Transcriptome analysis RNA-seq libraries were prepared using the NEXTFlex Rapid Directional qRNA-Seq Kit (Bioo Scientific Corp.) according to manufacturer’s instructions. Libraries were sequenced on the Illumina NextSeq550 platform (SR50). Reads were mapped to the human reference assembly and quantified with the STAR software (version 2.5). Based on these counts, statistical analysis of differential expression was carried out with DESeq2. Laminar flow adhesion assay The cell adhesion of MCC cell lines to selectins under physiological flow conditions was analyzed as described previously ????(Starzonek et al., 2020)?? using ibidi µ-slides VI 0.4 (ibiTreat, IBIDI) connected to a syringe pump (kdScientific, Holliston, MA, USA, Model 100 Series). (rh) E- and P-selectin FC-chimeras (R&D Systems, Minneapolis, MN, USA) were diluted in DPBS (Gibco) (5 µg/ml) and incubated for 30 min at 37°C. Tumor cells (1*105 cells/ml) were analyzed with a continuous flow rate of 8 ml/min and shear stress of 0.25 dyn/cm2. Cell attachment was assessed with an inverted microscope (Zeiss, Jena, Germany, Axiovert 200), data were analyzed with CapImage software (version 8.6., Dr. Heinrich Zeintl, Heidelberg). Each experiment was repeated three times. Phagocytic assay Differentiated macrophages were washed with PBS, detached using Accutase (Invitrogen) and seeded on glass coverslips in a 24-well plate (1x105). After 24 hrs, macrophages were co-cultured with Dox-/DMSO- treated WaGa shRNA sT cells (1:5 ratio). Additionally, macrophages were co-cultured with WaGa cells transfected with siRNA scr or siRNA targeting sT (both Horizon Discovery) using Viromer blue transfection reagent (Lypocalyx, Halle (Saale). Blocking experiments were performed in the presence of anti-CD47 antibody (B6.H12.2) or IgG control (Invitrogen). After 0.5, 1, 2, and 4 h cells were washed with PBS, fixed for 10 min using 4 % PFA and mounted in DAPI Mounting Medium. Samples were analyzed with a confocal laser scanning microscope (Leica TCS SP5) equipped with a 63x, NA1.4 oil immersion objective and Leica LAS AF software (Leica Microsystems, Wetzlar, Germany). The phagocytic index (no. of WaGa cells divided by no. of macrophages) was calculated, counting at least 100 macrophages per condition. Each condition was performed at least in triplicate. MACS Marker Screen WaGa shRNA sT/scrambled cells were induced with 1 µg/ml doxycycline or DMSO 3 days prior to each experiment. Cells were incubated with CellTrace™ Violet or Yellow (Invitrogen). After 24 h a surface marker screening (MACS Marker Screen Milteny Biotech) was performed according to manufacturer’s instructions using a FACS Fortessa flow cytometer (BD Biosciences). For data analysis, median fluorescence intensity (MFI) for each surface marker and cell line was determined via FlowJo software (Version X 10.0.7r2; Tree Star, Inc.). For general ligand expression, results of WaGa shRNA sT cells were compared to WaGa shRNA scrambled cells both inoculated with DMSO. All ligands with an MFI >100 in at least one of the cell lines were included in the analysis. Differences in surface marker expression (Log2Fold changes) were calculated by MFI of Dox-treated cells normalized to DMSO controls. Log2diff was determined by subtraction of Log2FoldChanges from WaGa shRNA scr from WaGa shRNA sT. Thresholds were set at Log2diff ± 0,1.