Mass cytometry (MC) is a high throughput multiparameter analytical technique for determining biomarker expression in cells.

We are interested in developing lanthanide nanoparticles (NPs) as high sensitivity tagging reagents for antibodies to analyze cells by mass cytometry (MC). Two key prerequisites for this application are that the NPs have to be colloidally stable in phosphate-containing buffers and the free NPs must have very low levels of nonspecific binding to cells. These are the issues we address here. We describe the synthesis of 30 nm diameter NaYF4:Yb,Er nanoparticles, their transfer to aqueous solution via citrate exchange, and their encapsulation in liposomes to minimize their interaction with live cells. The lipid coating consisted of a 2:2:1 mol ratio mixture of dioleoylphosphatidyl choline (DOPC), egg sphingomyelin (ESM), and ovine cholesterol (Chol), referred to as DEC221. Since encapsulating 30 nm NPs in liposomes is an unprecedented challenge, we added varying amounts of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG-2000] (mPEG2K-DSPE) to the lipid formulation, both to promote curvature of the lipid coating and to use the polyethylene glycol (PEG) chains to impart stealth and minimize interaction with cells. We succeeded in coating individual NPs with the lipid bilayer and showed that, after coating, the NPs were colloidally stable in PBS buffer for up to one month. We used MC to measure nonspecific binding of the lipid-coated NPs to three different suspension cell lines, Ramos, THP-1, and KG1a cells. For dosages of 50, 100, and 1000 NPs/cell, the measured signals were barely above background. For dosages of 10 000 and 30 000 NPs/cell, nonspecific binding levels were on the order of 10–15 NPs per cell, less than 0.1% of the applied dose. Dopant ions such as Yb also provide a measurable signal, indicating that NaYF4 NPs can serve as a useful host matrix for different lanthanide dopants for multiparameter experiments. These are very encouraging results for future experiments in which specific antibodies will be incorporated into the lipid coating.

In pharmaceutical research, high‐content screening is an integral part of lead candidate development. Measuring drug response in vitro by examining over 40 parameters, including biomarkers, signaling molecules, cell morphological changes, proliferation indices, and toxicity in a single sample, could significantly enhance discovery of new therapeutics. As a proof of concept, we present here a workflow for multidimensional Imaging Mass Cytometry™ (IMC™) and data processing with open source computational tools. CellProfiler was used to identify single cells through establishing cellular boundaries, followed by histoCAT™ (histology topography cytometry analysis toolbox) for extracting single‐cell quantitative information visualized as t‐SNE plots and heatmaps. Human breast cancer‐derived cell lines SKBR3, HCC1143, and MCF‐7 were screened for expression of cellular markers to generate digital images with a resolution comparable to conventional fluorescence microscopy. Predicted pharmacodynamic effects were measured in MCF‐7 cells dosed with three target‐specific compounds: growth stimulatory EGF, microtubule depolymerization agent nocodazole, and genotoxic chemotherapeutic drug etoposide. We show strong pairwise correlation between nuclear markers pHistone3 S28 , Ki‐67, and p4E‐BP1 T37/T46 in classified mitotic cells and anticorrelation with cell surface markers. Our study demonstrates that IMC data expand the number of measured parameters in single cells and brings higher‐dimension analysis to the field of cell‐based screening in early lead compound discovery.

Mass cytometry (MC) is a bioanalytical technique that uses metal-tagged antibodies (Abs) for high-dimensional single-cell immunoassays. Currently, this technology can measure over 40 parameters simultaneously on individual cells using metal-chelating polymer (MCP) based reagents. However, MC can in principle detect up to 135 parameters with the development of new elemental mass tags. Here we report the development of a tantalum oxide nanoparticle (NP)-based mass tag for MC immunoassays. Uniform-sized amine-functionalized tantalum oxide NPs (d ∼ 5.7 nm) were synthesized via a one-pot two-step reverse microemulsion method. These amine-functionalized NPs were further modified with azide groups by reacting with azide-PEG2k succinimidyl carboxymethyl ester (NHS-PEG2k -N3) cross-linkers. The Ab-NP conjugates were prepared by reacting azide-functionalized NPs with dibenzocyclooctyne (DBCO)-functionalized primary or secondary Abs (DBCO-Ab) followed by fast protein size exclusion liquid chromatography (FPLC) purification. Three Ab-NP conjugates (TaO2-PEG2k -goat antimouse, TaO2-PEG2k -CD25, TaO2-PEG2k -CD196) were fabricated and tested in MC immunoassays. For the TaO2-PEG2k -goat antimouse conjugate, we showed that it can effectively detect abundant CD20 biomarkers on Ramos cells. For TaO2-PEG2k -CD25 and TaO2-PEG2k -CD196 conjugates, we demonstrated that these Ab-NP conjugates could be integrated into the commercial Ab staining panels for high-dimensional single-cell immune profiling of human peripheral blood mononuclear cells.

