Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis

Mass cytometry is a single‐cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >104 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho‐protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single‐cell suspensions. This unit establishes a primer for single‐cell analysis of solid tumors and tissues, and has been tested with mass cytometry. The cells obtained from these protocols can be fixed for study, cryopreserved for long‐term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors. © 2017 by John Wiley & Sons, Inc.

[1]  Bo Wang,et al.  Visualization and analysis of single-cell RNA-seq data by kernel-based similarity learning , 2016, Nature Methods.

[2]  A. Greenplate,et al.  Dissecting Complex Cellular Systems with High Dimensional Single Cell Mass Cytometry , 2016 .

[3]  A Orfao,et al.  EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes , 2012, Leukemia.

[4]  S. Ansari,et al.  Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay. , 2011, Journal of visualized experiments : JoVE.

[5]  Erika Pastrana,et al.  Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche , 2014, Neuron.

[6]  Sean C. Bendall,et al.  Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes. , 2012, Immunity.

[7]  B. Wood,et al.  Considerations for the control of background fluorescence in clinical flow cytometry , 2009, Cytometry. Part B, Clinical cytometry.

[8]  Michael R. Green,et al.  Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling. , 2017, Blood.

[9]  Yuri Kotliarov,et al.  Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. , 2006, Cancer cell.

[10]  Y. Saeys,et al.  Computational flow cytometry: helping to make sense of high-dimensional immunology data , 2016, Nature Reviews Immunology.

[11]  Jonathan M Irish,et al.  Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells. , 2006, Blood.

[12]  J. Richards,et al.  Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[13]  Eli R. Zunder,et al.  Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm , 2015, Nature Protocols.

[14]  Alberto Orfao,et al.  Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies. , 2015, Blood.

[15]  Albert D Donnenberg,et al.  Rare event detection and analysis in flow cytometry: bone marrow mesenchymal stem cells, breast cancer stem/progenitor cells in malignant effusions, and pericytes in disaggregated adipose tissue. , 2011, Methods in molecular biology.

[16]  Sean C. Bendall,et al.  Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum , 2011, Science.

[17]  J. Paul Robinson Comparative Overview of Flow and Image Cytometry , 2005, Current protocols in cytometry.

[18]  R. Hardy,et al.  Demonstration of B-cell maturation in X-linked immunodeficient mice by simultaneous three-colour immunofluorescence , 1983, Nature.

[19]  R. Nussenblatt,et al.  Standardizing immunophenotyping for the Human Immunology Project , 2012, Nature Reviews Immunology.

[20]  Garry P. Nolan,et al.  Coordinate Analysis of Murine Immune Cell Surface Markers and Intracellular Phosphoproteins by Flow Cytometry1 , 2005, The Journal of Immunology.

[21]  J. Myklebust,et al.  Mass Cytometry of Follicular Lymphoma Tumors Reveals Intrinsic Heterogeneity in Proteins Including HLA‐DR and a Deficit in Nonmalignant Plasmablast and Germinal Center B‐Cell Populations , 2017, Cytometry. Part B, Clinical cytometry.

[22]  Michael Poidinger,et al.  High-dimensional analysis of the murine myeloid cell system , 2014, Nature Immunology.

[23]  Garry P Nolan,et al.  Single-cell Mass Cytometry for Analysis of Immune System Functional States This Review Comes from a Themed Issue on Host Pathogens Basic Concepts of Mass Cytometry , 2022 .

[24]  O. Ornatsky,et al.  Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. , 2008, Analytical chemistry.

[25]  Sean C. Bendall,et al.  viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia , 2013, Nature Biotechnology.

[26]  H. Maecker,et al.  Selecting fluorochrome conjugates for maximum sensitivity , 2004, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[27]  Josef Spidlen,et al.  Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) Compliant Manuscript Using the International Society for Advancement of Cytometry (ISAC) FCS File Repository (FlowRepository.org) , 2012, Current protocols in cytometry.

[28]  Ash A. Alizadeh,et al.  B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression , 2010, Proceedings of the National Academy of Sciences.

[29]  R. Henkelman,et al.  Identification of human brain tumour initiating cells , 2004, Nature.

[30]  A. Donnenberg,et al.  Flow cytometric determination of stem/progenitor content in epithelial tissues: An example from nonsmall lung cancer and normal lung , 2013, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[31]  Stuart B. Goodman,et al.  Clinical recovery from surgery correlates with single-cell immune signatures , 2014, Science Translational Medicine.

[32]  Mario Roederer,et al.  The Genetic Architecture of the Human Immune System: A Bioresource for Autoimmunity and Disease Pathogenesis , 2015, Cell.

[33]  S. Morrison,et al.  Prospective identification of tumorigenic breast cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Sean C. Bendall,et al.  Normalization of mass cytometry data with bead standards , 2013, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[35]  J. Irish,et al.  Cutting Edge: Redox Signaling Hypersensitivity Distinguishes Human Germinal Center B Cells , 2015, The Journal of Immunology.

[36]  S. Hunger,et al.  Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study. , 2008, Blood.

[37]  E. Newell,et al.  Mass cytometry: blessed with the curse of dimensionality , 2016, Nature Immunology.

[38]  H. Maecker,et al.  Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry. , 2015, Methods in molecular biology.

[39]  Rebecca A. Ihrie,et al.  Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry. , 2017, Cytometry. Part B, Clinical cytometry.

[40]  Jonathan M Irish,et al.  Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. , 2004, Clinical immunology.

[41]  J. Irish,et al.  Beyond the age of cellular discovery , 2014, Nature Immunology.

