News from… Part II

Department of Obstetrics and Gynecology, Ulm University, 89075 Ulm, Germany

Multiparametric mass cytometry (CyTOF) for exploring the sex-dependent crosstalk between DNA damage and inflammatory response during aging
The widely acknowledged sexual dimorphism in life expectancy (4 years), immunosenescence, and the incidences of age-related diseases provide evidence for sex differences in the aging process. Previously, we showed that peripheral blood lymphocytes (PBL) from older men and women differentially regulate multiple DNA damage and replication stress response pathways differentially contributing to the increase in genomic instability with age. Genomic instability and replication stress can lead to the release of DNA fragments from the nucleus into the cytoplasm, where these fragments activate the cyclic GMP-AMP synthase (cGAS) causing downstream activation of the stimulator of interferon genes (STING). Subsequent signaling involves NF-κB activation, senescence, and inflammation leading to immune cell recruitment. To correlate the crosstalk between replication stress, DNA damage response (DDR), and inflammation and to identify sex-differences within, we use multiparametric mass cytometry (CyTOF) as key method. To identify the immune cell subpopulations in cycling human PBL, we applied the Maxpar Direct Immune Profiling Assay (MDIPA) and added multiple markers for DDR, senescence, and cytoplasmatic DNA sensing. Ultimately, our panel consists of 47 markers including platinum-labeled antibodies. We successfully applied our panel to cycling human PBL after 72 h PHA-stimulating culture prior to staining. In total, we measured 85 samples from young (<26) and older (>60) men and women and have overcome obstacles caused by the experimental conditions required for DDR studies. Our ongoing data analysis shall reveal sex differences in the age-related amplification of DNA damage and inflammation.

German Rheumatology Research Center (DRFZ), a Leibniz Institute, Berlin, Germany

Comparative Performance Benchmarking of Lunarion vs. Helios and CyTOF XT Mass Cytometers
The Lunarion mass cytometer (Polaris Biology) is a recently developed platform for high-throughput, high-dimensional single-cell analysis. Installed at LIH (Luxembourg) and DRFZ (Berlin), its performance was evaluated in direct comparison with established Helios and CyTOF XT systems (Standard BioTools). Benchmarking was performed using standardized multi-element solutions, metal-labeled beads, and complex biological samples to assess the instruments under realistic operational conditions.

Sensitivity profiling using multi-element solutions revealed highly consistent performance across all channels, with near-identical signal curves between the two Lunarion instruments and approximately fivefold higher sensitivity relative to the CyTOF XT. Analysis of 46 single-stained, metal-labeled compensation beads spanning the mass range from ⁸⁹Y to ²⁰⁹Bi demonstrated an average one-log increase in signal intensity across most channels on the Lunarion compared with the Helios system. This gain was particularly pronounced in the lower atomic mass range (<150 amu). Importantly, it affected both positive and negative bead populations and was associated with a slightly higher staining index in most, though not all, channels. Coefficients of variation (CVs) were similar to those observed on the Helios and typically remained below 30%, indicating comparable measurement precision.

To evaluate performance on biological samples, phospho-signaling responses were analyzed in human whole blood from two donors following stimulation with innate immune ligands at multiple time points, which were barcoded using a combined beta-2-microglobulin and TeMal-based approach. Both platforms reliably delineated major immune populations and retained donor-specific phosphorylation profiles. Signal kinetics and fold-change dynamics for key markers such as p-p38 and pTBK1 (e.g., in classical monocytes) were closely aligned across instruments, supporting cross-platform reproducibility of cellular response measurements.

