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Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.

ABSTRACT: Tumor-associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE-1(+) M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow-derived macrophages (BMDMs) by combining M2 mediators (IL-4, glucocorticoids) and tumor-conditioned media (TCM). Admixture of MS4A8A(+) TCM/IL-4/GC-treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage-like cells displayed a special gene signature. Admixture of these MS4A8A(+) Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF-M-NM-:B activation and of the MAPK JNK and ERK did not show relevant effects, the p38M-NM-1/M-NM-2 MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A(+) TAM may serve as a novel cellular target for selective cancer therapy. A recombinant Ms4a8a cDNA was amplified by PCR (primer: Ms4a8a-SpeI-fw 5M-bM-^@M-^YATCGAATTCACTAGTAGCAAAGAGTTGGGAACCGGAGCAAGA3M-bM-^@M-^Y and Ms4a8a-NotI-rv: 5M-bM-^@M-^YATATGCGGCCGCTAGAGCATCTTTAT3M-bM-^@M-^Y) from Ms4a8a cDNA RZPDp981B0530D (IMAGE ID 905005), purified on agarose gel and subcloned after digestion with SpeI and NotI restriction enzymes into the expression vector pEF6/V5-His Topo (Invitrogen) according to standard molecular biology protocols. After confirming sequence identity, we transfected RAW264.7 cells with Ms4a8a vector DNA using Lipofectamine 2000 (Invitrogen) transfection reagent. Transfectants were selected by resistance to blasticidin (Invitrogen). Raw264.7Ms4a8a clone K8 with recombinant Ms4a4a expression were propagated. As a negative control, a vector-transfected RAW264.7mock (clone K3) was selected under parallel culture conditions.

ORGANISM(S): Mus musculus

SUBMITTER: Carsten Sticht

PROVIDER: E-GEOD-38171 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.

Schmieder Astrid A Schledzewski Kai K Michel Julia J Tuckermann Jan P JP Tome Lydia L Sticht Carsten C Gkaniatsou Cleopatra C Nicolay Jan P JP Demory Alexandra A Faulhaber Jörg J Kzhyshkowska Julia J Géraud Cyrill C Goerdt Sergij S

International journal of cancer 20101128 1

Tumor-associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE-1(+) M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow-derived macrophages (BMDMs) by combining M2 mediators (IL-4, gluco ...[more]

PMID: 20824698

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Project description:To compare tumor associated macrophage (TAM) from naïve and sepsis surviving mice we have employed Agilent microarrays slides with almost 60,000 genes (39,430 mRNA and 16,251 long non coding RNAs). Other experiments we conducted demonstrated TAM accumulation was increased in post-sepsis subjects. For this reason, we asked if TAM from post-sepsis mice could also exhibit a different gene expression profile. Sepsis was induced by cecal and ligation puncture. Naïve mice were used as control group. All animals were treated with ertapenem (20 mg/kg, i.p., 6 hours after surgery, and then each 12 hours for 3 days). B16-F10 melanoma (30,000 cells) were injected subcutaneously at day 14 after sepsis induction. Fourteen days after tumor inoculation, animals were killed and tumors were harvested and digested (collagenase and DNAse). TAM was isolated by a Percoll gradient (70/30) followed by a 1-hour adhesion protocol, reaching a purity of ~75%. For comparative reasons, we assessed TAM from post-sepsis (n = 4), TAM from naïve mice (n = 4), bone marrow derived macrophage from naïve (n = 4) and from post-sepsis (n = 4), M1-polarized macrophage (n = 4) and M2-polarized macrophage (n = 4). We found only minor gene expression differences between TAM from naïve and from post-sepsis mice (61 genes were up-regulated and 98 genes were down-regulated, fold-change > 0.58 or < -0.58, and p < 0.01). We found genes related to leukocyte activation were down-regulated in TAM from post-sepsis mice (e.g. Ccr7, Cd86, H2-Ab1), as well as genes related to antigen processing and presentation of peptide or polysaccharide antigen via MHC class II (H2-DMb1, Cd74, H2-Eb1, H2-Ob). A gene related to M2 polarization was up-regulated (Marco). Also, we found a down-regulation of Nfkbid in post-sepsis-derived TAM. This led us to hypothesize TAM from post-sepsis mice exhibit a more M2-like phenotype, which may in part contribute to post-sepsis tumor expansion. Three independent experiments were conducted for TAM obtaining, each experiment using n = 4 for naïve and n = 4 for post-sepsis. We selected the 4 best within a group of 12 samples, following A260/280 and A260/230 ratios. For bone marrow derived macrophage from naïve and from post-sepsis, and for M1 and M2-polarized macrophage, we conducted two independent experiments using n = 3 per group. The best 4 samples in each group was selected to microarray processing and analysis.

Dataset Information

Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.

Publications

Synergistic activation by p38MAPK and glucocorticoid signaling mediates induction of M2-like tumor-associated macrophages expressing the novel CD20 homolog MS4A8A.

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