Abstract
Background: Metastatic breast cancer cells recruit macrophages (metastasis-associated macrophages, or MAMs) to facilitate their seeding, survival and outgrowth. However, a comprehensive understanding of the gene expression program in MAMs and how this program contributes to metastasis remain elusive. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Some of the changes were validated using qRT-PCR and immunofluorescence. To probe the functional relevance to metastatic growth, a gene-targeting mouse model of female mice in the C57BL6/J background was used to study allograft inflammatory factor 1 (AIF1, also known as ionized calcium-binding adapter molecule 1 or IBA1). Results: Interferon signaling is one of the most activated pathways in MAMs, with strong upregulation of multiple components of the pathway and a significant enrichment for the gene signatures of interferon-alpha-treated human macrophages. Aif1, an interferon-responsive gene that regulates multiple macrophage activities, was robustly induced in MAMs. Aif1 deficiency in MAMs, however, did not affect development of lung metastases, suggesting that AIF1 indicates MAM activation but is dispensable for regulating metastasis. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting metastatic growth. Dissection of the underlying mechanisms and functional validation of potential targets in the profile may provide novel therapeutic strategies for the treatment of metastatic diseases.
Highlights
Tumor-associated macrophages are important in multiple steps of tumorigenesis and progression, including regulation of tumor cell invasion into the stroma, intravasation into blood vessels, seeding at distal organs, angiogenesis, inflammation and immune suppression (Qian & Pollard, 2010)
Gene expression profiling identifies strong activation of interferon signaling in metastasis-associated macrophages (MAMs) To analyze the gene expression profiles of MAMs and resident alveolar macrophages (RAMs), we dissected metastases from affected lungs 11 days after seeding and normal lungs before cell dissociation for fluorescence-activated cell sorting (FACS) sorting of MAMs and RAMs
gene set enrichment analysis (GSEA) revealed that MAMs were strongly enriched for three independent gene sets obtained from IFN-alpha-treated human macrophages (Figure 2D)
Summary
Tumor-associated macrophages are important in multiple steps of tumorigenesis and progression, including regulation of tumor cell invasion into the stroma, intravasation into blood vessels, seeding at distal organs, angiogenesis, inflammation and immune suppression (Qian & Pollard, 2010). Lung metastases resulting from mammary cancer have been shown to recruit MAMs that are CD11B+CD11C-, as compared to CD11B-CD11C+ resident alveolar macrophages (RAMs) (Qian et al, 2009). Despite the significance of these findings, they represent only a patchwork of a global program of MAMs, which remains elusive To this end, we performed deep RNA sequencing (RNA-seq) in MAMs isolated from micro-dissected lung metastases 11 days after tumor cell inoculation to gain unbiased insight into the global transcriptional program and to learn how MAMs may use this program to regulate metastatic growth in secondary organs. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting version 2
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