Gene expression profiling of tumor-associated macrophages and in-vitro cultured macrophages
ABSTRACT: The aim of this study was to decipher gene expression of tumor-associated macrophage in the 4T1 mouse model of breast cancer. Overall design: For M0 basal macrophages, Ly6C+ monocytes were isolated from splenocytes of 4T1-bearing mice using Miltenyi magnetic beads, seeded in a 6-wells plate at 1 million cells per wells in RPMI 10% FCS, penicillin/streptomycin, 50ng/ml M-CSF and cultured for 72h. F4/80+ TAMs were isolated from enzyme-digested 4T1 tumors using Miltenyi magnetic beads, and lysed directly in Ambion's PureLink™ RNA Mini Kit lysis buffer.
INSTRUMENT(S): Illumina MouseWG-6 v2.0 R2 expression beadchip [gene symbol version]
Project description:Tumor associated macrophages show signs of both, classical pro-inflammatory as well as alternative macrophage activation. The aim of this study was to compare TAMs across tumor types, to characterize their phenotype in detail and to identify the signaling nodules involved regulating classical and alternative activation traits. To identify gene expression profiles and pathways differentially activated in tumor-associated macrophages (TAMs) we isolated TAMs from in vivo animal models of EG7 thymoma (n=3), glioma (n=3) and neuroblastoma (n=3) by tumor digestion and subsequent enrichment of CD11b+ cells using Miltenyi magnetic beads. Thymomas grown in WT or Myd88-/- mice (n=5 per group) were used to identify TLR/IL1R dependent signaling in TAMs. RNA extracted from CD11b+ TAMs was analyzed using the Affymetrx HT_MG-430_PM arrays. For in-depth analysis of the role of inflammatory signaling by TNFR1 (Tnfrsf1a) and/or Myd88, EG7 TAM populations A (CD11b+, Ly6G-, Ly6C+, MHCII-) and C (CD11b+, Ly6C-, MHCII+) were isolated by flow cytometric sorting from WT, TNFR1-/-, Myd88flox/flox; Tie2-Cre and TNFR-/-; Myd88flox/flox; Tie2-cre mice and extracted RNA was analyzed by microarray using the Mouse Gene 2.0 GeneChip arrays (n=2 per genotype and population).
Project description:Hyperglycemia is an essential factor leading to micro- and macrovascular diabetic complications. Macrophages are key innate immune regulators of inflammation that undergo 2 major directions of functional polarization: classically (M1) and alternatively (M2) activated macrophages. The aim of the study was to examine the effect of hyperglycemia on transcriptional activation of M0, M1 and M2 human macrophages. Overall design: Monocytes were isolated from buffy coats by magnetic cell sorting using CD14 beads and cultivated in the presence of 5mM (NG) and 25mM (HG) glucose for 6 days under stimulation with IFNg (M1), IL4 (M2) or without cytokine (M0). M0, M1 and M2 macrophages from 4 individual donors were used
Project description:Different human mTEC subsets (MUC1, CEACAM5 and SGLT1) were purified by sequential enzymatic digestion (collagenase/dispase, trypsin) followed by enrichment using magnetic beads (CD45 beads, Miltenyi Biotech) and FACS sorting. Cells of the surface phenotype CD45-, CDR2-, EpCAM+ were further subdivided into MUC1+/MUC1-, CEACAM5+/CEACAM5- and SGLT1+/SGLT1- fractions. RNA was isolated using μMACS™ SuperAmp™ protocol (Miltenyi Biotec) and hybridized to Illumina Whole-Genome Expression Beadchips. Gene expression of Antigen-positive and Antigen-negative mTEC subsets was compared. Total RNA was isolated from ex-vivo isolated human mTEC subsets using μMACS™ SuperAmp™ protocol (Miltenyi Biotec)
Project description:The development and progression of CLL are dependent upon a complex microenvironmental network of cellular and molecular signals. As an example the in vitro survival of CLL cells is supported by nurse-like cells, which have been identified as a CLL-specific tumor-associated macrophage (TAM) population. However little is known regarding the role of TAMs in CLL development and progression. In the CLL xenograft model based on the engraftment of MEC1 human CLL cell line into Rag2-/-γc-/-, we analyzed the whole-genome transcriptome of TAMs isolated from the bone marrow. We found an enrichment of genes involved in inflammation and interaction between TAMs and leukemic cells. Rag2-/-γc-/- were injected intravenously with MEC1 cells and sacrificed at early stage of leukemia with age-matched wt Rag2-/-γc-/- mice (n=3/group). RNA was isolated from bone marrow murine monocytes/macrophages purified by magnetic separation and processed for illumina whole-genome gene expression direct-hybridization assay.
Project description:The goal of this study was to determine if blood circulating monocytes of metastatic breast cancer patient would express a different activation profile compared to healthy donors, in order to use this specific changesas biomarkers to monitor then response to therapy Overall design: CD11b+ cells were extracted from all blood of 4 healthy donors and 4 metastatic breast cancer patients using magnetic beads separation (Miltenyi). CD11b+ cells were then lysed and mRNA was extracted to perform RNASeq.
