High-content Live-cell Imaging Research Articles

The phenol red compound: A potential artifact in pharmacological induction of ferroptosis

Phenol red (PR) is a commonly used compound in culture media as a pH indicator. However, it is unknown whether this compound can interfere with the pharmacological induction of ferroptosis. Here, using high-content live-cell imaging death analysis, we determined that the presence of PR in the culture medium preconditioned normal and tumor cells to ferroptosis induced by system xc− inhibition mediated by imidazole ketone erastin (IKE) or GPX4 blockade in response to RSL-3, but had no significant effects against treatment with the endoperoxide FINO2. Mechanistically, we revealed that PR decreases the levels of the antiferroptotic genes Slc7a11, Slc3a2, and Gpx4, while promoting the overexpression de Acls4, a key inducer of ferroptosis. Additionally, through superresolution analysis, we determined that the presence of PR mislocalizes the system xc− from the plasma membrane. Thus, our results show that the presence of PR in the culture medium can be a problematic artifact for the accurate interpretation of cell sensitivity to IKE or RSL-3-mediated ferroptosis induction.

Computational label-free microscope through a custom-built high-throughput objective lens and Fourier ptychography

Label-free microscopy directly images live samples without fluorescent markers, providing a panoramic view of biological structures and functions. However, pursuing high-throughput and high-content live cell imaging requires both higher spatial resolution and larger field of view, which are limited by the optical system. In this study, we custom-built a 5▪/0.35 objective with a field number of 28 mm, providing a space-bandwidth product of 34 megapixels, which is four times greater than that of commercial high-throughput objectives such as the Nikon Lambda series (4▪/0.2, 10▪/0.45, 20▪/0.75). Furthermore, we incorporated Fourier ptychography microscopy (FPM) into the system, achieving a synthetic numerical aperture of 0.72, suitable for digital pathology and long-term live cell observation. To improve the FPM performance, we proposed a phantom-based calibration method for quantitative correction of illumination angle errors. Additionally, this method can also serve as an initial step for the on-line calibration. The remarkable capabilities of our 5▪/0.35 objective have been demonstrated, as well as the effectiveness of computational label-free microscopy. Furthermore, by combining our original FPM with more advanced hardware and algorithms, our FPM can achieve higher performance.

Multimodal assessment of mitochondrial function in Parkinson's disease.

The heterogenous aetiology of Parkinson's disease is increasingly recognized; both mitochondrial and lysosomal dysfunction have been implicated. Powerful, clinically applicable tools are required to enable mechanistic stratification for future precision medicine approaches. The aim of this study was to characterize bioenergetic dysfunction in Parkinson's disease by applying a multimodal approach, combining standardized clinical assessment with midbrain and putaminal 31-phosphorus magnetic resonance spectroscopy (31P-MRS) and deep phenotyping of mitochondrial and lysosomal function in peripheral tissue in patients with recent-onset Parkinson's disease and control subjects. Sixty participants (35 patients with Parkinson's disease and 25 healthy controls) underwent 31P-MRS for quantification of energy-rich metabolites [ATP, inorganic phosphate (Pi) and phosphocreatine] in putamen and midbrain. In parallel, skin biopsies were obtained from all research participants to establish fibroblast cell lines for subsequent quantification of total intracellular ATP and mitochondrial membrane potential (MMP) as well as mitochondrial and lysosomal morphology, using high content live cell imaging. Lower MMP correlated with higher intracellular ATP (r = -0.55, P = 0.0016), higher mitochondrial counts (r = -0.72, P < 0.0001) and higher lysosomal counts (r = -0.62, P = 0.0002) in Parkinson's disease patient-derived fibroblasts only, consistent with impaired mitophagy and mitochondrial uncoupling. 31P-MRS-derived posterior putaminal Pi/ATP ratio variance was considerably greater in Parkinson's disease than in healthy controls (F-tests, P = 0.0036). Furthermore, elevated 31P-MRS-derived putaminal, but not midbrain Pi/ATP ratios (indicative of impaired oxidative phosphorylation) correlated with both greater mitochondrial (r = 0.37, P = 0.0319) and lysosomal counts (r = 0.48, P = 0.0044) as well as lower MMP in both short (r = -0.52, P = 0.0016) and long (r = -0.47, P = 0.0052) mitochondria in Parkinson's disease. Higher 31P-MRS midbrain phosphocreatine correlated with greater risk of rapid disease progression (r = 0.47, P = 0.0384). Our data suggest that impaired oxidative phosphorylation in the striatal dopaminergic nerve terminals exceeds mitochondrial dysfunction in the midbrain of patients with early Parkinson's disease. Our data further support the hypothesis of a prominent link between impaired mitophagy and impaired striatal energy homeostasis as a key event in early Parkinson's disease.

