Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Flow Cytometry

2019 ◽  
pp. 181-194 ◽  
Author(s):  
Irene van der Haar Àvila ◽  
Patricia Marmol ◽  
Jeannette Cany ◽  
Rolf Kiessling ◽  
Yago Pico de Coaña
Keyword(s):  
Flow Cytometry ◽  
Dependent Cell

The Future of Flow Cytometry in Biotechnology: The Response to Diversity and Complexity

2005 ◽  
Author(s):  
Larry A. Sklar
Keyword(s):  
Flow Cytometry ◽  
Scattered Light ◽  
National Laboratory ◽  
Modern World ◽  
Light Sources ◽  
Main Function ◽  
Hydrodynamic Focusing ◽  
Los Alamos National Laboratory ◽  
The Future ◽  
Dependent Cell

Flow cytometry is a mature technology: Instruments recognizable as having elements of modern flow cytometers date back at least 30 years. There are many good sources for information about the essential features of flow cytometers, how they operate, and how they have been used. For the purposes of this book, it is necessary to know that flow cytometers have fluidic, optical, electronic, computational, and mechanical features. The main function of the fluidic components is to use hydrodynamic focusing to create a stable particle stream in which particles are aligned in single file within a sheath stream, so that the particles can be analyzed and sorted. The main functions of the optical components are to allow the particles to be illuminated by one or more lasers or other light sources and to allow scattered light as well as multiple fluorescence signals to be resolved and be routed to individual detectors. The electronics coordinate these functions, from the acquisition of the signals (pulse collection, pulse analysis, triggering, time delay, data, gating, detector control) to forming and charging individual droplets, and to making sort decisions. The computational components are directed at postacquisition data display and analysis, analysis of multivariate populations and multiplexing assays, and calibration and analysis of time-dependent cell or reaction phenomena. Mechanical components are now being integrated with flow cytometers to handle plates of samples and to coordinate automation such as the movement of a cloning tray with the collection of the droplets. The reader is directed to a concise description of these processes in Robinson’s article in the Encyclopedia of Biomaterials and Biomedical Engineering. This book was conceived of to provide a perspective on the future of flow cytometry, and particularly its application to biotechnology. It attempts to answer the question I heard repeatedly, especially during my association with the National Institutes of Health–funded National Flow Cytometry Resource at Los Alamos National Laboratory: What is the potential for innovation in flow cytometer design and application? This volume brings together those approaches that identify the unique contributions of flow cytometry to the modern world of biotechnology.

scholarly journals Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

2020 ◽  
Vol 8 (1) ◽  
pp. e000195 ◽  
Author(s):  
Johannes Laengle ◽  
Julijan Kabiljo ◽  
Leah Hunter ◽  
Jakob Homola ◽  
Sophie Prodinger ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Valproic Acid ◽  
Cancer Cells ◽  
Histone Deacetylase ◽  
Breast Cancer Cells ◽  
Histone Deacetylase Inhibitors ◽  
Combined Treatment ◽  
Dependent Cell ◽  
The Impact

BackgroundThe monoclonal antibody (mAb) trastuzumab is part of the standard of care for patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer. Antibody-dependent cell-mediated phagocytosis (ADCP) and cytotoxicity (ADCC) are major mechanisms of action of the mAb trastuzumab. Histone deacetylase inhibitors (HDACi), such as valproic acid (VPA) or vorinostat (SAHA), exert several immunostimulatory properties, which contribute at least in part to their anticancer effect. However, the impact of HDACi-induced immunostimulatory effects on trastuzumab-mediated anti-tumor immune response is not well characterized.MethodsWe analyzed the ADCP and ADCC activity of peripheral blood mononuclear cells (PBMCs) from age and gender-matched healthy volunteers (n=5) against HDACi-treated HER2-overexpressing breast cancer cells (SKBR3), using a well-established in vitro three-color imaging flow cytometry and flow cytometry approach.ResultsVPA and SAHA enhanced trastuzumab-mediated ADCP and trastuzumab-independent cytotoxicity. Mechanistically, VPA upregulated the activating antibody-binding receptor Fc-gamma receptor (FcγR) IIA (CD32A) on monocytes (CD14+). Moreover, VPA and SAHA downregulated the anti-apoptotic protein myeloid leukemia cell differentiation 1 (MCL1) in breast cancer cells. Additionally, VPA and SAHA induced an immunogenic cell death, characterized by the exposure of calreticulin (CALR), as well as decreased the “do not eat me” signal CD47 on tumor cells.ConclusionsHDACi VPA and SAHA increase trastuzumab-mediated phagocytosis and trastuzumab-independent cytotoxicity. The immunomodulatory activities of those HDACi support a rationale combined treatment approach with mAb for cancer treatment.

