identified in a zebrafish model were applied in rat models of PKD. The administration of an anthelmintic agent had a positive impact on the progression of polycystic kidney disease. The beneficial effect appears to be sex specific. If further corroborated, our findings may allow repurposing of a drug approved for another indication to slow disease progression in patients with cystic kidney diseases.

An efficient cellular image-based platform for high-content screening of neuroprotective agents against chemotherapy-induced neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting side effect, with no approved therapy for prevention or treatment. Here, we aimed to establish a high-content image platform based on the neurite outgrowth of dorsal root ganglia (DRG)-derived neuron cells for the discovery of neuroprotective agents against paclitaxel-induced CIPN. ND7/23 cells, an immortalized hybrid DRG cell line, were maturely differentiated by induction with nerve growth factor and upregulation of intracellular cAMP levels. High-content image analyses of the neurofilament-stained neurite network showed that paclitaxel disrupted the neurite outgrowth of well-differentiated ND7/23 DRG neuron cells, recapitulating characteristic effects of paclitaxel on primary cultured DRG neurons. This process coincided with the upregulated activity of store-operated Ca2+ entry, similar to those found in rodent models of paclitaxel-induced CIPN. The previously identified neuroprotective agents, minoxidil and 8-Br-cyclic adenosine monophosphate ribose (8-Br-cADPR), attenuated the reduction in total neurite outgrowth in paclitaxel-damaged ND7/23 cells. Additionally, the total neurite outgrowth of well-differentiated ND7/23 cells was concentration-dependently reduced by the neurotoxic chemotherapeutic agents, oxaliplatin and bortezomib, but not the less neurotoxic 5-fluorouracil. We demonstrated that high-content analyses of neurite morphology in well-differentiated DRG neuron-derived cells provide an effective, reproducible, and high-throughput strategy for developing therapeutics against CIPN.

Abstract 1844: Development of a high content screening (HCS) platform for novel cancer immunotherapeutics

Abstract There has been dramatic progress in the understanding of fundamental causes of cancer, giving rise to novel treatments which harness the power of the immune system to combat cancer progression. Despite this, no two cancers have the same pathogenesis and there is significant variation in patient response rates. Understanding mechanisms of carcinogenesis in model cell culture systems, such as aberrant signaling, as well as how tumor immunity can be initiated, retargeted or reinvigorated upon incorporation of the immune system will fuel drug discovery. High-Content Screening (HCS) platforms allow single-cell imaging analysis and are used to interrogate these models to enable drug development and understand mechanisms of action. They provide an opportunity to test novel cancer therapeutics in a translationally relevant setting that integrates primary immune effector function. Here we outline several macrophage-based HCS assays which allow assessment of therapeutics targeting;1) early aspects of immune cell activation 2) macrophage mitochondrial health 3) macrophage activation and re-polarization 4) immune cell infiltration and tumor killing. To test efficacy of drugs targeting immune signaling or activation, macrophages were treated with a canonical NF-ƙB activator +/- TCPA-1, a known inhibitor for benchmarking as a model therapeutic. Robust NF-ƙB translocation was observed and reduced in the presence of TCPA-1 rendering this an appropriate assay to screen therapeutic cytokines or TLR agonists activating NF-ƙB signaling. A macrophage inflammasome activation assay was developed to analyze levels of ASC and speck formation. CRID3i, an established inflammasome inhibitor was validated as a robust control therapeutic that prevented NLRP3 activation suggesting this is a relevant platform to assess cancer drug efficacy. Interrogating mitochondrial health and dynamics in primary macrophages is vital to understanding the impact of accumulating ROS species or metabolites in the tumor microenvironment. Assays modelling the effects of these molecules on macrophage polarization and activation as well as mitochondrial integrity were used to test macrophage repolarizing therapeutics, a key approach in immuno-oncology. Finally, a complex multi-cellular 3D spheroid tumor killing assay was optimized to model immune cell infiltration following exposure to IO-targeted therapeutics and robust enhancement of tumor killing was directly observed. Overall, these assays demonstrate the power of imaging in performing HCS to assess the efficacy and mechanisms of action of a range of IO-targeted therapeutics. This suite of assays is applicable to IO drug screens targeting macrophage function, immune cell activation and immune cell infiltration. Citation Format: Rebecca Roberts, Henry Leonard, Ilona Aylott, Douglas Best, Dan Rocca, Ben Thompson, Nunan Robert, Louise Brackenbury. Development of a high content screening (HCS) platform for novel cancer immunotherapeutics [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 1844.