A machine learning and live-cell imaging tool kit uncovers small molecules induced phospholipidosis

Drug-induced phospholipidosis (DIPL), characterized by excessive accumulation of phospholipids in lysosomes, can lead to clinical adverse effects. It may also alter phenotypic responses in functional studies using chemical probes. Therefore, robust methods are needed to predict and quantify phospholipidosis (PL) early in drug discovery and in chemical probe characterization. Here, we present a versatile high-content live-cell imaging approach, which was used to evaluate a chemogenomic and a lysosomal modulation library. We trained and evaluated several machine learning models using the most comprehensive set of publicly available compounds and interpreted the best model using SHapley Additive exPlanations (SHAP). Analysis of high-quality chemical probes extracted from the Chemical Probes Portal using our algorithm revealed that closely related molecules, such as chemical probes and their matched negative controls can differ in their ability to induce PL, highlighting the importance of identifying PL for robust target validation in chemical biology.

Single-cell imaging and transcriptomic analyses of firm adhesion between patient-derived cancer and endothelial cells under shear stress

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is a key event during tumour progression and metastasis formation. However, the analysis of its underlying cellular and molecular mechanisms is largely limited by the intrinsic difficulties to study the interactions between circulating cancer cells and endothelial cells in vivo, and in vitro under conditions that mimic the in vivo blood flow. Here, we developed a method to study cell:cell firm adhesion under shear-stress conditions coupled to high-content live-cell imaging, and single-cell RNAseq analysis. As the model system, we used cancer cells freshly isolated from patient-derived xenografts (PDXs) and human primary endothelial cells. Breast cancer is the most common cancer in women worldwide and the leading cause of cancer-related deaths among women. Therefore, we set up protocols for breast cancer PDX tumour dissociation, isolation and purification to obtain freshly isolated PDX-derived human cancer single cell suspension. We then implemented an in vitro assay to study cancer to endothelial cells firm adhesion under shear-stress, using an all–human microfluidic model coupled to time-lapse and live-cell imaging. Finally, we developed a method to successfully retrieve, separate and enrich alive endothelial and cancer cells from the flow-based firm adhesion assay. Most notably, we used retrieved cells for single-cell RNAseq analysis and showed that samples quality, number of cells and transcripts per cell were consistent and optimal for downstream discovery analyses. In conclusion, we developed a workflow method that can provide insights into the mechanisms of cancer adhesion to endothelial cells, and identify new targets for personalized treatments development for the clinic to prevent and/or treat breast cancer metastasis formation.

Viability Analysis and High-Content Live-Cell Imaging for Drug Testing in Prostate Cancer Xenograft-Derived Organoids

Tumor organoids have been pushed forward as advanced model systems for in vitro oncology drug testing, with the eventual goal to direct personalized cancer treatments. However, drug testing efforts suffer from a large variation in experimental conditions for organoid culturing and organoid treatment. Moreover, most drug tests are restricted to whole-well viability as the sole read-out, thereby losing important information about key biological aspects that might be impacted due to the use of administered drugs. These bulk read-outs also discard potential inter-organoid heterogeneity in drug responses. To tackle these issues, we developed a systematic approach for processing organoids from prostate cancer (PCa) patient-derived xenografts (PDXs) for viability-based drug testing and identified essential conditions and quality checks for consistent results. In addition, we generated an imaging-based drug testing procedure using high-content fluorescence microscopy in living PCa organoids to detect various modalities of cell death. Individual organoids and cell nuclei in organoids were segmented and quantified using a dye combination of Hoechst 33342, propidium iodide and Caspase 3/7 Green, allowing the identification of cytostatic and cytotoxic treatment effects. Our procedures provide important insights into the mechanistic actions of tested drugs. Moreover, these methods can be adapted for tumor organoids originating from other cancer types to increase organoid-based drug test validity, and ultimately, accelerate clinical implementation.