National Cytometry Platform, Luxembourg Institute of Health, Luxembourg

Democratization of mass cytometry and data analysis (#BI4Flow)
The recent deployment of the Lunarion benchtop mass cytometer represents a transformative shift toward the democratization of mass cytometry in research and clinical settings. Historically, mass cytometry has been confined to specialized core facilities requiring dedicated operators and substantial infrastructure, limiting accessibility for individual research groups. The Lunarion’s user-friendly design and simplified maintenance protocols enable scientists to operate the instrument directly, effectively transferring analytical capacity from core facility specialists to end-users. This democratization parallels the paradigm shift initiated by Business Intelligence (BI) tools in the corporate analytics landscape, where domain experts gain direct access to sophisticated analytical capabilities without requiring specialized technical expertise. By integrating Lunarion’s operational accessibility with BI principles—including interactive data visualization, intuitive interfaces, and seamless data sharing mechanisms—we can establish a framework that empowers immunologists and cell biologists to conduct complex multidimensional analyses independently. This integration not only accelerates scientific discovery through rapid iteration and hypothesis testing but also fosters collaborative science through interactive visualizations that enable non-expert stakeholders to explore and interpret high-dimensional single-cell data. We propose that this convergence of accessible hardware, user-centric software design, and business intelligence methodologies will fundamentally reshape how single-cell analysis is conducted across academic, clinical, and industrial research environments.

Institute of Pathology, University Medicine Halle, Martin Luther University Halle-Wittenberg, 06112 Halle (Saale), Germany
Section of Immunopathology, Institute of Pathology, Martin Luther University Halle-Wittenberg, 06112 Halle (Saale), Germany

Establishment of Imaging Mass Cytometry for High-Dimensional Spatial Profiling of Decalcified Bone Marrow Biopsies

Background: Comprehensive spatial characterization of the bone marrow microenvironment in myeloproliferative neoplasms (MPN) is technically challenging due to tissue complexity and bone decalcification. Imaging mass cytometry (IMC) enables high-dimensional, spatially resolved single-cell analysis; however, its implementation in decalcified bone marrow biopsies remains limited.

Methods: We established an IMC workflow for decalcified bone marrow biopsies using a panel of 32 metal-conjugated antibodies targeting immune, stromal, and proliferative markers. IMC was performed on 22 biopsies from MPN patients before treatment and after therapy with the JAK inhibitors Ruxolitinib or Fedratinib. Following image acquisition, robust single-cell segmentation and unsupervised clustering were applied to define cell (sub) populations. Distribution of cell (sub)populations and marker expression profiles were compared across treatment conditions to demonstrate the applicability of the approach.

Results: IMC analysis of decalcified bone marrow biopsies (whole slide) enabled the detection of major immune and stromal cell populations and identified 11 distinct cellular clusters based on the marker expression. Spatially resolved analyses revealed cell type-specific expression patterns and treatment-associated shifts in the cellular composition. Bone marrow samples from Ruxolitinib-treated patients showed increased CD68+CD163- macrophage and myofibroblast populations accompanied by reduced expression of (immune) signaling markers (NFkB, pERK, pSTAT3) and proliferation (Ki-67) markers. In contrast, bone marrow samples from patients treated with Fedratinib had a decreased CD14+ monocyte abundance and increased expression of Ki-67 and NFkB compared to biopsies prior to treatment.

Conclusions: Here, we established a high-dimensional, spatial single-cell profiling of decalcified bone marrow biopsies. The presented workflow enables detailed characterization of the bone marrow microenvironment and provides a basis for future studies investigating treatment-induced remodeling in MPN and other hematologic diseases.

Institute of Immunology, Core Facility Cytometry, Jena University Hospital, Friedrich-Schiller-University Jena, Jena, Germany

Introducing UriTOF: A Preservation Workflow for Urine CyTOF
Objective: Mass cytometry (CyTOF) allows high-dimensional single-cell analysis but typically requires immediate sample processing, which limits its use for fragile, low-cell clinical specimens such as urine.

Methods: We developed UriTOF, a preservation strategy that enables delayed processing by combining cisplatin viability staining with gentle fixation and cryopreservation. The protocol was validated using PBMCs and applied to urine samples from kidney transplant recipients.