Bone Marrow Interaction in Multiple Myeloma Pathogenesis: Phenotypical Analysis, Kinetics and Novel Therapy Approaches Based On CXCR4 Inhibition.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2450-2450
Author(s):  
Johannes Waldschmidt ◽  
Dagmar Wider ◽  
Marie Follo ◽  
Josefina Udi ◽  
Martina Kleber ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Cell Trafficking ◽  
Newly Diagnosed ◽  
Infiltration Rates ◽  
Dependent Cell ◽  
And Migration

Abstract Abstract 2450 Introduction: The interaction between malignant plasma cells and their microenvironment is central in multiple myeloma (MM) pathogenesis. Binding of MM cells to bone marrow (BM) stroma cells triggers the expression of adhesion molecules and secretion of chemo- and cytokines, promoting MM cell growth, drug resistance and migration. Stromal-derived factor-1 (SDF-1) and its receptor CXCR4 are essential for normal hematopoietic progenitor cell movement and adherence within the BM microenvironment. In leukemia and lymphoma, oncoproteins may inhibit SDF-1-dependent cell trafficking within the BM through a mechanism that is not fully understood. For that reason, understanding SDF-1-dependent cell trafficking within the BM and targeting MM-cell - host-BM interactions display a promising approach for the development of novel therapeutic strategies. Methods: BM samples of MM patients (n=59) were analysed using flow cytometry and compared to MGUS patients (n=3) and healthy volunteers (n=7). We compared patient samples with low BM infiltration (≤5%; n=13) intermediate (5–30%; n=29) and high infiltration rates (≥30%; n=17). We also assessed expression of adhesion molecules in MM patients with long-term disease control (n=20) vs. both newly diagnosed (n=16) and symptomatic MM patients (n=23) as previously grouped by San Miguel et al. (Haematologica July 6,2012). We also sought to elucidate in vitro, whether specific anti-MM agents (bortezomib, vorinostat, pomalidomide, EGCG), with and without M210B4 stroma support, and with and without the CXCR4 inhibitor AMD3100, target the interaction of MM cells. Experiments were performed using MM cell lines (U266, RPMI8226, L363, NCI-H929), the control T-cell line MOLT-4 and MM-patient BM samples. Cell viability was assessed via Trypan Blue- and AnnexinV/PI-staining. CD138, CXCR4 (SDF1-receptor), CD49d (VLA-4), CD11a (LFA-1) and CD44 (HERMES antigen) expression was evaluated by flow cytometry and ScanR microscopy. Results: In BM samples of MM patients as compared to MGUS and healthy volunteers, the CXCR4/CD138- (p=.036), CD49d/CD138- (p=.0013) and CD44/CD138-expression (p=.0072) was significantly amplified and correlated with increasing BM infiltration rates (p=.001). Both newly diagnosed and symptomatic MM patients confirmed significantly increased CXCR4/CD138-, CD49d/CD138- (p=.0013) and CD44/CD138-expression as compared to patients with long-term disease control. Of note, in newly diagnosed patients, the expression of adhesion molecules was even more enhanced than in symptomatic myeloma patients, underlining their critical and future potential role as targets for novel therapeutics. Comparison of MM cell lines' adhesion and migration markers with that of MM-patient BM specimens revealed U266 as the cell line most closely resembling human specimens. Cytotoxic effects with use of MM cell lines and bortezomib, vorinostat and pomalidomide confirmed prior cytotoxic concentrations. Cocultivation with stroma substantially reduced apoptosis and induced tumor protective effects. Additional AMD3100 treatment restored sensitivity to bortezomib, vorinostat and pomalidomide. CXCR4 expression was substantially reduced after AMD3100 treatment, while that of CD49d, CD44 and CD11a remained widely unchanged. Toxic or therapeutic effects of AMD3100 monotherapy were excluded for used doses of 50μM. Additional use of ScanR microscopy visualized co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. ScanR microscopy results correlated with flow cytometry-determined CXCR4 expression. Ongoing analyses of both ScanR microscopy and flow cytometry will allow the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Conclusions: Our findings underline the critical role of adhesion and migration molecules in MM and may pave the way for novel therapeutic approaches targeting these microenvironmental mediators. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Microsphere cytometry to interrogate microenvironment-dependent cell signaling

2017 ◽  
Vol 9 (2) ◽  
pp. 123-134 ◽  
Author(s):  
Henriette Christie Ertsås ◽  
Garry P. Nolan ◽  
Mark A. LaBarge ◽  
James B. Lorens
Keyword(s):  
Flow Cytometry ◽  
Cell Signaling ◽  
Single Cell ◽  
Adherent Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Dependent Cell

A novel microsphere-based flow cytometry approach to study adherent cell signaling responses in different microenvironmental contexts at the single cell level.

scholarly journals A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal

PLoS ONE ◽  
2011 ◽  
Vol 6 (4) ◽  
pp. e18762 ◽  
Author(s):  
Nadia Korfali ◽  
Vlastimil Srsen ◽  
Martin Waterfall ◽  
Dzmitry G. Batrakou ◽  
Vanja Pekovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Nuclear Envelope ◽  
Transmembrane Proteins ◽  
Dependent Cell ◽  
Stress Dependent

Antibody-dependent cell-mediated cytotoxicity: detection by automated flow cytometry with ultramicro techniques

Science ◽  
1980 ◽  
Vol 209 (4454) ◽  
pp. 404-406 ◽  
Author(s):  
A. Attallah ◽  
T. Yeatman ◽  
P. Noguchi ◽  
J. Johnson
Keyword(s):  
Flow Cytometry ◽  
Dependent Cell

Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores

2001 ◽  
Vol 258 (1-2) ◽  
pp. 183-191 ◽  
Author(s):  
Robert W Wilkinson ◽  
Alice E Lee-MacAry ◽  
Derek Davies ◽  
David Snary ◽  
Elizabeth L Ross
Keyword(s):  
Flow Cytometry ◽  
Dependent Cell
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