Bioactive Constituents of Verbena officinalis Alleviate Inflammation and Enhance Killing Efficiency of Natural Killer Cells.

Natural killer (NK) cells play key roles in eliminating pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine for its anti-tumor and anti-inflammatory activities, but its effects on immune responses remain largely elusive. This study aimed to investigate the potential of V. officinalis extract (VO extract) to regulate inflammation and NK cell functions. We examined the effects of VO extract on lung injury in a mouse model of influenza virus infection. We also investigated the impact of five bioactive components of VO extract on NK killing functions using primary human NK cells. Our results showed that oral administration of VO extract reduced lung injury, promoted the maturation and activation of NK cells in the lung, and decreased the levels of inflammatory cytokines (IL-6, TNF-α and IL-1β) in the serum. Among five bioactive components of VO extract, Verbenalin significantly enhanced NK killing efficiency in vitro, as determined by real-time killing assays based on plate-reader or high-content live-cell imaging in 3D using primary human NK cells. Further investigation showed that treatment of Verbenalin accelerated the killing process by reducing the contact time of NK cells with their target cells without affecting NK cell proliferation, expression of cytotoxic proteins, or lytic granule degranulation. Together, our findings suggest that VO extract has a satisfactory anti-inflammatory effect against viral infection in vivo, and regulates the activation, maturation, and killing functions of NK cells. Verbenalin from V. officinalis enhances NK killing efficiency, suggesting its potential as a promising therapeutic to fight viral infection.

Abstract 634: The impact of IL-4 on migration in triple negative breast cancer cell lines

Abstract In the United States, triple-negative breast cancer (TNBC), an aggressive form of breast cancer (BCa), disproportionately affects women of African descent and contributes to racial disparities in BCa mortality. Various risk factors are thought to drive this disparity, including socioeconomic status, tumor characteristics, comorbidities, and quality of treatment. Complex diseases and environmental insults all invoke and/or disrupt host inflammation, with individual immune and variable responses to therapeutic agents being dictated in part by genetic variants. This latter has seldom been studied with regards to precision diagnosis and prognosis of individual patient responses, in the frame of geographical ancestry and TNBC. Interleukin-4 receptors (IL4R) have been shown to be upregulated in many epithelial cancers and could be a promising target for metastatic tumor therapy. Yet, IL4R overexpression and/or IL4R variants have not been evaluated in TNBC cell lines of varying genetic ancestry. In view of this, we investigated the effects of IL-4 treatment in four TNBC cell lines varying in proportions of West African Ancestry (WAA) in each cell line: MDA-MB-231 (0.019), HCC-1143 (0.012), HCC-1806 (0.807), MDA-MB-468 (0.803). The frequency of IL4R variants also differed in each cell line. We used high content live cell imaging to evaluate the effect of IL-4 treatment on cellular migration by measuring wound healing. We show that IL-4 treatment on TNBC cells enhances migration as indicated by increased wound healing when compared to non-treated cells. High glucose, at a concentration of 10 g/L, delays cell migration in IL4-treated TNBC cells. This data suggests that comorbidities, such as high glucose levels (i.e., uncontrolled diabetes) could impact tumor behavior and mortality disparities between populations. Citation Format: Portia Andrews, Kevin S. Kimbro. The impact of IL-4 on migration in triple negative breast cancer cell lines [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 634.

Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction

Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.

118 (PB108) - Standardization of viability assays and high-content live-cell imaging protocols for large-scale drug testing in prostate cancer PDX-derived organoids

Background: Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, standardized drug testing procedures are generally lacking, introducing experimental variability that complicates reproducibility, both within and between institutions. Moreover, common viability assays are restricted to endpoint measurements and viability as the sole read-out, losing important information on key biological aspects that might be exclusive to 3D organoids and that may be impacted by the administered drugs.

In-cytoplasm mitochondrial transplantation for mesenchymal stem cells engineering and tissue regeneration.

Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly “tuned” for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose‐derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high‐content live‐cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5′‐triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs, which are mainly linked to signaling pathways associated with DNA replication and cell division. We hypothesize that increase in ATP and cyclin‐dependent kinase 1 and 2 expression might be responsible for promoting enhanced proliferation, migration, and differentiation of ADSCs in vitro. More importantly, mito‐transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full‐thickness skin defect rat model with improved skin repair compared with nontreated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.

Human amyotrophic lateral sclerosis excitability phenotype screen: Target discovery and validation.

SUMMARYDrug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders.

Scale-Up Engineering Super-Energetic Adipose-Derived Mesenchymal Stem Cells with Mitochondrial Transplantation for Tissue Regeneration

Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly “tuned” for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose-derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high-content live-cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5′-triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage, and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs which are mainly involved in signaling pathways related to DNA replication and cell cycle. We propose that increase in ATP and cyclin-dependent kinase (CDK) 1 and 2 expression might be responsible for promoting enhanced proliferation, migration and differentiation of ADSCs in vitro. More importantly, mito-transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full-thickness skin defect rat model with improved skin repair compared with non-treated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.

Abstract B116: Evaluating the ability of clinically approved chemotherapeutics to decrease race-associated breast cancer mortality disparities via targeting the tumor-promoting effect of Crystallin Beta B2

Abstract In the United States, breast cancer (BC) remains the second leading cause of cancer death in women. The unequal burden in the mortality of BC in African American (AA) women compared to Caucasian American (CA) women goes without full explanation. When socioeconomic factors such as education and income are accounted for, this mortality disparity persists. Therefore, to acquire an understanding of how to end BC disparities, it is crucial to examine the underlying cellular and molecular mechanisms driving this disproportionate outcome. Studies on gene expression analyses have identified a particular gene, CRYβB2, with higher expression in AA breast tumors. Expression of CRYβB2 was also found to be increased in studies comparing AA and CA colorectal, renal, glioblastoma, and prostate cancers, and has been linked with decreased survival. Although the precise mechanisms of elevated CRYβB2 expression remain unknown, overexpression is significantly associated with BC tumor promotion. In view of this, we investigated the biological effects of CRYβB2 in basal-like, triple negative breast cancer (TNBC) cell lines. CRYβB2 expression was manipulated via CRIPSR/Cas9-mediated knockout and lentiviral overexpression systems. Our preliminary data show enhanced proliferation and invasion in 3D cultures with cells overexpressing CRYβB2 compared to controls, suggesting two mechanisms by which CRYβB2-overexpression may enhance tumor promotion and promote poor patient outcome. To identify drugs that may specifically target cells overexpressing CRYβB2, we subjected one of our CRYβB2 model systems (SUM159 cells overexpressing CRYβB2, with CRYβB2 knocked out, and the parental SUM159 cells with low CRYβB2 expression) to increasing doses of 125 clinically approved chemotherapeutics. High content live cell imaging was used to evaluate the effects of the drugs by measuring 3D spheroid BC cell growth, and IC50 were generated to determine whether cells with altered CRYβB2 expression were more or less sensitive to particular classes of drugs. Drugs identified with increased cytotoxicity to cells overexpressing CRYβB2 were confirmed using two additional TNBC models with altered CRYβB2 levels. Additional in vitro mechanistic studies evaluating the effect of the drugs on proliferation, cytotoxicity and invasion of CRYβB2-overexpressing were executed in 3D cultures with live cell imaging. Drugs that significantly affect the proliferation or invasion of CRYβB2-overexpressing cells will be evaluated in future studies for efficacy and toxicity in conjunction with other chemotherapeutics via in vivo AA-specific xenografts and PDX models. This information will be used to explore the potential use of these chemotherapeutics for neoadjuvant or adjuvant therapy in BC patients with tumors that overexpress CRYβB2. The identification of clinically approved drugs that inhibit CRYβB2 function may quickly be applied in the clinic to increase survival rates until more effective and less toxic, targeted-therapies are developed. Citation Format: Portia L. Andrews, Denise K. Reaves, Megan E. Martin, Michael Tarpley, Kevin P. Williams, Jodie M. Fleming. Evaluating the ability of clinically approved chemotherapeutics to decrease race-associated breast cancer mortality disparities via targeting the tumor-promoting effect of Crystallin Beta B2 [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B116.

