Gene expression profiling of tumor cells and M2 macrophages from WT mice and mice with deletion of integrin beta3 in macrophage lineage cells (b3KOM)
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ABSTRACT: To better understand the impact of integrin beta3 signaling in myeloid cells on the tumor microenvironment, we compared the gene expression profiles of FACS isolated GFP+ PyMT-BO1 MFP tumor cells and also M2 TAMs (CD11b+Gr1-F4/80+CD206+) from tumor tissue of WT mice and b3ΚΟΜ mice.
Project description:To better understand the impact of integrin beta3 signaling in myeloid cells on the tumor microenvironment, we compared the gene expression profiles of FACS isolated GFP+ PyMT-BO1 MFP tumor cells and also M2 TAMs (CD11b+Gr1-F4/80+CD206+) from tumor tissue of WT mice and b3ÎÎÎ mice. PyMT-BO1-GFP-Luc tumor cells (100,000) were injected into mammary fat pad (MFP) tissue from WT and b3KOM mice. PyMT-BO1-GFP-Luc cells and CD206hi tumor-associated macrophages (TAMs) were FACS-sorted from day 10 MFP tumor tissue. FACS-sorted cells directly used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:To understand the underlying cause for reduced lung metastasis, we compared global gene expression profiles of F4/80+ FACS sorted tumor-associated macrophages (TAMs) PyMT;E2f3 f/f (control) and PyMT;Lys Cre:E2f3 f/f (experimental) mice. We compared gene expression profile between TAMs isolated from mammary tumors of PyMT;E2f3 f/f and PyMT;Lys Cre:E2f3 f/f mice
Project description:Comparison of gene expression data between control and MFP-treated C4-HD tumors. The role of active antitumor immunity in hormone receptor positive (HR+) breast cancer has been historically underlooked. The aim of this study was to determine the contribution of the immune system to antiprogestin-induced tumor growth inhibition using a hormone-dependent breast cancer model. BALB/c-GFP+ bone marrow (BM) cells were transplanted into immunodeficient NSG mice to generate an immunocompetent NSG/BM-GFP+ (NSG-R) mouse model. Treatment with the antiprogestin Mifepristone (MFP) inhibited growth of 59-2-HI tumors with similar kinetics in both animal models. Interestingly, MFP treatment reshaped the tumor microenvironment, enhancing the production of proinflammatory cytokines and chemokines. Tumors in MFP-treated immunocompetent mice showed increased infiltration of F4/80+ macrophages, NK, and CD8 T cells, displaying a central memory phenotype. Mechanistically, MFP induced immunogenic cell death in vivo and in vitro, as depicted by the expression and subcellular localization of the alarmins calreticulin and HMGB-1 and the induction of an immunogenic cell death gene program. Moreover, MFP-treated tumor cells efficiently activated immature dendritic cells, evidenced by enhanced expression of MHC-II and CD86, and induced a memory T cell response, attenuating tumor onset and growth after re-challenge. Finally, MFP treatment increased the sensitivity of HR+ 59-2-HI tumor to PD-L1 blockade, suggesting that antiprogestins may improve immunotherapy response rates. These results contribute to a better understanding of the mechanisms underlying the antitumor effect of hormonal treatment and the rational design of therapeutic combinations based on endocrine and immunomodulatory agents in HR+ breast cancer.
Project description:Six-weeks old (C57Bl6, Cx3cr1gfp/+) mice were intraperitonealy infected with a low number (1.104) of L. monocytogenes (EGDe strain) in exponential growth phase (bacteria were grown in BHI at 108/ml, and diluted 10.000x in PBS immediately before injection). Group of three mice were euthanized, before infection. Peritoneal cells were recovered by peritoneal lavage. Cells from individual mice were stained with antibodies to CD11b (PECy7), Gr1 (APC), NK1.1, B220 and CD3 (PE), and F4/80 (biotin-conjugated followed by streptavidinpacific blue) for sorting. Gr1- monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1-, gfphigh; Gr1+ monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1+, gfpint; and polymorphonuclear cells were purified as NK1.1- CD3- B220- CD11b+ F4/80- Gr1high, gfp-. 1.103 cells from each mice, time point, and phenotype were purified by facs sorting according to their phenotype. Samples were kept at 4°C before and during the sort. Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences).
Project description:To determine the molecules involved in beta3 integrin signaling in HSCs, we carried out DNA microarray analyses using HSCs stimulated with ligand-mimic antibody for intetegrin beta3 in the presence of stem cell factor(SCF) and/or thrombopoietin (TPO). All experiments were performed in duplicate or triplicate using sorted cells that were independently prepared in separate experiments.
