VB, JW, TB, FA, and LT were involved in manuscript writing and the final approval. macrophages. MOL2-14-571-s005.csv (257K) GUID:?B4DAB7AF-E529-4CE8-9DD7-7EA86DA7BFBC Table S5 . Differential protein abundance of macrophages comparing DLBCL\CM and M\CSF differentiated macrophages. MOL2-14-571-s006.csv (257K) GUID:?E0405417-DBFC-475E-A20A-E89273DE07B8 Abstract Macrophages (M) are abundantly present in the tumor microenvironment and may predict outcome in solid tumors and defined lymphoma subtypes. M heterogeneity, the mechanisms of their recruitment, and their differentiation into lymphoma\promoting, alternatively activated M2\like phenotypes are still not fully understood. Therefore, further functional studies are required to understand biological mechanisms associated with human tumor\associated M (TAM). Here, we show that the global mRNA expression and protein abundance of human M differentiated in Hodgkin lymphoma (HL)\conditioned medium (CM) differ from those of M educated by conditioned media from diffuse large B\cell lymphoma (DLBCL) cells or, classically, by macrophage colony\stimulating factor (M\CSF). Conditioned media from HL cells support TAM differentiation through upregulation of surface antigens such as CD40, CD163, CD206, and PD\L1. In particular, RNA and cell GP9 surface protein expression of (models show that co\cultures 5-Iodo-A-85380 2HCl of HL cells with monocytes or M support dissemination of lymphoma cells via lymphatic vessels, while tumor size and vessel destruction are decreased in comparison with 5-Iodo-A-85380 2HCl lymphoma\only tumors. Immunohistology of human HL tissues reveals a fraction of cases feature large numbers of CD206\positive cells, with high expression being characteristic of HL\stage IV. In summary, the lymphoma\TAM interaction contributes to matrix\remodeling and lymphoma cell dissemination. for 10?min at 4?C, sterile\filtered, and stored at 4?C for a maximum of 2?weeks. 2.1.1. Monocyte isolation Peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated from fresh buffy coats by density\gradient centrifugation over Biocoll separating solution (Biochrom, Berlin, Germany). CD14+ monocytes were obtained from PBMCs by magnetic cell separation using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturers instructions. Purity of CD14+ cells after magnetic cell separation was determined by staining with specific markers and quantification by flow cytometry using a FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA). 2.1.2. Macrophage differentiation Monocyte isolation and macrophage differentiation were performed as described previously (Menck microcomputed tomography (micro\CT) QuantumFX (Perkin Elmer Health Sciences, Hopkinton, MA, USA) and the following acquisition parameters: 90\kV tube voltage, 200\A tube current, FOV 20??20?mm2, 2\min total acquisition time resulting in 3D datasets with a voxel size of 40??40??40?m3. The software scry v6.0 (Kuchel & Sautter GbR, R?tenbach, Germany) was used for 3D rendering and volume measurement. For this purpose, the CAM around the tumor was manually removed using a virtual scalpel and the tumor mass was segmented based on a brightness threshold. 2.3. Transcriptomics The samples were analyzed by RNA\Seq. Read quality was assessed with fastQC (Andrews (2010): FastQC: a quality control tool for high\throughput sequence data. Available online at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), FastqPuri and QoRTs (Hartley and Mullikin, 2015; Perez\Rubio genome (release 87). The mean pseudo\alignment rate was 87.42%. In order to mitigate the donor effect, the combat function of the R\package sva was applied (Leek J.T. (2018): Available online at: http://bioconductor.org/packages/release/bioc/html/sva.html). DESeq2 was used for differential gene expression (GE) analysis and 5-Iodo-A-85380 2HCl and 60 subsequent SWATH windows of variable size for 35?ms each (mass range, 230C1500?test to correct for multiple comparisons as indicated. Normal distribution and homogeneity of variance were tested using the KolmogorovCSmirnov test and the test was performed for nonparametric testing. Significance levels are indicated as *(DC\SIGN), and were lowly expressed. More importantly, probably the most striking.