Human Amyotrophic Lateral Sclerosis Excitability Phenotype Screen: Target Discovery and Validation

Drug development is hampered by poor target selection. Phenotypic screens using cells differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we designed a multi-step screening funnel using patient-derived motor neurons. High content live cell imaging was used to evaluate neuronal excitability, and from a chemogenomic library of 2899 target-annotated compounds, 67 reduced hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identified thirteen targets that modulate motor neuron excitability, including two known ALS excitability modulators; AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identified D2 dopamine receptors as contributing to ALS motor neuron excitability, a novel discovery. This screen demonstrates the power of human disease cell based phenotypic screens for identifying clinically relevant targets for neurological disorders.

Single-Cell and Population-Level Analyses Using Real-Time Kinetic Labeling Couples Proliferation and Cell Death Mechanisms.

Quantifying cytostatic and cytotoxic outcomes are integral components of characterizing perturbagens used as research tools and in drug discovery pipelines. Furthermore, data-rich acquisition, coupled with robust methods for analysis, is required to properly assess the function and impact of these perturbagens. Here, we present a detailed and versatile method for single-cell and population-level analyses using real-time kinetic labeling (SPARKL). SPARKL integrates high-content live-cell imaging with automated detection and analysis of fluorescent reporters of cell death. We outline several examples of zero-handling, non-disruptive protocols for detailing cell death mechanisms and proliferation profiles. Additionally, we suggest several methods for mathematically analyzing these data to best utilize the collected kinetic data. Compared to traditional methods of detection and analysis, SPARKL is more sensitive, accurate, and high throughput while substantially eliminating sample processing and providing richer data.

Abstract 4977: Dynamic transcriptional programs controlling single NK cell killing dynamics

Abstract NK cells have a vast heterogeneity in function and phenotype in vivo and their healthy function is critical in the prevention of nascent cancers. NK cell-based immunotherapies, such as adoptive NK cell therapy, are emerging and greater insights into the mechanism of NK cell activation could increase their efficacy. In vitro cytotoxicity assays have revealed that a small subset of NK cells kill the majority of cancer target cells, yet little is known about the dynamic state or triggers of these highly cytotoxic cells. Here, we present methodology for discovery of the molecular precursors and drivers of single NK cell killing events. We study the NK-92 cell line both as a model of primary NK cells and to gain direct insights for improving NK-92 based adoptive cell therapies. In our method, we first performed single cell RNA sequencing on NK-92 cells seeded sparsely on a monolayer of HeLa cancer cells along with a control NK-92 cell population. Clustering cells by principal component analysis revealed groups of NK cells that upregulated genes associated with NK cell cytotoxicity in the HeLa cell activated sample that are absent in the control group. Landmark genes associated with the cytotoxic NK cell clusters included PRF1, GZMK, and ITGB2. Utilizing live cell imaging, we identified categories of dynamic functional phenotypes in NK-92 cells exposed to a HeLa cell monolayer including non-motile and non-killing, highly motile but non-killing, single killing, serial killing, and exhausted. In order to link the systems-level single cell gene expression data to functional NK cell behaviors, we next performed high content live cell imaging of NK-92 cells bearing cas9-based endogenous genetic reporters as they surveilled and killed HeLa cells. Utilizing a custom analysis algorithm, we tracked the motility and killing activity of individual NK-92 cells, and correlated this activity to the dynamic gene expression. Next, we aim to identify the causality and sequence of transcriptional changes associated with NK cell killing and extend our results to the analysis of the critical transcription factor networks governing dynamic NK cell functional phenotypes. We note that the single cell functional discovery strategy that we have shown can be applied more broadly to other model systems of the cancer microenvironment. Citation Format: Matthew S. Hall, Joseph T. Decker, Emanuelle I. Grody, Rachel B. Blaisdell, Jacqueline S. Jeruss, Lonnie D. Shea. Dynamic transcriptional programs controlling single NK cell killing dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4977.

