A holistic understanding of tissue and organ structure and function requires the detection of molecular constituents in their original three-dimensional (3D) context. Imaging mass cytometry (IMC) enables simultaneous detection of up to 40 antigens and transcripts using metal-tagged antibodies but has so far been restricted to two-dimensional imaging. Here we report the development of 3D IMC for multiplexed 3D tissue analysis at single-cell resolution and demonstrate the utility of the technology by analysis of human breast cancer samples. The resulting 3D models reveal cellular and microenvironmental heterogeneity and cell-level tissue organization not detectable in two dimensions. 3D IMC will prove powerful in the study of phenomena occurring in 3D space such as tumor cell invasion and is expected to provide invaluable insights into cellular microenvironments and tissue architecture.

Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO 4 and RuO 4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO 4 and RuO 4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO 4 and RuO 4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO 4 and RuO 4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. V C 2016 International Society for Advancement of Cytometry Key terms mass tag cellular barcoding; CyTOF; mass cytometry; osmium tetroxide; ruthenium tetroxide ELEMENTAL mass spectrometry-based cytometry, or mass cytometry (1-4), is a recently developed technology platform that enables characterization of single cells using affinity reagents, such as antibodies covalently linked to polymeric, metalloaded chelators (4). Use of elemental metal isotopes, as opposed to fluorochromes, enables quantification of up to 46 epitope abundances simultaneously on individual cells (1-3). The detection modality in mass cytometry is an inductively coupled plasma time of flight mass spectrometer (ICP-TOF MS). In this instrument, metal isotope labeled cells are atomized and ionized in an argon plasma and the abundance and distribution of the isotopes is measured in a TOF mass analyzer. With the current generation of mass cytometry instruments, 135 isotope channels can be recorded simultaneously at an acquisition rate of $2,000 cells/ second (5,6).Recently we introduced a multiplexing strategy called MCB, in which metal isotopes are used to label samples with unique metal isotope barcodes (6,7). After sample mixing, cells are labeled with antibodies and analyzed on the mass cytometer. In a first data analysis step, the single cells are then assigned to their sample based on their metal isotope barcode. The main advantages of this approach are that all cells are stained homogeneously with the same antibody mix, the fluidics system of the mass cytometer does not have to be cleaned for individual samples, and all MCB samples are subjected to the same instrument drift. Using this strategy, up to 96 samples can be barcoded and 39 different parameters analyzed on each single cell (7).Both lanthanide isotopes and palladium isotopes have been used to barcode cells (6,7). In both methods ion chelating bifunctional compounds that react with cells

Methotrexate (MTX) is an antiproliferative drug used for treating inflammatory diseases, including psoriasis. Nevertheless, its use in localized therapy is hindered because of poor transdermal penetration. We show that MTX coupled with gold nanoparticles (GNPs) demonstrates superior antiinflammatory efficacy than MTX alone in an imiquimod-induced mouse model, significantly reducing gd T cells, CD4 þ T cells, and neutrophils. Furthermore, it was well tolerated upon systemic and topical administration. In an AGR129 human xenograft mouse model, two-week topical treatment with MTX-GNPs inhibited skin hyperplasia significantly better than topical calcipotriol-betamethasone and led to profound tissue remodeling, involving the upregulation of extracellular matrix reorganization and the downregulation of cornification and keratinization processes. The number of resident T cells in the grafts, as well as interleukin-17 production, drastically decreased upon MTX-GNP treatment. While both MTX and MTX-GNPs directly prevented the proliferation and induced apoptosis of T cells, the suppression of cytokine production was a shared mechanism of GNP and MTX-GNPs. In conclusion, MTX-GNPs influence immune and stromal components of the skin, leading to the potent inhibition of pathogenesis in preclinical psoriasis. MTX-GNPs surpass the efficacy of conventional MTX and standard of care, emerging as a non-steroidal, topical alternative for psoriasis treatment.

Neutrophils are the first nonresident effector immune cells that migrate to a site of infection or inflammation; however, improper control of neutrophil responses can cause considerable tissue damage. Here, we found that neutrophil responses in inflamed or infected skin were regulated by CCR7-dependent migration and phagocytosis of neutrophils in draining lymph nodes (dLNs). In mouse models of Toll-like receptor–induced skin inflammation and cutaneous Staphylococcus aureus infection, neutrophils migrated from the skin to the dLNs via lymphatic vessels in a CCR7-mediated manner. In the dLNs, these neutrophils were phagocytosed by lymph node–resident type 1 and type 2 conventional dendritic cells. CCR7 up-regulation on neutrophils was a conserved mechanism across different tissues and was induced by a broad range of microbial stimuli. In the context of cutaneous immune responses, disruption of CCR7 interactions by selective CCR7 deficiency of neutrophils resulted in increased antistaphylococcal immunity and aggravated skin inflammation. Thus, neutrophil homing to and clearance in skin-dLNs affects cutaneous immunity versus pathology.