Automated high-content imaging in iPSC-derived neuronal progenitors.

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

High-content image-based CRISPR screening reveals regulators of 3D genome architectures.

The spatial organization of chromatin critically impacts many essential genomic functions, from the regulation of gene expression to the replication of the genome. Changes in chromatin organization are associated with aging and a wide range of diseases including cancer. Despite the fundamental importance of correct chromatin organization and its effects on cellular behavior, very little is known regarding the precise folding arrangement of chromatin in various cell states, nor the molecular mechanisms that control chromatin organization in the cell nucleus. Current approaches are severely limited by a lack of experimental tools to directly trace chromatin folding, and to efficiently screen for molecular regulators of chromatin organization in the genome. To address these needs, we previously developed an image-based 3D genomics technique termed chromatin tracing, which enables direct 3D tracing of chromatin folding along individual chromosomes in single cells. Recently, we further developed Multiplexed Imaging of Nucleome Architectures (MINA), which enables simultaneous measurements of multiscale chromatin folding, associations of genomic regions with nuclear lamina and nucleoli, and copy numbers of numerous RNA species in the same cells in mammalian tissue. Here we report our latest technology development - an image-based high-content CRISPR screening platform - to systematically uncover new regulators of 3D genome organization. We performed a pooled loss-of-function screen of hundreds of selected genes in human cells, identified target genes by inventing a cellular barcoding technique termed BARC-FISH, and visualized genome organization by chromatin tracing in the same single cells. Using 1.4 million imaged 3D-positions along chromosome traces, we identified tens of novel regulators of chromatin architectures at different length scales. Loss of the ATP-dependent helicase CHD7, which causes the congenital syndrome CHARGE, promotes multi-scale chromatin decompaction. This method enables scalable, high-throughput identification of chromatin topology regulators in diverse contexts.

U-IMPACT: a universal 3D microfluidic cell culture platform

The development of organs-on-a-chip has resulted in advances in the reconstruction of 3D cellular microenvironments. However, there remain limitations regarding applicability and manufacturability. Here, we present an injection-molded plastic array 3D universal culture platform (U-IMPACT) for various biological applications in a single platform, such as cocultures of various cell types, and spheroids (e.g., tumor spheroids, neurospheres) and tissues (e.g., microvessels). The U-IMPACT consists of three channels and a spheroid zone with a 96-well plate form factor. Specifically, organoids or spheroids (~500 μm) can be located in designated areas, while cell suspensions or cell-laden hydrogels can be selectively placed in three channels. For stable multichannel patterning, we developed a new patterning method based on capillary action, utilizing capillary channels and the native contact angle of the materials without any modification. We derived the optimal material hydrophilicity (contact angle of the body, 45–90°; substrate, <30°) for robust patterning through experiments and theoretical calculations. We demonstrated that the U-IMPACT can implement 3D tumor microenvironments for angiogenesis, vascularization, and tumor cell migration. Furthermore, we cultured neurospheres from induced neural stem cells. The U-IMPACT can serve as a multifunctional organ-on-a-chip platform for high-content and high-throughput screening.

Multicellular regulation of miR-196a-5p and miR-425-5 from adipose stem cell-derived exosomes and cardiac repair.

Cardiac transplantation of adipose-derived stem cells (ASC) modulates the post-myocardial infarction (post-MI) repair response. Biomolecules secreted or shuttled within extracellular vesicles, such as exosomes, may participate in the concerted response. We investigated the exosome's microRNAs due to their capacity to fine-tune gene expression, potentially affecting the multicellular repair response. We profiled and quantified rat ASC-exosome miRNAs and used bioinformatics to select uncharacterized miRNAs down-regulated in post-MI related to cardiac repair. We selected and validated miR-196a-5p and miR-425-5p as candidates for the concerted response in neonatal cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages using a high-content screening platform. Both miRNAs prevented cardiomyocyte ischemia-induced mitochondrial dysfunction and reactive oxygen species production, increased angiogenesis, and polarized macrophages toward the anti-inflammatory M2 immunophenotype. Moreover, miR-196a-5p reduced and reversed myofibroblast activation and decreased collagen expression. Our data provide evidence that the exosome-derived miR-196a-5p and miR-425-5p influence biological processes critical to the concerted multicellular repair response post-MI.