Real-Time Integration of Cell Death and Proliferation Kinetics at the Single-Cell and Population-Level Using High-Throughput Live-Cell Imaging

Quantifying cytostatic and cytotoxic outcomes are integral components of characterizing perturbagens used as research tools and/or in drug discovery pipelines. Furthermore, data-rich acquisition coupled with robust methods for analysis is required to properly assess the function and impact of these perturbagens. Here, we present a detailed and versatile method for Single-cell and Population-level Analyses using Real-time Kinetic Labeling (SPARKL). SPARKL integrates high-content live-cell imaging with automated detection and analysis of fluorescent reporters of cell death. We outline several examples of zero-handling, non-disruptive protocols for detailing cell death mechanisms and proliferation profiles. Additionally, we suggest several methods for mathematically analyzing these data to best utilize the collected kinetic data. Compared to traditional methods of detection and analysis, SPARKL is highly sensitive, accurate, and extremely high-throughput while substantially eliminating sample processing and providing richer data.

Abstract 5559: Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy

Abstract Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely due to the fact that these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the fact that the ECDs are in opposite orientation. Here we report the generation of a two-sided fusion protein incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain. Initial confirmation of the structural integrity of TIM3-Fc-OX40L was performed using functional ELISA assays, wherein simultaneous binding to GAL9 and OX40 was observed. A potential advantage of utilizing the intact ECD of TIM3 is the ability to bind all candidate TIM3 ligands, and the TIM3 end of the fusion protein binds both GAL9 and phosphatidylserine (PS) on the surface of human tumor cells. The OX40L end of the fusion protein binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in cells engineered to overexpress OX40 and an NFκB-luciferase reporter in the absence of Fc receptor cross-linking. Additionally, the TIM3-Fc-OX40L ARC added to primary human PBMCs along with the super-antigen Staphylococcal enterotoxin B, induced secretion of the cytokines IL2 and TNFα. In vitro human T cell/tumor cell co-culture studies indicated that TIM3-Fc-OX40L stimulated enhanced T cell mediated killing of GAL9 positive human tumor cells as compared to OX40L-Fc controls. In vivo, TIM3-Fc-OX40L stimulated significant expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells and vaccinated with Ova/Alum. Time-lapse high content live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L functionally tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was significantly superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. Importantly, a pharmacodynamic biomarker of tumor rejection was identified by the elevation in serum cytokines post-treatment. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is uniquely advantageous because the construct links those two signals in the same microenvironmental context, at the time in which T cells are engaging cognate tumor antigen. Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5559.

Abstract 5567: Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy

Abstract Current attempts at combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule. This is likely because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. SIRPα-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of SIRPα (CD172a) and the ECD of CD40L, adjoined by a central Fc domain. The SIRPα end of the ARC binds immobilized CD47 at 3.59 nM affinity and binds CD47 on the surface of human tumor cells both in vitro and in vivo, but does not bind human platelets or RBCs. Further, no hemolytic activity was observed with the human SIRPα-Fc-CD40L ARC in vitro as compared to CD47 mAbs. The CD40L end of the ARC binds immobilized CD40 at 756 pM affinity and binds CD40 on primary macrophages. The SIRPα-Fc-CD40L ARC stimulates functional activity (independent of Fc receptor cross-linking) in NFκB-luciferase reporter cells (CD40 driven activation of NFκB). Addition of SIRPα-Fc-CD40L to an ex vivo super-antigen (SEB) cytokine release assay stimulated secretion of IL2 and TNFα from human PBMC. Furthermore, time-lapse high content live cell imaging has shown that when activated human macrophages were co-cultured with CD47 positive human tumor cells, SIRPα -Fc-CD40L was shown to enhance phagocytosis. Finally, the therapeutic activity of SIRPα-Fc-CD40L in established murine MC38 and CT26 tumors was superior to either CD47 blocking antibody, CD40 agonist antibody or combination antibody therapy. Notably, the superior anti-tumor immunity observed with SIRPα-Fc-CD40L was not accompanied by significant mucosal inflammation and weight loss stimulated by murine CD40 agonist antibodies. Finally, treatment of cynomolgus macaques with SIRPα-Fc-CD40L was safe, and no evidence of hemolysis or thrombocytopenia was observed. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule. Signal replacement of CD47 by CD40L may uniquely poise macrophages in the tumor microenvironment for activation and cross-presentation of tumor antigens following enhanced tumor cell phagocytosis. Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist redirected checkpoint (ARC), SIRPα-Fc-CD40L, for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5567.