Flow Cytometry-based Methods Research Articles

In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods

Although cellular senescence was originally defined as an irreversible form of cell cycle arrest, in therapy-induced senescence models, the emergence of proliferative senescence-escaped cancer cells has been reported by several groups, challenging the definition of senescence. Indeed, senescence-escaped cancer cells may contribute to resistance to cancer treatment. Here, to study senescence escape and isolate senescence-escaped cells, we developed novel flow cytometry-based methods using the proliferation marker Ki-67 and CellTrace CFSE live-staining. We investigated the role of a novel senescence marker (DPP4/CD26) and a senolytic drug (azithromycin) on the senescence-escaping ability of MCF-7 and MDA-MB-231 breast cancer cells. Our results show that the expression of DPP4/CD26 is significantly increased in both senescent MCF-7 and MDA-MB-231 cells. While not essential for senescence induction, DPP4/CD26 contributed to promoting senescence escape in MCF-7 cells but not in MDA-MB-231 cells. Our results also confirmed the potential senolytic effect of azithromycin in senescent cancer cells. Importantly, the combination of azithromycin and a DPP4 inhibitor (sitagliptin) demonstrated a synergistic effect in senescent MCF-7 cells and reduced the number of senescence-escaped cells. Although further research is needed, our results and novel methods could contribute to the investigation of the mechanisms of senescence escape and the identification of potential therapeutic targets. Indeed, DPP4/CD26 could be a promising marker and a novel target to potentially decrease senescence escape in cancer.

Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry.

Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.

Deciphering metabolic crosstalk in context: lessons from inflammatory diseases.

Metabolism plays a crucial role in regulating the function of immune cells in both health and disease, with altered metabolism contributing to the pathogenesis of cancer and many inflammatory diseases. The local microenvironment has a profound impact on the metabolism of immune cells. Therefore, immunological and metabolic heterogeneity as well as the spatial organization of cells in tissues should be taken into account when studying immunometabolism. Here, we highlight challenges of investigating metabolic communication. Additionally, we review the capabilities and limitations of current technologies for studying metabolism in inflamed microenvironments, including single-cell omics techniques, flow cytometry-based methods (Met-Flow, single-cell energetic metabolism by profiling translation inhibition (SCENITH)), cytometry by time of flight (CyTOF), cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), and mass spectrometry imaging. Considering the importance of metabolism in regulating immune cells in diseased states, we also discuss the applications of metabolomics in clinical research, as well as some hurdles to overcome to implement these techniques in standard clinical practice. Finally, we provide a flowchart to assist scientists in designing effective strategies to unravel immunometabolism in disease-relevant contexts.

Universal ddPCR-based assay for the determination of lentivirus infectious titer and lenti-modified cell vector copy number

The translation of cell-based therapies from research to clinical setting requires robust analytical methods that successfully adhere to current good manufacturing practices and regulatory guidelines. Lentiviral vectors are commonly used for gene delivery to generate genetically modified therapeutic cell products. For some cell therapy products, standardized characterization assays for potency and safety have gained momentum. Translational applications benefit from assays that can be deployed broadly, such as for lentiviral vectors with various transgenes of interest. Development of a universal method to determine lentivirus infectious titer and vector copy number (VCN) of lenti-modified cells was performed using droplet digital PCR (ddPCR). Established methods relied on a ubiquitous lenti-specific target and a housekeeping gene that demonstrated comparability among flow cytometry-based methods. A linearized plasmid control was used to determine assay linearity/range, sensitivity, accuracy, and limits of quantification. Implementing this assay, infectious titer was assessed for various production runs that demonstrated comparability to the flow cytometry titer. The ddPCR assay described here also indicates suitability in the determination of VCN for genetically modified CAR-T cell products. Overall, the development of these universal assays supports the implementation of standardized characterization methods for quality control.

Staining of activated ß2-integrins in combination with CD137 and CD154 for sensitive identification of functional antigen-specific CD4+ and CD8+ T cells.

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations.

Evaluation of the antitumor activity of moronecidin (Piscidin)-like peptide in combination with anti-PD-1 antibody against melanoma tumor.