Fingerprinting Small Molecule Modulators of Nucleolar Biophysics

Ribosomal RNA (rRNA) is the most abundant RNA in cells, reflecting its fundamental role in enabling protein synthesis in all living organisms. Nucleolus is a membraneless and multicompartmentalized nuclear body that governs rRNA biogenesis, a process that is misregulated in various diseases. Recent research has identified several small molecule therapeutics that alter rRNA biogenesis and subsequently nucleolar morphology, challenging the accepted mechanisms of action for these compounds and simultaneously establishing the nucleolus as an attractive therapeutic target. Nevertheless, there are currently no comprehensive and quantitative frameworks that capture and relate various drug‐induced changes in nucleolar morphology and its material properties to its function. We hypothesized that we can establish such a structure‐function framework by screening a panel of nucleolus‐perturbing small molecule drugs and measuring changes in biophysical properties of the nucleolus in cells from microscopy images. Here, we combine morphometric, thermodynamic, and kinetic measurements of drug‐treated nucleoli to reveal “fingerprints” for drugs that modulate distinct stages of rRNA biogenesis. We anticipate that this framework provides a starting point for robustly determining and predicting structure‐function relationships for nucleolar phenotype perturbations as well as for developing high‐content screening platforms to expedite the discovery of novel nucleolus‐targeting therapeutics.

Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening.

Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.

Dual-color imaging of DNA and RNA simultaneously with an aggregation/monomer-based deep-red fluorescent probe

Since many of the pivotal questions confronting chemical biology concern the interaction of more than one analyte, it is essential to investigate multiple analytes simultaneously. While commercial fluorescent dyes capable of detecting and quantitating DNA or RNA are widely used in biological research, few probes can be applied in DNA and RNA together. Distraction from each other is also a puzzling question for individual detection. Here, we have discovered a dual-responsive probe, namely, DR1, which can selectively recognize DNA by emitting out red fluorescence in the nucleus, while an entirely different stain pattern by labeling cytoplasm and nuclei with distinct cyanine fluorescence is observed owing to binding with RNA. Mechanism study found that DR1 binds to DNA in a monomer state with red fluorescence, and therefore the cyanine fluorescence may derive from its aggregated state while it binds to RNA. Then, DR1 was applied to quantify DNA and RNA contents and evaluate the effects of several inhibitors comprehensively on high-content screening platforms. Our study provided a new tool for simultaneously monitoring cellular DNA and RNA, and gaining new insights into developing dual-channel probes for multiple biomolecules.

Discovery of Potential Neuroprotective Agents against Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is a common adverse effect of cancer treatment. No medication has been shown to be effective in the prevention or treatment of chemotherapy-induced neurotoxicity. Using minoxidil as an initial template for structural modifications in conjunction with an in vitro neurite outgrowth assay, an image-based high-content screening platform, and mouse behavior models, an effective neuroprotective agent CN016 was discovered. Our results showed that CN016 could inhibit paclitaxel-induced inflammatory responses and infiltration of immune cells into sensory neurons significantly. Thus, the suppression of proinflammatory factors elucidates, in part, the mechanism of action of CN016 on alleviating paclitaxel-induced peripheral neuropathy. Based on excellent efficacy in improving behavioral functions, high safety profiles (MTD > 500 mg/kg), and a large therapeutic window (MTD/MED > 50) in mice, CN016 might have great potential to become a peripherally neuroprotective agent to prevent neurotoxicity caused by chemotherapeutics as typified by paclitaxel.

Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics.

Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.

A versatile, automated and high-throughput drug screening platform for zebrafish embryos.

ABSTRACTZebrafish provide a unique opportunity for drug screening in living animals, with the fast-developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan® Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft® Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and X-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high-content screening in zebrafish. This article has an associated First Person interview with the first author of the paper.

