Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding.

Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.

Mass Cytometry Tags: Where Chemistry Meets Single-Cell Analysis.

Mass cytometry is a highly multiparametric proteomic technology that allows the measurement and quantification of nearly 50 markers with single-cell resolution. Mass cytometry reagents are probes tagged with metal isotopes of defined mass and act as reporters. Metals are detected using inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS). Many different types of mass-tag reagents have been developed to afford myriad applications. We have classified these compounds into polymer-based mass-tag reagents, nonpolymer-based mass-tag reagents, and inorganic nanoparticles. Metal-chelating polymers (MCPs) are widely used to profile and quantify cellular biomarkers; however, both the range of metals that can be detected and the metal signals have to be improved. Several strategies such as the inclusion of chelating agents or highly branched polymers may overcome these issues. Biocompatible materials such as polystyrene and inorganic nanoparticles are also of profound interest in mass cytometry. While polystyrene allows the inclusion of a wide variety of metals, the high metal content of inorganic nanoparticles offers an excellent opportunity to increase the signal from the metals to detect low-abundance biomarkers. Nonpolymer-based mass-tag reagents offer multiple applications: cell detection, cell cycle property determination, biomarker detection, and mass-tag cellular barcoding (MCB). Recent developments have been achieved in live cell barcoding by targeting proteins (CD45, b2m, and CD298), by using small and nonpolar probes or by ratiometric barcoding. From this perspective, the principal applications, strengths, and shortcomings of mass-tag reagents are highlighted, summarized, and discussed, with special emphasis on mass-tag reagents for MCB. Thereafter, the future perspectives of mass-tag reagents are discussed considering the current state-of-the-art technologies.

Drug "Clicking" on Cell-Penetrating Fluorescent Nanoparticles for In Cellulo Chemical Proteomics.

Chemical proteomics approaches are widely used to identify molecular targets of existing or novel drugs. This manuscript describes the development of a straightforward approach to conjugate azide-labeled drugs via click chemistry to alkyne-tagged cell-penetrating fluorescent nanoparticles as a novel tool to study target engagement and/or identification inside living cells. A modification of the Baeyer test for alkynes allows monitoring the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, guaranteeing the presence of the drug on the solid support. As a proof of concept, the conjugation of the promiscuous kinase inhibitor dasatinib to Cy5-labeled nanoparticles is presented. Dasatinib-decorated fluorescent nanoparticles efficiently inhibited its protein target SRC in vitro, entered cancer cells, and colocalized with SRC in cellulo.

Multiparameter single-cell proteomic technologies give new insights into the biology of ovarian tumors.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnosis at advanced stage compounded with its excessive genomic and cellular heterogeneity make curative treatment challenging. Two critical therapeutic challenges to overcome are carboplatin resistance and lack of response to immunotherapy. Carboplatin resistance results from diverse cell autonomous mechanisms which operate in different combinations within and across tumors. The lack of response to immunotherapy is highly likely to be related to an immunosuppressive HGSOC tumor microenvironment which overrides any clinical benefit. Results from a number of studies, mainly using transcriptomics, indicate that the immune tumor microenvironment (iTME) plays a role in carboplatin response. However, in patients receiving treatment, the exact mechanistic details are unclear. During the past decade, multiplex single-cell proteomic technologies have come to the forefront of biomedical research. Mass cytometry or cytometry by time-of-flight, measures up to 60 parameters in single cells that are in suspension. Multiplex cellular imaging technologies allow simultaneous measurement of up to 60 proteins in single cells with spatial resolution and interrogation of cell-cell interactions. This review suggests that functional interplay between cell autonomous responses to carboplatin and the HGSOC immune tumor microenvironment could be clarified through the application of multiplex single-cell proteomic technologies. We conclude that for better clinical care, multiplex single-cell proteomic technologies could be an integral component of multimodal biomarker development that also includes genomics and radiomics. Collection of matched samples from patients before and on treatment will be critical to the success of these efforts.

Measuring trogocytosis between ovarian tumor and natural killer cells.

Trogocytosis is an active transport mechanism by which one cell extracts a plasma membrane fragment with embedded molecules from an adjacent cell in a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting key experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. For complete details on the use and execution of this protocol, please refer to Gonzalez et al. (2021).

High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment.

Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy.

PCR-free and chemistry-based technology for miR-21 rapid detection directly from tumour cells.

miRNAs are well known for being implicated in a myriad of biological situations, including those related to serious diseases. Amongst miRNAs, miRNA-21 has the spotlight as it is reported to be up-regulated in multiple severe pathological conditions, being its quantification a key point in medicine. To date, most of the techniques for miRNA quantification have shown to be less effective than expected; thus, we herein present a novel, rapid, cost-effective, robust and PCR-free approach, based on dynamic chemistry, for the identification and quantification of miRNA directly from tumour cells using both FACS and a fluorescent microplate. This dynamic chemistry novel application involves bead based reagents and allows quantifying the number of miR-21 molecules presented in MDA-MB-468 and H1975 tumour cells.

Metallofluorescent Nanoparticles for Multimodal Applications.

Herein, we describe the synthesis and application of cross-linked polystyrene-based dual-function nano- and microparticles containing both fluorescent tags and metals. Despite containing a single dye, these particles exhibit a characteristic dual-band fluorescence emission. Moreover, these particles can be combined with different metal ions to obtain hybrid metallofluorescent particles. We demonstrate that these particles are easily nanofected into living cells, allowing them to be used for effective fingerprinting in multimodal fluorescence-based and mass spectrometry-based flow cytometry experiments. Likewise, the in situ reductions of the metal ions enable other potential uses of the particles as heterogeneous catalysts.