High-content Flow Cytometry Research Articles

A high-content flow cytometry and dual CRISPR-Cas9 based platform to quantify genetic interactions.

Probing epistasis between two genes can be a critical first step in identifying the molecular players in a cellular pathway. The advent of CRISPR-Cas mediated genetic screen has enabled studying of these genetic interactions at a genomic scale. However, when combining depletion of two genes using CRISPR Cas9, reduced targeting efficiencies due to competition for Cas loading and recombination in the cloning step have emerged as key challenges. Moreover, given conventional CRISPR screens typically involve comparison between the initial and final time point, it is difficult to parse the time kinetics with which a perturbed genetic interaction impacts viability, and it also becomes challenging to assess epistasis with essential genes. Here, we discuss a high-throughput flow-based approach to study genetic interactions. By utilizing two different Cas9 orthologs and monitoring viability at multiple time points, this approach helps to effectively mitigate the limitations of Cas9 competition and enables assessment of genetic interactions with both essential and non-essential genes at a high temporal resolution.

Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells

BackgroundDespite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action.MethodsThis work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers.ResultsSignificant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media.ConclusionsThese data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.

Abstract 4213: Targeting the cMet inhibitors combined with anti-PD-1 therapy: Preclinical approach to turn the resistance into opportunities for overcoming tumor evasion

Abstract Liver cancer is the fourth most common cause of cancer-related mortality world-wide, and Hepatocellular carcinoma (HCC) is the most common form of liver cancer accounting for 90% of cases. Therapeutic strategies include surgical resection, radiotherapy, liver transplantation and pharmacological treatment with small-molecules multikinase inhibitors and/or immune-checkpoint blockade (ICB); however, outcomes remain poor and improvements to systemic therapy are desperately needed. Recently, Cabozantinib a kinase inhibitor with activity against VEGFR2, AXL and cMET, has been approved for second line use in advanced HCC; however, monotherapy achieves less than a 4-month extension of overall survival. Previously, our collaborators demonstrated that the efficacy of cMet inhibition is self-limited by induction of high PD-L1 expression. Given this data, the goal of our study was to investigate the efficacy of combining cMet inhibition with either capmatinib or cabozantinib with antibody blockade of the PD-1 immune checkpoint receptor for therapy of HCC. Using multiple syngeneic, immunocompetent models of HCC (Hepa1-6, HCA) in two different strains of mice (C57BL6, C3H), we investigated the combination potential of each of these cMet inhibitors with anti-PD-1 antibodies. Further, we profiled the changes to the tumor infiltrating myeloid and T cell repertoires which correlated to mono- as well as combination therapy responses in these animals using high content flow cytometry on a BD X-30. We found Hepa1-6 to be exquisitely anti-PD-1 sensitive with 100% of animals being cured by the combination. In the less immunotherapy-sensitive HCA-1 model, we also found that the cMet inhibitors cooperate with PD-1 blockade to promote rejection of HCA-1 HCC model. Again, we found that combination therapy could cure nearly all animals of pre-implanted HCA-1 tumors. Mechanistically, we found that concomitant cMet inhibition enhances the efficacy of PD-1 therapy increasing the number of tumor-infiltrating CD4 Teff cells while decreasing the CD4 Treg population. We also found that combined therapy improves CD8+ T cell infiltration, memory CD8 T cell fraction and NK cytotoxic potential within the tumor microenvironment. Within the HCA-1 myeloid compartment, we discovered a significant decrease in the tumor-associated neutrophil (N2-TAN) population (PMN-MDSC). Further, we found that combination therapy promotes pro-inflammatory M1 conversion of stromal macrophages. Collectively, these data provide a rationale for combining anti-PD-1 therapy with cMet inhibitors as a potential strategy to overcome resistance to immune checkpoint blockade therapy in HCC. Citation Format: Ricardo Alexandre de Azevedo, Hsieh Cheng-En, Priyamvada Jayaprakash, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Brittany Morrow, Michelle Winkler, Shweta Mahendra Hedge, Arthur Liu, Michael A. Curran. Targeting the cMet inhibitors combined with anti-PD-1 therapy: Preclinical approach to turn the resistance into opportunities for overcoming tumor evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4213.

Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-soluble liver antigen (SLA) autoantibodies is associated with reduced overall survival, but the associated autoreactive CD4 T cells have not yet been characterised. Herein, we isolated and deeply characterised SLA-specific CD4 T cells in patients with AIH. We used brief exvivo restimulation with overlapping SLA peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterise their transcriptome and T-cell receptor (TCR) repertoire. Autoreactive TCRs were cloned and used to identify dominant SLA-derived epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing to identify their phenotypic niche. We further characterised disease-associated peripheral blood T cells by high-content flow cytometry in 42 patients with AIH and 17 controls with non-alcoholic steatohepatitis. Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5-CCR6-CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-helper profile. SLA81-100 and SLA177-204 contain dominant T-cell epitopes. Autoreactive TCR clonotypes were predominantly found in the memory PD-1+CXCR5-CD4 T cells, which were significantly increased in the blood of patients with AIH and supported B-cell differentiation through IL-21. Finally, we identified specific T-cell phenotypes linked to disease activity and IgG level during AIH. We provide a deep characterisation of rare circulating autoreactive CD4 T cells and identify their peripheral reservoir in AIH. We also propose a specific phenotype of autoreactive T cells related to AIH disease activity, which will be essential to track, delineate, and potentially target these pathogenic cells. One principal characteristic of autoimmune hepatitis (AIH), like for many other autoimmune diseases, is the accumulation of autoantibodies produced by B lymphocytes following their interaction with autoreactive CD4 T lymphocytes. In this study, we identified and characterised with high resolution these CD4 T cells. This will be essential to track, delineate, and potentially target them during AIH.

