Abstract
BackgroundComprehensive 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.ResultsInitial 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-DRdim and CD14− phenotype, were undetectable in frozen samples.ConclusionThese results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.
Highlights
Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies
The T-cell compartment was further delineated into helper T-cells (CD3 + CD4+) and cytotoxic T-cells (CD3 + CD8+), whereas B-cell were identified by co-expression of CD19+ and CD45RA+
Average values for helper-T-cells, cytotoxic-T-cells and natural killer (NK)-cells represented 38.1 ± 3.2, 20.0 ± 3.4 and 16.3% ± 3.1% of CD45+ cells respectively, while B-cells, monocytes and MDCs made up a smaller fraction of Peripheral blood mononuclear cells (PBMC) with 7.4 ± 2.1, 10.3 ± 2.5 and 4.1% ± 1.0% respectively
Summary
Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Cancer represents a multifaceted disease characterized by extreme genetic and epigenetic heterogeneity of the transformed cells, and by clonal progression of treatment-resistant tumors as a result of targeted therapies. Most recent are targeting two immuno-checkpoint receptors; CTLA4 (cytotoxic T-lymphocyte-antigen 4), PD-1 (programmed cell death 1) and its ligand PDL1, and show a remarkable efficacy against certain tumors, through re-activating cytotoxic T (Tc) cell mediated tumor killing [5, 6]. These positive clinical outcomes support further investment in targeting additional immunomodulatory receptors on T-cytotoxic (Tc) cells, and expanding these approaches to other immune cells. For example natural killer (NK) cells can be recruited to kill tumor cells, without potential concern for causing acute cytokine storm or long-term autoimmune responses [7, 8], while suppression of Tregulatory (Treg) cells can be used to enhance immune response against tumor cells [9]
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