Project description:Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor Spic is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80+VCAM+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor Bach1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Further, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insight into iron homeostasis. Global gene expression pattern of Spic+ monocytes, Spic- monocytes, and Spic high red pulp macrophages were compared by sorting these cells from Spic(igfp/+) splenocytes and performing microarray-based gene expression profiling. Splenocytes were prepared from Spic(igfp/+) mice and were first negatively selected for CD4, CD8, and B220 by MACS (Miltenyi Biotech) purification using the respective microbeads. Negatively selected splenocytes were then stained with anti-CD11b and anti-Ly6c and sorted for Spic+ monocytes (CD11b+Ly6c+Spic+) and Spic- monocytes (CD11b+Ly6c+Spic-). Purified RPM were obtained by staining splenocytes with anti-F4/80 and sorting for F4/80 hi Spic-EGFP hi cells.
Project description:The ability of Entamoeba histolytica to phagocytose host cells correlates to observed virulence in vivo. To better understand the mechanism of phagocytosis we used paramagnetic beads coated with host ligand and sorted trophozoites based on phagocytic ability. Gene expression was then measured in both the sorted phagocytic and non-phagocytic populations using a custom Affymetrix chip for E. histolytica. Feed forward regulation of phagocytosis by Entamoeba histolytica. Infection and Immunity. PMID 23045476 M280 Streptavidin Dynabeads (Invitrogen) were labeled with 20ug/mL biotinylated Human C1q (Quidel). Entamoeba histolytica (strain HM1) were washed twice with PBS then resuspended with the labeled beads at a 10:1 ratio of beads to trophozoites. Samples were incubated at 37°C for 45 minutes, washed twice with agitation to remove adherent beads, then resuspended in MACS buffer. Samples were loaded into magnetic columns (Miltenyi Biotec) and trophozoites were seperated according to manufacturer's protocols. phagocytic vs. non-phagocytic Entamoeba histolytica populations
Project description:Genes expression in Ly6C+/F4/80+ inflammatory macrophages, CX3CR1+/F4/80+ tissue resident macrophages and Ly6G+/F4/80- neutrophils which were isolated from day 3 wounds in C57/B6 mice aged 8 weeks by cell sorting Ly6C+ macrophages expressed higher (over 5 folds) levels of 241 genes compared to CX3CR1+ macrophages, and 3382 genes compared to neutrophils
Project description:Macrophages from RA synovial fluids were compared to primary human blood-derived macrophages. Keywords: disease state analysis Overall design: Macrophages from synovial fluids of five RA patients were isolated by ficoll density gradient centrifugation, followed by positive selection of CD14+ cells using magnetic beads. The patients’ diagnoses were determined by their physicians, in each case a Board-certified rheumatologist at the Hospital for Special Surgery, and were definite RA according to American College of Rheumatology criteria. De-identified samples were processed in an anonymous manner using a protocol approved by the Institutional Review Board of the Hospital for Special Surgery. Human peripheral blood mononuclear cells were isolated from venous blood of independent healthy donors by centrifugation on a Ficoll density gradient. Monocytes were obtained from PBMCs using anti-CD14 magnetic beads, and were >96% pure as verified by flow cytometry. Monocytes were differentiated into macrophages by culturing in RPMI 1640 medium supplemented with 10% fetal bovine serum and M-CSF (20 ng/ml). The viability and purity of macrophages was comparable in all conditions Gene expression was analyzed using Affymetrix microarrays and protocols.
Project description:Hematopoietic stem/progenitor cells (HS/PCs) were transduced with lentiviral vectors overexpressing OFP and either miR-511-3p or a control, mutated miRNA sequence (miR-511-3p-mut). The transduced HS/PCs were then transplanted in recipient C57BL/6 mice. Tumors (Lewis lung carcinomas, LLC) were injected s.c. 4 weeks after HS/PC transplant. Lentiviral vector-transduced (OFP+), tumor-associated macrophages (TAMs) were isolated 4 weeks after LLC injection by fluorescence-activated cell sorting. Gene expression profiles of TAMs overexpressing either miR-511-3p or miR-511-3p-mut were obtained from 3 independent biological samples/each. Gene expression profiles of miRNA-overexpressing TAMs were then compared with the gene expression profiles of wild-type TAMs isolated from LLCs grown in nontransplanted C57BL/6 mice; the latter TAMs were subfractioned into MRC1+CD11c(low) or CD11c+MRC1(low) subsets before RNA isolation and analysis. Comparison of gene expression profiles of TAMs revealed that miR-511-3p overexpression tunes down the expression of multiple genes that are classically upregulated in protumoral MRC1+CD11c(low) TAMs. TAMs were isolated from Lewis lung carcinomas grown s.c. in C57BL/6 mice.