Immunotherapy has changed the landscape of oncology over the last decade and has become a standard of care for various cancers. Researchers previously demonstrated that B16-F10 melanoma in C57Bl6 mice is resistant to immune checkpoint inhibitors. The goal of this study was to investigate how anti-PD1 antibodies functioned in combination with a new antimicrobial peptide (AMP) called moronecidin-like peptide (MLP). We studied the cytotoxic effect of AMP on the B10-F16 tumor cell line with the MTT experiment. The necrotic and apoptotic cells were determined by Presidium iodide (PI) /Annexin V staining and flow cytometry-based methods. Mice were inoculated subcutaneously with B10-F16 tumor cells in the mammary gland. Each group was sacrificed two weeks after the last injection to examine tumor-specific CD8+ T cell responses using flow cytometry. Annexin V and PI staining assay revealed that MPL significantly induces apoptosis in B16F10 cells. It should be noted that MLP in combination with anti-PD-1 improved antigen-specific T-cell responses synergistically (P=0.01) when compared with respective monotherapy. Furthermore, when compared with the respective monotherapies, combination therapy significantly controlled tumor growth in B10-F16 tumor cells and increased survival rate. Treatments with anti-PD-1 inhibitors alone had only a minor effect on tumor size, whereas combination therapy resulted in significant tumor growth control and increased animal survival. MLP therapy combined with anti-PD-1 antibody improves anti-tumor immune response in addition to inducing tumor cell apoptosis. As a result, the evidence suggests that intratumoral injection of MPL can improve anti-PD-1 antibody antitumor response.

Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through pattern recognition receptor (PPR) interaction with pathogen-associated molecular patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition and phagocytosis by neutrophils. Therefore, phagocytosis assays performed using neutrophils in whole blood, compared to isolated cells, will differ due to the presence of opsonizing blood serum components, as well as other blood components like platelets. Powerful and sensitive flow cytometry-based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

Abstract B002: A CRISPR/Cas9 screening strategy for identifying modifiers of the viral mimicry response

Abstract Viral mimicry is the induction of a cellular antiviral response triggered by endogenous nucleic acids rather than exogenous viral infection. Such nucleic acids are sensed by cytosolic pattern recognition receptors and interpreted by the cell as an infection, leading to activation of an antiviral signaling cascade that results in interferon (IFN) signaling and subsequent upregulation of interferon-stimulated genes (ISGs). While downstream effects of ISGs are diverse and vary from direct induction of apoptosis to adaptive immune system engagement, the end result of viral mimicry is the culling of the ‘infected’ cancer cell. The viral mimicry response was first described as a mechanism of the FDA-approved DNA demethylating agents azacytidine (AZA) and decitabine (DAC). By removing transcriptionally repressive DNA methylation, these compounds allow normally-silenced repetitive elements of the genome to be expressed as double-stranded RNA (dsRNA) species that trigger viral mimicry. Since then, a body of literature has emerged implicating viral mimicry as a common mechanism underlying many different therapies and drug targets, including histone modifiers (e.g., LSD1, EZH2) as well as non-epigenetic targets such as cell cycle regulators (e.g., CDK4/6). The diversity of viral mimicry-inducing targets highlights the need for a systematic and discovery-based method to identify novel drug targets. Here, we accomplish this through the development of a viral mimicry cell reporter system composed of a green fluorescent protein (GFP) reporter coupled to the interferon-stimulated response element (ISRE) for CRISPR/Cas9-based pooled library screening. Using ISRE stimulation and subsequent GFP expression as a readout for viral mimicry activation, flow cytometry-based methods can be used to quantify the population of cells undergoing viral mimicry. Exogenous treatments of type I IFN or the synthetic dsRNA analogue polyinosinic-polycytidylic acid in the absence or presence of the JAK/STAT inhibitor ruxolitinib were used in conjunction with flow cytometry to validate the robustness and specificity of the reporter. To date, two preliminary screens in colorectal and lung cancer cell lines (LIM1215 and A549, respectively) have been performed using this system with a pooled single guide RNA library targeting epigenetic modifiers. These proof-of-principle experiments revealed two candidate hits – the histone methyltransferase SETDB1 and the lysine demethylase KDM5C. Follow-up validation experiments confirm GFP expression and viral mimicry activation in cells with single-gene knockouts for SETDB1 and KDM5C and demonstrate enhanced activation in combination with decitabine, as well as provide rationale for further study of these hits as therapeutic targets. Our work thus far demonstrates the feasibility of our approach and indicates the utility of our system as a novel tool for studying viral mimicry biology. Citation Format: Raymond Chen, Ilias Ettayebi, Daniel D. De Carvalho. A CRISPR/Cas9 screening strategy for identifying modifiers of the viral mimicry response. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B002.