BONE MARROW MESENCHYMAL STEM CELLS DERIVED FROM HEALTHY DONORS BUT NOT THOSE DERIVED FROM ACUTE MYELOID LEUKEMIA PATIENTS ARE ABLE TO AFFECT PROLIFERATION AND DEATH OF LEUKEMIC CELL LINEAGES K562 AND K562 LUCENA

<h3>Background</h3> In bone marrow (BM), mesenchymal stem cells (MSC) are a rare cell population. Beyond that, BM-MSCs play an important role in the maintenance of medullary niche and in supporting hematopoiesis. However, BM MSCs from patients with leukemia seems to participate in the disease progression and relapse. In this study we have evaluated in vitro some aspects of the influence of BM MSCs derived from healthy donors and acute myeloid leukemia (AML) patients on leukemic cell lines K562 (sensitive to vincristine) and K562 Lucena (multidrug resistance phenotype - MDR), as measured by the proliferation rates and death characteristics. <h3>Methods</h3> BM-MSCs were obtained from 3 healthy donors and 3 AML patients after written informed consent. K562 and K562-Lucena cell lines were co-cultured with BM-MSCs in a transwell system by 48 h. After incubation, leukemic cells (K562 and K562-Lucena) were evaluated for proliferation, viability, apoptosis, and necrosis by fluorescent methodology in a High-content Screening platform. <h3>Results</h3> Healthy BM-MSCs were shown to decrease proliferation of both K562 and K562-Lucena cell lines (37% and 42%, respectively) and viability (42% and 28%, respectively), while increased cell death rates through apoptosis (80% and 140%, respectively) and necrosis (28 fold and 32 fold, respectively) mechanisms, when compared with their respective controls. However, these effects were not observed when leukemic cells K562 and K562-Lucena were co-cultured with AML-BM-MSC. <h3>Conclusion</h3> BM-MSC derived from healthy donors decreased K562 and K562-Lucena cell lines proliferation and viability, while increased their apoptosis and necrosis rates. However, AML-BM-MSC did not affected proliferation, viability, and cell death rates of K562 and K562-Lucena cell lines. In conclusion, AML-BM-MSCs seem to lose the capacity to control proliferation and death capacity of leukemic cells. Information on ethical approval and funding support: This work was approved by Comitê de Ética em Pesquisa (CEP) of HC-FMUSP and supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Instituto Nacional de Ciência e Tecnologia – Fluidos Complexos (INCT-FCx); Instituto Nacional de Ciência e Tecnologia em Medicina Regenerativa (INCT-Regenera).

High-Content Analysis-Based Sensitivity Prediction and Novel Therapeutics Screening for c-Met-Addicted Glioblastoma.

Simple SummaryReal-time ex vivo drug testing tailors individual therapeutics based on predicted drug responses. Most technologies to date rely on conventional drug screening that provides low confidence data. Here, we present high-content analysis-based drug testing of glioblastoma patients to identify the right glioblastoma patients for a given drug. This generates multi-parameter biomarker and phenotype readouts providing a better reliability of the assay. Additionally, we showed a high-content drug repurposing screen and defined a new c-Met-inhibiting function of the CDK4/6 inhibitor Abemaciclib. Large-scale high throughput screening results demonstrate that Abemaciclib sensitivity in glioblastoma patients is highly correlated with the c-Met inhibitors sensitivity, further supporting the accuracy of the platform and important new clinical implications regarding multiple functions of Abemaciclib.(1) Background: Recent advances in precision oncology research rely on indicating specific genetic alterations associated with treatment sensitivity. Developing ex vivo systems to identify cancer patients who will respond to a specific drug remains important. (2) Methods: cells from 12 patients with glioblastoma were isolated, cultured, and subjected to high-content screening. Multi-parameter analyses assessed the c-Met level, cell viability, apoptosis, cell motility, and migration. A drug repurposing screen and large-scale drug sensitivity screening data across 59 cancer cell lines and patient-derived cells were obtained from 125 glioblastoma samples. (3) Results: High-content analysis of patient-derived cells provided robust and accurate drug responses to c-Met-targeted agents. Only the cells of one glioblastoma patient (PDC6) showed elevated c-Met level and high susceptibility to the c-Met inhibitors. Multi-parameter image analysis also reflected a decreased c-Met expression and reduced cell growth and motility by a c-Met-targeting antibody. In addition, a drug repurposing screen identified Abemaciclib as a distinct CDK4/6 inhibitor with a potent c-Met-inhibitory function. Consistent with this, we present large-scale drug sensitivity screening data showing that the Abemaciclib response correlates with the response to c-Met inhibitors. (4) Conclusions: Our study provides a new insight into high-content screening platforms supporting drug sensitivity prediction and novel therapeutics screening.