Mass cytometry is a highly multiplexed single-cell analysis platform that uses metal-tagged reagents to identify multiple cellular biomarkers. The current metal-tagged reagent preparation employs thiol-maleimide chemistry to covalently couple maleimide-functionalized metal-chelating polymers (MCPs) with antibodies (Abs), a process that requires partial reduction of the Ab to form reactive thiol groups. However, some classes of Abs (for example, IgM) as well as biomolecules lacking cysteine residues have been challenging to label using this method. This inherent limitation led us to develop a new conjugation strategy for labeling a wide range of biomolecules and affinity reagents. In this report, we present a metal tagging approach using a new class of azide- or transcyclooctene-terminated MCPs with copper(I)-free strain-promoted alkyne-azide cycloaddition or tetrazine-alkene click chemistry reactions, in which biomolecules with -NH functional groups are selectively activated with a dibenzocyclooctyne or tetrazine moiety, respectively. This approach enabled us to generate highly sensitive and specific metal-tagged IgGs, IgMs, small peptides, and lectins for applications in immunophenotyping and glycobiology. We also created dual-tagged reagents for simultaneous detection of markers by immunofluorescence, mass cytometry, and imaging mass cytometry using a two-step conjugation process. The Helios mass cytometer was used to test the functionality of reagents on suspension human leukemia cell lines and primary cells. The dual-tagged Abs, metal-tagged lectins, and phalloidin staining reagent were used to visualize target proteins and glycans on adherent cell lines and frozen/FFPE tissue sections using the Hyperion Imaging System. In some instances, reagents produced by click conjugation showed superior sensitivity and specificity compared to those of reagents produced by thiol-maleimide chemistry. In general, the click chemistry-based conjugation with new MCPs could be instrumental in developing a wide range of highly sensitive metal-containing reagents for proteomics and glycomics applications.

In pharmaceutical research, high-content screening is an integral part of lead candidate development. Drug response in vitro over 40 parameters including biomarkers, signaling molecules, cell morphological changes, proliferation indexes and toxicity in a single sample could significantly enhance discovery of new therapeutics. As a proof of concept, we present a workflow for multidimensional Imaging Mass Cytometry™ (IMC™) and data processing with open source computational tools. CellProfiler was used to identify single cells through establishing cellular boundaries, followed by histoCAT™ (histology topography cytometry analysis toolbox) for extracting single-cell quantitative information visualized as t-SNE plots and heatmaps. Human breast cancer-derived cell lines SKBR3, HCC1143 and MCF-7 were screened for expression of cellular markers to generate digital images with a resolution comparable to conventional fluorescence microscopy. Predicted pharmacodynamic effects were measured in MCF-7 cells dosed with three target-specific compounds: growth stimulatory EGF, microtubule depolymerization agent nocodazole and genotoxic chemotherapeutic drug etoposide. We show strong pairwise correlation between nuclear markers pHistone3 S28 , Ki-67 and p4E-BP1 T37/T46 in classified mitotic cells and anti-correlation with cell surface markers. Our study demonstrates that IMC data expands the number of measured parameters in single cells and brings higherdimension analysis to the field of cell-based screening in early lead compound discovery.

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.

The synthesis of a polylysine polymer functionalized with the previously reported astonishingly inert [In(cb-te2pa)] + chelate was performed. A biotin end group allowed the conjugation to biotinylated beads by the intermediary of a Fluorescein isothiocyanate / neutravidin receptor. High quality Imaging Mass Cytometry TM trials, based on 115 In detection were performed to highlight the behavior of the material. Anti-CD20 antibody was labeled by the so obtained In(III) modified polylysine using the biotin/neutravidin interaction. Ramos (CD20[+]) and HL-60 (CD20[-]) cell lines were co-stained with the In(III) modified bioconjugate by finding the best staining conditions. Both immunofluorescence IF-M and Mass Cytometry analyses confirmed the specific binding of anti-CD20 onto Ramos cells. CyTOF R histograms constructed on the 115 In detection allowed to define and to separate, with a good signal-to-noise ratio, two populations (Ramos and HL-60). The inertness of In(III)-MCP-Nav over a three-month storage period was proved by performing new functionality tests involving Jurkat cells (CD20[-]) as well as the possibility of multiplexing trials involving the 115 In channel. The results assure a promising future use of the previously announced [In(cb-te2pa)] + complex based polymers for mass cytometry. Electronic Supplementary Information (ESI) available: [1H NMR of TFA-Lys-NCA, Poly(TFA-L-Lys)-Btn, Poly(L-Lys)-Btn and (PEGpoly(L-Lys)-Btn); GPC chromatogram of Poly(L-Lys)-Btn; NMR integration for PEG number evaluation;. Selected areas of blanks' and beads' 115 In integral intensities on IMC images of indium beads; Average 154 Sm counts per cell for Jurkat (CD45[+]) and Ramos (CD45[+]) cells; Overlayed histograms of Jurkat and Ramos cells].