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High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors.

ABSTRACT: Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be associated with invasive carcinoma cells. The gene expression signature of this population was compared with a similar population derived from spleens of non-tumor-bearing mice using high-density oligonucleotide arrays. Using stringent selection criteria, transcript abundance of 460 genes was shown to be differentially regulated between the two populations. Bioinformatic analyses of known functions of these genes indicated that formerly ascribed TAM functions, including suppression of immune activation and matrix remodeling, as well as multiple mediators of tumor angiogenesis, were elevated in TAMs. Further bioinformatic analyses confirmed that a pure and valid TAM gene expression signature in mouse tumors could be used to assess expression of TAMs in human breast cancer. The data derived from these more physiologically relevant autochthonous tumors compared with previous studies in tumor xenografts suggest tactics by which TAMs may regulate tumor angiogenesis and thus provide a basis for exploring other transcriptional mediators of TAM trophic functions within the tumor microenvironment.

SUBMITTER: Ojalvo LS

PROVIDER: S-EPMC2665764 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors.

Ojalvo Laureen S LS King William W Cox Dianne D Pollard Jeffrey W JW

The American journal of pathology 20090213 3

Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be ...[more]

PMID: 19218341

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Project description:The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high-throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage-independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene expression in primary human breast tumors was interrogated. Twenty of the human orthologues of MMTV CIS associated genes are deregulated and/or mutated in human breast tumors.

Project description:UNLABELLED:Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model. METHODS:Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry. RESULTS:The phospholipid- and apoA-I-labeled rHDL were produced at 79% ± 13% (n = 6) and 94% ± 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 ± 2.8 and 8.6 ± 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively. CONCLUSION:We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment.

Dataset Information

High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors.

Publications

High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors.

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