[42]  Iuis-Who Nomenclature Subcommittee Nomenclature for clusters of differentiation (CD) of antigens defined on human leukocyte populations. IUIS-WHO Nomenclature Subcommittee. , 1984, Bulletin of the World Health Organization.

[43]  J. Sosman,et al.  Myelodysplastic Syndrome Revealed by Systems Immunology in a Melanoma Patient Undergoing Anti–PD-1 Therapy , 2016, Cancer Immunology Research.

[44]  Sean C. Bendall,et al.  A deep profiler's guide to cytometry. , 2012, Trends in immunology.

[45]  Jonathan M Irish,et al.  High-dimensional single-cell cancer biology. , 2014, Current topics in microbiology and immunology.

[46]  L. Herzenberg,et al.  Identification of B-cell subsets: an exposition of 11-color (Hi-D) FACS methods. , 2004, Methods in molecular biology.

[47]  S. Weiss,et al.  A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[48]  Jonathan M Irish,et al.  Single Cell Profiling of Potentiated Phospho-Protein Networks in Cancer Cells , 2004, Cell.

[49]  Peter O. Krutzik,et al.  Single‐Cell Phospho‐Protein Analysis by Flow Cytometry , 2007, Current protocols in immunology.

[50]  P. Chattopadhyay,et al.  Cytometry: today's technology and tomorrow's horizons. , 2012, Methods.

[51]  G. Nolan,et al.  Mapping normal and cancer cell signalling networks: towards single-cell proteomics , 2006, Nature Reviews Cancer.

[52]  Kirsten E Diggins,et al.  Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data. , 2015, Methods.

[53]  F. Craig,et al.  Flow cytometric immunophenotyping for hematologic neoplasms. , 2008, Blood.

[54]  Rebecca A. Ihrie,et al.  Single cell analysis of human tissues and solid tumors with mass cytometry , 2017, Cytometry. Part B, Clinical cytometry.

[55]  Eli R. Zunder,et al.  Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining , 2014, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[56]  P. Chattopadhyay,et al.  Seventeen-colour flow cytometry: unravelling the immune system , 2004, Nature Reviews Immunology.

[57]  Dmitry Bandura,et al.  Highly multiparametric analysis by mass cytometry. , 2010, Journal of immunological methods.

[58]  O. Ornatsky,et al.  Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. , 2009, Analytical chemistry.

[59]  Sean C. Bendall,et al.  Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis , 2015, Cell.

[60]  G. Nolan,et al.  Mass Cytometry: Single Cells, Many Features , 2016, Cell.

[61]  Sean C. Bendall,et al.  Extracting a Cellular Hierarchy from High-dimensional Cytometry Data with SPADE , 2011, Nature Biotechnology.

[62]  Nikesh Kotecha,et al.  Web‐Based Analysis and Publication of Flow Cytometry Experiments , 2010, Current protocols in cytometry.

[63]  Erika Pastrana,et al.  Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny , 2009, Proceedings of the National Academy of Sciences.

[64]  G. Mufti,et al.  Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment. , 2016, Blood.

[65]  Peter O. Krutzik,et al.  Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events , 2003, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[66]  J. Irish,et al.  High-Dimensional Analysis of Acute Myeloid Leukemia Reveals Phenotypic Changes in Persistent Cells during Induction Therapy , 2016, PloS one.

[67]  Mario Roederer,et al.  Single-cell technologies for monitoring immune systems , 2014, Nature Immunology.

[68]  S. Kalams,et al.  Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry , 2016, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[69]  Douglas B. Johnson,et al.  Systems immune monitoring in cancer therapy. , 2016, European journal of cancer.

[70]  Mario Cazzola,et al.  The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. , 2016, Blood.

[71]  Kenneth D Cole,et al.  Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[72]  Sean C. Bendall,et al.  Single-Cell Trajectory Detection Uncovers Progression and Regulatory Coordination in Human B Cell Development , 2014, Cell.

[73]  M. Roederer,et al.  Optimizing a multicolor immunophenotyping assay. , 2007, Clinics in laboratory medicine.

[74]  J. Irish,et al.  Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry. , 2015, Methods in molecular biology.

[75]  A. Boyd,et al.  Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines , 2015, Anatomy & cell biology.

[76]  K. M. May,et al.  Mammalian Cell Tissue Culture Techniques , 2017, Current protocols in molecular biology.

[77]  Mario Roederer,et al.  Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients , 1999, Nature Medicine.

[78]  R. Hardy,et al.  Three-color immunofluorescence analysis of mouse B-lymphocyte subpopulations. , 1984, Cytometry.

[79]  Mark M. Davis,et al.  Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE) , 2013, Proceedings of the National Academy of Sciences.

[80]  Kirsten E Diggins,et al.  Characterizing cell subsets in heterogeneous tissues using marker enrichment modeling , 2016, Nature Methods.

[81]  D. Nixon,et al.  Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach , 2008, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[82]  J. Bluestone,et al.  Harnessing the plasticity of CD4+ T cells to treat immune-mediated disease , 2016, Nature Reviews Immunology.

[83]  Bruce Greig,et al.  2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: Optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia , 2007, Cytometry. Part B, Clinical cytometry.

[84]  Leonore A. Herzenberg,et al.  Identification of B-Cell Subsets , 2004 .

[85]  Russ B. Altman,et al.  Towards a Cytokine-Cell Interaction Knowledgebase of the Adaptive Immune System , 2008, Pacific Symposium on Biocomputing.

[86]  Howard Y. Chang,et al.  Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells , 2009, Proceedings of the National Academy of Sciences.