Single cell analysis of human tissues and solid tumors with mass cytometry.
Mass cytometry measures 36 or more markers per cell and is an appealing platform for comprehensive phenotyping of cells in human tissue and tumor biopsies. While tissue disaggregation and fluorescence cytometry protocols were pioneered decades ago, it is not known whether established protocols will be effective for mass cytometry and maintain cancer and stromal cell diversity.



Tissue preparation techniques were systematically compared for gliomas and melanomas, patient derived xenografts of small cell lung cancer, and tonsil tissue as a control. Enzymes assessed included DNase, HyQTase, TrypLE, collagenase (Col) II, Col IV, Col V, and Col XI. Fluorescence and mass cytometry were used to track cell subset abundance following different enzyme combinations and treatment times.



Mechanical disaggregation paired with enzymatic dissociation by Col II, Col IV, Col V, or Col XI plus DNase for 1 h produced the highest yield of viable cells per gram of tissue. Longer dissociation times led to increasing cell death and disproportionate loss of cell subsets. Key markers for establishing cell identity included CD45, CD3, CD4, CD8, CD19, CD64, HLA-DR, CD11c, CD56, CD44, GFAP, S100B, SOX2, nestin, vimentin, cytokeratin, and CD31. Mass and fluorescence cytometry identified comparable frequencies of cancer cell subsets, leukocytes, and endothelial cells in glioma (R = 0.97), and tonsil (R = 0.98).



This investigation establishes standard procedures for preparing viable single cell suspensions that preserve the cellular diversity of human tissue microenvironments. © 2016 International Clinical Cytometry Society.
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.
Nalin
N
Leelatian
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.
Deon B
DB
Doxie
Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Allison R
AR
Greenplate
Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Bret C
BC
Mobley
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.|Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee.
Jonathan M
JM
Lehman
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.
Justine
J
Sinnaeve
Department of Surgical Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Rondi M
RM
Kauffmann
Department of Pediatric Otolaryngology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Jay A
JA
Werkhaven
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.|Department of Neurological Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee.
Akshitkumar M
AM
Mistry
Department of Neurological Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee.
Kyle D
KD
Weaver
Department of Neurological Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee.
Reid C
RC
Thompson
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.|Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee.
Pierre P
PP
Massion
Department of Surgical Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Mary A
MA
Hooks
Department of Surgical Oncology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Mark C
MC
Kelley
Department of Neurological Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee.
Lola B
LB
Chambless
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.|Department of Neurological Surgery, Vanderbilt University School of Medicine, Nashville, Tennessee.
Rebecca A
RA
Ihrie
Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee.|Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Jonathan M
0000-0001-9428-8866
JM
Irish
D015703:Antigens, CD / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D019169:Jurkat Cells / Q000166:cytology; D017493:Leukocyte Common Antigens / Q000032:analysis; D009369:Neoplasms / Q000473:pathology*; D059010:Single-Cell Analysis / Q000379:methods
D015703:Antigens, CD; D006684:HLA-DR Antigens; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
Key terms: solid tumor; glioma; human tissue; mass cytometry (CyTOF); melanoma; single cell biology; small cell lung cancer (SCLC); tonsil