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Triple‐negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer that lacks targeted therapy. TNBC manifests characteristic, extensive intratumoral heterogeneity that promotes disease progression and influences drug response. Single‐cell techniques in combination with next‐generation computation provide an unprecedented opportunity to identify molecular events with therapeutic potential. Here, we describe the generation of a comprehensive mass cytometry panel for multiparametric detection of 23 phenotypic markers and 13 signaling molecules. This single‐cell proteomic approach allowed us to explore the landscape of TNBC heterogeneity, with particular emphasis on the tumor microenvironment. We prospectively profiled freshly resected tumors from 26 TNBC patients. These tumors contained phenotypically distinct subpopulations of cancer and stromal cells that were associated with the patient's clinical status at the time of surgery. We further classified the epithelial‐mesenchymal plasticity of tumor cells, and molecularly defined phenotypically diverse populations of tumor‐associated stroma. Furthermore, in a retrospective tissue‐microarray TNBC cohort, we showed that the level of CD97 at the time of surgery has prognostic potential.

Background:Background: Human Leukocyte Differentiation Antigen (HLDA) workshops are organized by the Human Cell Differentiation Molecules (HCDM) consortium to test and validate the reactivity of particular antibody clones to specific targets. Thereby the consortium provides the scientific community validated antibody clones reactive to particular cluster of differentiation (CD) markers. Although this approach has been used since the 1980s, quantitative profiling of CD markers at "single-cell" level and benchmarking of reagents are currently lacking. Aims:Aims: We aimed to develop a flow cytometric procedure allowing CD marker expression profiling in a standardized way in time and place Methods: Methods: First, we developed and titrated two antibody panels with a free position in the phycoerythrin (PE) channel.The panels enable identification of 27 innate and adaptive leukocyte cell populations present in peripheral blood. The panels were custom dried in 96-well plates, and the Quantibrite™ PE Beads were used for quantification of the PE signal. Subsequently, we developed a high content framework to evaluate the titration of PE conjugated monoclonal antibodies using fluorescently barcoded cell lines and peripheral blood cells. The selected titer and critical antibody information (such as clone, catalogue number, vendor, gene and CDname etc.) were centrally stored in an inventory table, which was expanded into an experiment master table (EMT) following inclusion of experimental details (e.g. the position of individual mAbs in 96-well plate, experiment name, operator etc.). The EMT was used to generate an experimental protocol with automated calculation of reagent amounts and volumes. Post acquisition, the fcs files were annotated using all relevant experimental information from the EMT table. Results: Results:To validate our approach, we quantified protein expression of four selected CD markers (CD11b, CD31, CD38 and CD40) with well-known expression pattern on peripheral blood leukocytes that showed high reproducibility across centers. We also performed benchmarking of four anti-CD3 clones, of which the titration curves revealed variable performance: from high-performance TB3 clone, through intermediate-performance UCHT1 and SK7 clones to lowperformance MEM-57 clone. Three out of four anti-CD3 clones showed similar pattern of staining, whereas the MEM-57 clone showed decreased intensity on CD4 and CD8 T cells while retaining comparable intensity to the other clones for TCRγ δ + T cells. Our pilot results on childhood T-ALL patient samples (n=7) revealed potential targets for minimal residual disease monitoring.Summary/Conclusion: Summary/Conclusion: In summary, we optimized a procedure for quantitative expression profiling of surface antigens on subsets of blood leukocyte and proved its feasibility with inter-laboratory comparison in three different laboratories. The presented workflow enables (i) to map the expression patterns of HLDA-approved antibody clones to CD markers, (ii) to benchmark new antibody clones to...

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