On the Relevance of Soft Tissue Sarcomas Metabolic Landscape Mapping.

Soft tissue sarcomas (STS) prognosis is disappointing, with current treatment strategies being based on a “fit for all” principle and not taking distinct sarcoma subtypes specificities and genetic/metabolic differences into consideration. The paucity of precision therapies in STS reflects the shortage of studies that seek to decipher the sarcomagenesis mechanisms. There is an urge to improve STS diagnosis precision, refine STS classification criteria, and increase the capability of identifying STS prognostic biomarkers. Single-omics and multi-omics studies may play a key role on decodifying sarcomagenesis. Metabolomics provides a singular insight, either as a single-omics approach or as part of a multi-omics strategy, into the metabolic adaptations that support sarcomagenesis. Although STS metabolome is scarcely characterized, untargeted and targeted metabolomics approaches employing different data acquisition methods such as mass spectrometry (MS), MS imaging, and nuclear magnetic resonance (NMR) spectroscopy provided important information, warranting further studies. New chromatographic, MS, NMR-based, and flow cytometry-based methods will offer opportunities to therapeutically target metabolic pathways and to monitorize the response to such metabolic targeting therapies. Here we provide a comprehensive review of STS omics applications, comprising a detailed analysis of studies focused on the metabolic landscape of these tumors.

High-purity Isolation for Genotyping Rare Cancer Cells from Blood Using a Microfluidic Chip Cell Sorter

A multistep sorting method for enrichment of rare cells, such as circulating tumor cells, in the blood without cumbersome pretreatments required by most flow cytometry-based methods, which lead to high cost and decreased detection efficiency, was developed. After only hemolysis and cell staining, cancer cells are enriched by repetitive sorting (3×) based on nuclear-positive, cytokeratin-positive, and CD45-negative expression. Experiments using spikes of PC-9 cells showed a mean recovery of 65% and mean purity of 83%, which was retained up to 72 hours after blood draw using preservative tubes. Significant differences in expression level of programmed death-ligand 1 or vimentin were observed between high- and low-expressing cell lines, concurrently with enrichment. Next-generation sequencing analysis of recovered PC-9, A549, and MDA-MB231 cells successfully detected all known mutations. This novel isolation method applicable for preserved samples with sufficient recovery and purity may be substantially beneficial for recovering cells for subsequent molecular analysis.

Breast cancer cell line toxicity of a flavonoid isolated from Baccharis densiflora

BackgroundFlavonoids are compounds of interest in the search for new anti-cancer therapies. We have previously isolated the methoxyflavones 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone (8-methoxycirsilineol), 5,4′-dihydroxy-6,7,8-trimethoxyflavone (xanthomicrol), and 5,4,'3′-trihydroxy-6,7,8-trimethoxyflavone (sideritoflavone) from Baccharis densiflora. Herein, we investigate the toxicity of these methoxyflavones in human breast-derived cell line. Our main aim was to focus on the cancer stem cell (CSC) sub-population of JIMT-1 breast cancer cells.MethodsInitially, dose response experiments yielding inhibitory concentration 50 (IC50) values were performed using MCF-7, HCC1937, and JIMT-1 breast cancer, and the MCF-10A normal-like breast cell lines to get an understanding of toxic ranges. Due to a clear difference in the toxicity of the flavones, only sideritoflavone was selected for further studies using the JIMT-1 cell line. Effects on the CSC sub-population was investigated using flow cytometry-based methods. A wound healing assay and digital holographic microscopy were used to investigate effects on cell movement. A reporter assay was used to study effects on signal transduction pathways and Western blot for protein expression.ResultsThe dose response data showed that 8-methoxycirsilineol was non-toxic at concentrations below 100 μM, that the IC50 of xanthomicrol was between 50 and 100 μM, while sideritoflavone was highly toxic with a single digit μM IC50 in all cell lines. Treatment of the JIMT-1 cells with 2 μM sideritoflavone did not selectively effect the CSC sub-population. Instead, sideritoflavone treatment inhibited the proliferation of both the non-CSC and the CSC sub-populations to the same extent. The inhibition of cell proliferation resulted in an accumulation of cells in the G2 phase of the cell cycle and the treated cells showed an increased level of γ-H2A histone family member X indicating DNA double strand breaks. Analysis of the effect of sideritoflavone treatment on signal transduction pathways showed activation of the Wnt, Myc/Max, and transforming growth factor-β pathways. The level of p65/nuclear factor kappa-light-chain-enhancer of activated Β cells was increased in sideritoflavone-treated cells. Cell movement was decreased by sideritoflavone treatment.ConclusionsAltogether our data show that the methoxyflavone sideritoflavone has favourable anti-cancer effects that may be exploited for development to be used in combination with CSC specific compounds.