Project description:The immune system cellular response to tissue damage and infection requires the recruitment of blood leukocytes to the target tissue. This process is mediated through a classical multistep mechanism which involves transient rolling on the endothelium and recognition of inflammation followed by extravasation. We show here, by direct examination of blood monocyte functions in vivo, that resident monocytes monitor the endothelium of healthy tissues through patrolling, a new mechanism which allows extravasation in the absence of rolling.Patrolling depends on the integrin LFA1 and the chemokine receptor CX3CR1, and is required for rapid tissue invasion and initiation of an early immune response by monocytes that differentiate into macrophages at the site of tissue damage and infection. The main goal of the experiment was to compare expression levels of genes in Gr1- monocytes in the blood and after recruitment in the peritoneum during experimental infection with Listeria monocytogenes (time course analysis). Experiment Overall Design: Six-weeks old (C57Bl6, Cx3cr1gfp/+) mice were intraperitonealy infected with a low number (1.104) of L. monocytogenes (EGDe strain) in exponential growth phase (bacteria were grown in BHI at 108/ml, and diluted 10.000x in PBS immediately before injection). Group of three mice were euthanized, before infection (time â??0â??) and 2 and 8 hours after infection (time â??2â?? and â??8â??). Peripheral blood cells were recovered at time â??0â??, and peritoneal cells were recovered at time â??0â??, â??2hâ??, and â??8hâ?? by peritoneal lavage. Cells from individual mice were stained with antibodies to CD11b (PECy7), Gr1 (APC), NK1.1, B220 and CD3 (PE), and F4/80 (biotin-conjugated followed by streptavidinâ??pacific blue) for sorting. Gr1- monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1-, gfphigh; Gr1+ monocytes were purified as NK1.1- CD3- B220- CD11b+ F4/80low Gr1+, gfpint; and polymorphonuclear cells were purified as NK1.1- CD3- B220- CD11b+ F4/80- Gr1high, gfp-. 1.103 cells from each mice, time point, and phenotype were purified by facs sorting according to their phenotype. Samples were kept at 4°C before and during the sort. Cells were directly sorted in the SuperAmp Lysis Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) using a FACS Aria cell-sorter (BD biosciences).
Project description:We sought to identify genes that are differentially regulated in CD11b+Gr1+ cells after tumor challenging. Mice were challenged with LLC-RFP cells (5×10^6 cells, subcutaneous injection) for 9 days. Single cell suspensions were prepared from lungs of both tumor challenged and control wild type mice and stained with antibodies against CD11b and Gr1. Approximately 1×10^5 CD11b+GR1+ myeloid progenitor cells were sorted by FACS (Aria II, BD Bioscience). Total RNA was extracted from the sorted cells for microarray analysis. In this dataset, we include the expression data of CD11b+Gr1+ cells obtained from both control wild type and tumor challenged mice. These data were used to obtain 22 genes that are upregulated in response to tumor challenging.
Project description:The aim of these experiments was designed to compare gene expression in human myeloma cell lines expressing beta 3 integrin vs counterpart cell lines that do not express beta 3 integrin. Keywords: Gene expression,cell lines, siRNA Phenotype characterization of both primary and cultured myeloma plasma cells was assessed by flow cytometry (FACScanto, Becton Dickinson, San Jose, CA) using a panel of MoAbs or antisera to the following markers: CD138, CD38, CD56, k/l chains, CD20, CD44, CD54, alphav and beta3 chains. The microarray analysis was performed on: myeloma bone resorbing cell lines alphav-beta3 positive and the same cell line silenced for the integrins; myeloma non bone resorbing cell lines negative for the expression of avb3.
Project description:To determine the molecules involved in beta3 integrin signaling in HSCs, we carried out DNA microarray analyses using HSCs stimulated with ligand-mimic antibody for intetegrin beta3 in the presence of stem cell factor(SCF) and/or thrombopoietin (TPO). All experiments were performed in duplicate or triplicate using sorted cells that were independently prepared in separate experiments. After CD34-KSL HSCs were cultured with 2C9.G2 or control IgG for 5 days in S-Clone SF-03 medium supplemented with 50 ng/ml SCF and/or 50 ng/ml TPO, gene expression profiles were examimed using 5,000 sorted CD48-KSL cells from each sample.
Project description:Interleukin-27 (IL-27) is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells and has a potential to be used as a therapeutic for cancer. We have recently demonstrated that systemic delivery of IL-27 using adeno-associated virus (AAV-IL-27) exhibits potent inhibition of tumor growth in mouse models. In this work, we demonstrate that AAV-IL-27 treatment leads to significant expansion of CD11b+Gr1+ myeloid cells (MCs). AAV-IL-27-induced expansion of CD11b+Gr1+ cells is IL-27R-dependent, requires Stat3 signaling but is inhibited by Stat1 signaling. AAV-IL-27 treatment does not increase the self-renewal capacity of CD11b+Gr1+ cells but induces significant expansion of hematopoietic stem cells (HSCs) and granulocyte-monocyte progenitor (GMP) cells. IL-27-induced Ly6G+ MCs upregulate MHC class I/II molecules but are less immune suppressive in vitro, and their expansion does not promote, but rather inhibit tumor growth in vivo. In the tumor microenvironment, IL-27 gene therapy appears to promote Ly6G+ MCs to differentiate into MHC class I/IIhigh and F4/80high macrophages. Thus, IL-27 gene therapy induces expansion of CD11b+Gr1+ myeloid cells and promotes their differentiation into anti-tumor M1 macrophages in tumor microenvironment.Bone marrow myeloid cells of tumor-bearing C57BL/6 mice treated with AAV-IL-27/ctrl for 2 weeks were sepreated by MACS. Transcriptome profiling (RNA-seq) of these purified Ly6G+ cells (>95%) was completed by BGI. Changing in gene express profiling resulted by IL-27 was analyzed in this study.