Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.
Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens.



Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b+and CD15+, HLA-DRdimand CD14-phenotype, were undetectable in frozen samples.



These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.
Department of Pharmacology, Cellular Pharmacology, Merck and Co. Inc, 33 Avenue Louis Pasteur, Boston, 02115, MA, USA.
Elma
E
Kadić
Department of Biology-Discovery, Immunooncology, Merck and Co. Inc, Boston, MA, USA.
Raymond J
RJ
Moniz
Department of Pharmacology, Cellular Pharmacology, Merck and Co. Inc, 33 Avenue Louis Pasteur, Boston, 02115, MA, USA.
Ying
Y
Huo
Department of Chemistry, Capabilities Enhancement, Merck and Co. Inc, Boston, MA, USA.
An
A
Chi
Department of Pharmacology, Cellular Pharmacology, Merck and Co. Inc, 33 Avenue Louis Pasteur, Boston, 02115, MA, USA. Ilona_Kariv@merck.com.
Ilona
0000-0002-0171-0456
I
Kariv
D063646:Carcinogenesis / Q000276:immunology*; D002292:Carcinoma, Renal Cell / Q000473:pathology*; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation; D015179:Colorectal Neoplasms / Q000473:pathology*; D015925:Cryopreservation; D005240:Feasibility Studies; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D000072737:Myeloid-Derived Suppressor Cells / Q000276:immunology*; D010641:Phenotype; D059010:Single-Cell Analysis; D059016:Tumor Microenvironment
Cryopreservation; CyTOF™; Flow cytometry (FC); Immunophenotyping; Mass Cytometry (MC); Tumor infiltrating lymphocytes (TILs); Tumor microenvironment (TME); Tumor samples; ViSNE