A multi-dimensional, time-lapse, high content screening platform applied to schistosomiasis drug discovery

Approximately 10% of the world’s population is at risk of schistosomiasis, a disease of poverty caused by the Schistosoma parasite. To facilitate drug discovery for this complex flatworm, we developed an automated high-content screen to quantify the multidimensional responses of Schistosoma mansoni post-infective larvae (somules) to chemical insult. We describe an integrated platform to process worms at scale, collect time-lapsed, bright-field images, segment highly variable and touching worms, and then store, visualize, and query dynamic phenotypes. To demonstrate the methodology, we treated somules with seven drugs that generated diverse responses and evaluated 45 static and kinetic response descriptors relative to concentration and time. For compound screening, we used the Mahalanobis distance to compare multidimensional phenotypic effects induced by 1323 approved drugs. Overall, we characterize both known anti-schistosomals and identify new bioactives. Apart from facilitating drug discovery, the multidimensional quantification provided by this platform will allow mapping of chemistry to phenotype.

Abstract 5697: Image based quantification of myeloid cell repolarization and their interplay with the tumor microenvironment

Abstract BACKGROUND The myeloid cell compartment plays an important role in anti-tumor immune responses and represents a heterogeneous population with both cancer-promoting and cancer-restraining actions. Unleashing the full potential of cancer immunotherapies requires an understanding of the cellular mechanisms that govern these opposite actions. We previously described a high-content screening platform to assess effects of immune modulators on the infiltration and killing of tumor organoids. Here, we incorporated the myeloid cell compartment to have a better representation of the human immune system in the tumor microenvironment (TME). In-house developed software was trained on a set of features that enabled discrimination between undifferentiated monocytes and polarized M1 and M2 macrophages and dendritic cells in 3D. This assay is well suitable to test cancer immunotherapies that target multiple cell types involved in anti-tumor immune responses and to understand in general the suppressive tumor environment. MATERIAL and METHODS Different myeloid populations were generated in 3D from monocytes derived from healthy donor PBMCs. Polarized M1 and M2 macrophages, DCs and undifferentiated monocytes were then co-cultured with either tumor conditioned media or spheroids derived from different cancer cell lines or colon rectal cancer organoids, growing in protein hydrogel. The cellular interactions were visualized using high-content microscopy and quantified with multiparametric morphometric analysis with OMinerTM software. RESULTS 3D imaging and phenotypic analysis was used to classify different myeloid cell populations. This classification was confirmed functionally by profiling of released cytokines. In addition, our analysis revealed the different effects of tumor cells and the immunosuppressive microenvironment that they generated on the myeloid cells phenotypes. We also used this approach to demonstrate a repolarisation of M2 type macrophages into M1 type macrophages upon treatment with CSF1R inhibitor and a preventive effect of this compound on macrophage skewing induced by conditioned media from tumor cells of different origin. CONCLUSIONS The assay presented here enables visualization and quantification of effects of immunotherapies on myeloid cells using 3D phenotypic analysis. This co-culture system provides means to elucidate the bi-directional interplay between tumor and immune cells, allowing for analysis of functional reprograming of the suppressive population towards a M1 phenotype induced by drug candidates. This advanced platform for testing cancer immunotherapies also combines the complexity of the TME with the robustness of a high throughput screening platform. Citation Format: Gera Goverse, Nataliia Beztsinna, Benjamin Visser, Emma Spanjaard, Kuan Yan, Leo Price, Lidia Daszkiewicz. Image based quantification of myeloid cell repolarization and their interplay with the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5697.