Results: Cell integrity, surface marker expression, and viability discrimination were maintained across freeze–thaw cycles and low cell inputs. Urine immune profiles were comparable to standard CyTOF workflows and enabled identification of urinary CD8⁺CD38⁺ T cells as a marker of T cell–mediated rejection, validated across multiple orthogonal platforms.

Conclusion: UriTOF enables delayed and standardized CyTOF analysis of low-yield urine samples, supporting clinical and translational immune profiling and facilitating non-invasive biomarker discovery in kidney transplantation.

Department of Internal Medicine III with Haematology, Medical Oncology, Haemostaseology, Infectiology and Rheumatology, Oncologic Center, Paracelsus Medical University, Salzburg, Austria

Salzburg Cancer Research Institute – Laboratory for Immunological and Molecular Cancer Research (LIMCR), Austria

Cancer Cluster Salzburg, Salzburg, Austria

Department of Biosciences and Medical Biology, Paris-Lodron University Salzburg, Salzburg, Austria

Institute of Pathology, Paracelsus Medical University Salzburg, 5020 Salzburg, Austria

Dept. of Artificial Intelligence and Human Interfaces, Paris-Lodron-University Salzburg, AustriaAbstract:

Interactive Cell Gating and ultra-specific detection of RNA: New IMC tools for focused microenvironment analysis

Image Mass Cytometry (IMC) enables the study of complex biological environments such as tumor-immune landscapes, where the spatial context of expanding tumor and immune cell clones is of high relevance. Current workflows cluster cells by their expression to cellular patches or neighborhoods, but intuitive tools for selecting individual cells based on their spatial location are currently lacking. Moreover, visualizing individual clones by their unique CDR3
region, which necessitates the detection of ultra-specific RNA, is currently not supported by standard IMC workflows. To address these problems, we present two novel tools.
Spatialgater provides an interactive web interface for spatial gating of cells in IMC data. Users can visualize cells on zoomable tissue images and select specific cells by drawing polygon gates. Cell identifiers from selected cells can be exported as CSV files or written back as logical gate columns in colData() of the SpatialExperiment for downstream analysis. We provide the function as an R package available at https://github.com/Mark-Ste/spatialgater.
While simultaneous detection of mRNA (>300 bp) is possible, there is currently no technique for the detection of RNA with single-base pair precision, required for the study of subclonal tumor evolution and identification of clonal expansion in situ. Therefore, we introduce a protocol for combined microscopic and IMC staining on single slides, enabling the integration of single base pair-specific RNA information and spatial protein multiplex data.
We showcase the usability and benefit for microenvironment analysis of the gating tool andultra-specific RNA-protein co-detection in a leukemic setting.

Department I of Internal Medicine, Medical Faculty of the University of Cologne, and University Hospital Cologne

From TeLEV to MULTI-TeLEV – Simultaneous profiling of EV uptake and recipient cell signaling from six EV sources
Abstract

Extracellular vesicles (EVs) are potent modulators of tumor immune microenvironments. To study these interactions at single-cell resolution, we previously developed TeLEV (Tellurium-based labeling of extracellular vesicle proteomes), utilizing the metabolic incorporation of L-2-tellurienylalanine (TePhe) to create mass-tagged EVs. Using this approach, we identified the specific induction of an interleukin receptor triad (CD25, CD123, CD127) and downstream STAT5 signaling as a functional consequence of B-cell malignant EV uptake.

In this update, we present MULTI-TeLEV (Multiplexed isotopically enriched TeLEV), a significant methodological advancement designed to interrogate complex EV environments. By synthesizing TePhe variants with distinct enriched tellurium isotopes, MULTI-TeLEV enables simultaneous tracking of EVs from up to six sources within the same experimental setting.

Leveraging this capability, we provide new insights into the rules of EV entry. We demonstrate that EV uptake is highly competitive. When recipient cells are exposed to multiple EV sources simultaneously, uptake is not stochastic; rather, we observe distinct uptake patterns where specific EV sources are preferentially internalized over others. MULTI-TeLEV thus enables the precise deconvolution of source-specific uptake hierarchies that are invisible in conventional single-source assays.