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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0192-1) contains supplementary material, which is available to authorized users.

The discovery of a potent selective low dose Janus kinase 1 (JAK1) inhibitor suitable for clinical evaluation is described. As part of an overall goal to minimize dose, we pursued a medicinal chemistry strategy focused on optimization of key parameters that influence dose size, including lowering human Cl and increasing intrinsic potency, bioavailability, and solubility. To impact these multiple parameters simultaneously, we used lipophilic ligand efficiency as a key metric to track changes in the physicochemical properties of our analogs, which led to improvements in overall compound quality. In parallel, structural information guided advancements in JAK1 selectivity by informing on new vector space, which enabled the discovery of a unique key amino acid difference between JAK1 (Glu966) and JAK2 (Asp939). This difference was exploited to consistently produce analogs with the best balance of JAK1 selectivity, efficacy, and projected human dose, ultimately culminating in the discovery of compound 28.

The systematic profiling of cellular functions and phenotypes, especially for cancer and inflammation diseases, enables patient stratification strategies and personalized medicine approaches. These studies include quantification of cell surface phenotypic and activation markers, and detection of secreted proteins and peptides. This information can aid discovery of both clinical biomarkers, as well as Ex Vivo/ In Vitro read-outs to guide development of novel therapeutics. The focus of this review is to highlight the value of the precision medicine data generated using novel high-dimensional technologies, such as mass cytometry (MC) and imaging MC, and to review emerging data tools which enable comprehensive data analysis and elucidation. Integration of these novel multiplex read-outs and corresponding mathematical analyses has facilitated discovery of previously unrecognized prognostic clinical biomarkers. During the last two decades our knowledge of cellular homeostasis during disease origination and progression has significantly advanced due to the systematic profiling of cellular functions and phenotypes, especially for immune-cell mediated pathologies [1,2]. In fact, many clinical trials now include cellular immunophenotyping as a biomarker component [3,4]. Patient specimens, often as small as a core needle biopsies or superfluous volumes of routinely collected fluids such as blood or sputum [5], are tested by a multitude of single cell profiling methods focusing on gaining a better understating of the cellular milieu. These studies include quantification of cell surface phenotypic and activation markers [6], ex-vivo response analysis to therapies [7], and detection of secreted proteins and peptides [8]. Multiplexed analyses throughout clinical trials have enabled the collection of valuable information from precious and limited patient specimens applied to investigate cellular disease perturbations. Ultimately these data aid in the development of disease diagnostic and prognostic tools, and more importantly determine patient stratification strategies which enable personalized medicine approaches. The focus of this review is to highlight the value of the translational data generated using novel technologies, such as mass cytometry (MC) and imaging MC, which has brought single cell immunophenotyping and imaging of the tumor microenvironment and other tissues to a new forefront, and to review emerging data tools which enable comprehensive data analysis and interpretation, often identifying previously unrecognized connections between cellular phenotypes and functions in healthy and diseased states [9][10][11][12].Although still in early development, MC based single cell immunophenotyping and analysis are steadily gaining recognition and becoming a mainstay in not only immune-mediated disease research, but in interrogating basic biology of other therapeutic targets [13][14][15][16]. MC or cytometry by time of flight (CyTOF) merges concepts of both flow cytometry and mass spectrometry [17,18]. This method r...

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