Novel fluorochrome development enables 34-color high-content flow cytometry

Abstract Flow cytometry is an essential technology to identify and quantify cell populations. Here we examined the development of new fluorochromes that could enhance flow cytometry’s capability. The latest spectral flow cytometers have 3 excitation lasers (405nm, 488nm, and 640nm) and incorporate the Avalanche Diodes Photodetector technology, which demonstrates significant improvement in sensitivity for the fluorescent emission signal longer than 800nm. However, there is no commercially available fluorochrome which could be excited by the above lasers and has peak emission signal beyond 800nm. To address the gap in technology, here we engineered 6 new fluorochromes: PE-750, PE-810, PE-830 for blue laser and APC-750, APC-810, APC-830 for red laser. These were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Then, the fluorochromes were conjugated with antibodies using Click chemistry, which is a simple, robust reaction with high-yield and high-reaction sensitivity. Also, we demonstrated long-term stability at −20°C with protein stabilizing cocktails. Most importantly, in order to show the utility of these novel fluorochromes, we created a 34-color flow cytometry panel to measure broad human immune function with high sensitivity on a 3-laser Aurora. The additional UV laser upgrade will increase the instrument capacity to more than 40 color. This panel will be applied to identify the expression patterns of many different receptors involved in trafficking on multiple different cell types from dissociated tumors and blood samples. In conclusion, this high-content flow cytometry panel will improve analysis and diagnosis in Immuno-oncology and facilitate innovation in biomarker discovery.

Toward deterministic and semiautomated SPADE analysis.

SPADE stands for spanning-tree progression analysis for density-normalized events. It combines downsampling, clustering and a minimum-spanning tree to provide an intuitive visualization of high-dimensional single-cell data, which assists with the interpretation of the cellular heterogeneity underlying the data. SPADE has been widely used for analysis of high-content flow cytometry data and CyTOF data. The downsampling and clustering components of SPADE are both stochastic, which lead to stochasticity in the tree visualization it generates. Running SPADE twice on the same data may generate two different tree structures. Although they typically lead to the same biological interpretation of subpopulations present in the data, robustness of the algorithm can be improved. Another avenue of improvement is the interpretation of the SPADE tree, which involves visual inspection of multiple colored versions of the tree based on expression of measured markers. This is essentially manual gating on the SPADE tree and can benefit from automated algorithms. This article presents improvements of SPADE in both aspects above, leading to a deterministic SPADE algorithm and a software implementation for semiautomated interpretation. © 2017 International Society for Advancement of Cytometry.

The Endosomal Sorting Complex ESCRT-II Mediates the Assembly and Architecture of ESCRT-III Helices

The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that mediate topologically similar membrane-sculpting processes, including cytokinesis, retroviral egress, and multivesicular body (MVB) biogenesis. Although ESCRT-III drives membrane remodeling that creates MVBs, its structure and the mechanism of vesicle formation are unclear. Using electron microscopy, we visualize an ESCRT-II:ESCRT-III supercomplex and propose how it mediates vesicle formation. We define conformational changes that activate ESCRT-III subunit Snf7 and show that it assembles into spiraling ~9 nm protofilaments on lipid monolayers. A high-content flow cytometry assay further demonstrates that mutations halting ESCRT-III assembly block ESCRT function. Strikingly, the addition of Vps24 and Vps2 transforms flat Snf7 spirals into membrane-sculpting helices. Finally, we show that ESCRT-II and ESCRT-III coassemble into ~65 nm diameter rings indicative of a cargo-sequestering supercomplex. We propose that ESCRT-III has distinct architectural stages that are modulated by ESCRT-II to mediate cargo capture and vesicle formation by ordered assembly.

Fluorescent cell barcoding for multiplex flow cytometry.

Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed.

High-Content Flow Cytometry and Temporal Data Analysis for Defining a Cellular Signature of Graft-Versus-Host Disease

Acute graft-versus-host disease (GVHD) is diagnosed by clinical and histologic criteria that are often nonspecific and typically apparent only after the disease is well established. Because GvHD is mediated by donor T cells and other immune effector cells, we sought to determine whether changes within a wide array of peripheral blood lymphocyte populations could predict the development of GvHD. Peripheral blood samples from 31 patients undergoing allogeneic blood and marrow transplant were analyzed for the proportion of 121 different subpopulations defined by 4-color combinations of lymphocyte phenotypic and activation markers at progressive time points posttransplant. Samples were processed using a newly developed high content flow cytometry technique and subjected to a spline- and functional linear discriminant analysis (FLDA)-based temporal analysis technique. This strategy identified a consistent posttransplant increase in the proportion and extent of fluctuation of CD3 +CD4 +CD8β + cells in patients who developed GVHD compared to those that did not. Although larger prospective clinical studies will be necessary to validate these results, this study demonstrates that high-content flow cytometry coupled with temporal analysis is a powerful approach for developing new diagnostic tools, and may be useful for developing a sensitive and specific predictive test for GVHD.