Droplet-based microfluidic platform for high-throughput screening of Streptomyces

Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2–111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.

Folliculin haploinsufficiency causes cellular dysfunction of pleural mesothelial cells

Birt–Hogg–Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms “cell–cell adhesion junction” and “cell adhesion molecule binding”. Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.

Human blood PIG-A mutation and micronucleated reticulocyte flow cytometric assays: Method optimization and evaluation of intra- and inter-subject variation.

We previously described flow cytometry-based methods for scoring the incidence of micronucleated reticulocytes (MN-RET) and PIG-A mutant phenotype reticulocytes (MUT RET) in rodent and human blood samples. The current report describes important methodological improvements for human blood analyses, including immunomagnetic enrichment of CD71-positive reticulocytes prior to MN-RET scoring, and procedures for storing frozen blood for later PIG-A analysis. Technical replicate variability in MN-RET and MUT RET frequencies based on blood specimens from 14 subjects, intra-subject variability based on serial blood draws from 6 subjects, and inter-subject variation based on up to 344 subjects age 0 to 73 years were quantified. Inter-subject variation explained most of the variability observed for both endpoints (≥77%), with much lower intra-subject and technical replicate variability. The relatively large degree of inter-subject variation is apparent from mean and standard deviation values for MN-RET (0.15 ± 0.10%) and MUT RET (4.7 ± 5.0 per million, after omission of two extreme outliers). The influences of age and sex on inter-subject variation were investigated, and neither factor affected MN-RET whereas both influenced MUT RET frequency. The lowest MUT RET values were observed for subjects <11 years old, and males had moderately higher frequencies than females. These results indicate that MN-RET and MUT RET are automation-compatible biomarkers of genotoxicity that bridge species of toxicological interest to include human populations. These data will be useful for appropriately designing future human studies that include these biomarkers of genotoxicity, and highlight the need for additional work aimed at identifying the sources of inter-individual variability reported herein.

Comparison of flow cytometry-based apoptosis detection methods for measuring of human native and cryopreserved spermatozoids viability

The aim of the study was to compare the ability of various media for cryopreservation of sperm to ensure their viability after thawing and to assess the possibility of using Narcissus pseudonarcissus lectin to determine the viability of native and cryopreserved human sperm by flow cytometry.&#x0D; Materials and methods. Used ejaculate 54 men aged 26 to 47 years, undergoing treatment for infertility. The control was a native ejaculate, which was also used for the in vitro fertilization procedure. Four parallel samples were frozen using various commercial media. After storage and thawing, spermatozoa viability was assessed by flow cytometry using three dyes and Narcissus pseudonarcissus lectin.&#x0D; Results. All assays showed that cryopreservation led to a twofold decrease of sperm viability, dye to the changes in the composition and properties of cell membrane, decrease in mitochondrial membrane potential, as well as the damages of acrosomal complex and nucleus. The lowest decrease in sperm viability was shown for Quinns advantage sperm freezing medium for cryopreservation.&#x0D; Conclusion. Flow cytometry makes it possible to evaluate with high efficiency sperm viability as the part of in vitro fertilization. The results of viability assessment using daffodil lectin make the prediction of in vitro fertilization outcome more accurate.

Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y12 Inhibition-Insights for Multi-Center Trials.

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate® whole-blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR-C 66096). Platelet function testing was carried out simultaneously by Multiplate® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA- and flow cytometry-based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate® was slightly higher for the flow cytometry-based VASP assay (r = 0.79, p < 0.0001) than for the ELISA-based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus-recommended assays with their standardized cut-offs should not be used interchangeably in multi-center clinical studies but, rather, they should be standardized throughout sites.

Women who received varicella vaccine versus natural infection have different long-term T cell immunity but similar antibody levels

BackgroundVaricella-zoster virus (VZV) infection during pregnancy is associated with serious fetal anomalies. The live-attenuated VZV vaccine was approved in 1995, so many vaccinated women are now of childbearing age. The question of long-term immunity to varicella is critical because breakthrough chickenpox can occur after vaccination. ObjectiveTo compare humoral and T cell immunity between women of childbearing age who were immunized by vaccination or chickenpox disease. Study designNon-pregnant females between 18 and 36 years old with a history of VZV immunization (n = 20) or prior chickenpox disease (n = 20) were recruited. IgG antibody titers and T cell responses were measured by flow cytometry-based methods in serum and peripheral blood, respectively. ResultsThere were no significant differences in median antibody titers between vaccinated and chickenpox groups (p = 0.34). The chickenpox group had significantly higher levels of VZV antigen-specific CD4 T cells (p = 0.004). ConclusionNatural infection induced higher VZV-specific T cell immune responses than vaccination.

Flow Cytometry-Based Assays to Quantify Complement Deposition and Neutrophil Uptake of Group A Streptococcus.

Quantification of complement deposition and subsequent neutrophil-mediated uptake provides an important way to assess the role of different bacterial factors in evasion of the host innate immune response. Here, we describe flow cytometry-based methods to allow quantification of deposition of the complement opsonin C3 on the bacterial surface and subsequent uptake by primary human neutrophils. The assays outlined below provide key methods to determine whether specific bacterial factors are involved in the evasion of complement-mediated immunity, using widely accessible reagents and equipment.

Abstract B142: Co-culture of tumor organoids with immune cells for immuno-oncology drug development

Abstract Hubrecht organoid technology (HUB) can in vitro preserve and propagate patient-derived epithelial cancer cells whilst still maintaining tumour heterogeneity and genetic features. Co-culture of tumor organoids with peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL) can potentially expand tumor-specific T cells that mediate explicit killing of tumor organoids. Recapitulating the tumor microenvironment (TME) by co-culture of tumor organoids with immune cells is highly attractive for immuno-oncology (IO) drug development, and will provide an efficient and refined approach for assessing the immune modulatory and tumor killing effects of IO drug candidates, such as monoclonal antibodies, CAR-T, CAR-NK and small molecules. In this study, we aimed to establish and validate IO assay platform using co-culture methods of organoids with various immune cells. We used the HUB organoid approach to create panels of patient-derived (PDO), patient-derived-xenograft (PDX)-derived organoids (PDXO) and murine tumor-derived tumor organoids, followed by co-culturing with corresponding cytotoxic T cells. We developed tumor organoid killing assays using both flow cytometry-based methods and real-time imaging by IncuCyte as readouts in 96-well format. Tumor-reactivity of T cells to organoids was analysed by T cell proliferation, IFNg ELISA in the culture medium, and intracellular staining of IFNg in T cells. Co-culture of tumor organoids expressing various antigens was also used to evaluate antibody dependent cell mediated cytotoxicity (ADCC) by NK cells, and complement dependent cytotoxicity (CDC) and antibody dependent phagocytosis by macrophages for monoclonal antibody drugs. We have profiled the expression of immune inhibitory molecules on various tumor organoids and assessing the effect of immune checkpoint blockade (e.g. anti-PD-1/PD-L1) in organoid-immune cell co-culture. Our preliminary data demonstrated the feasibility of in vitro patient-derived model system in the field of IO research using tumor organoid co-culture with immune cells, and their application in IO target and drug discovery. Citation Format: Zhongliang Li, Jun Zhou, Rui Zhang, Fei Wang, Shuzong Wang, Xiaoxi Xu, Yujun Huang, Henry Li. Co-culture of tumor organoids with immune cells for immuno-oncology drug development [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B142. doi:10.1158/1535-7163.TARG-19-B142

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals.

Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.