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.2009 Mar;174(3):1048-64.
doi: 10.2353/ajpath.2009.080676. Epub 2009 Feb 13.

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

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

Laureen S Ojalvo et al. Am J Pathol.2009 Mar.

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.

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Figures

Figure 1
Figure 1
Csf1r-EGFP expression and Texas-Red conjugated 70kd dextran identify a pure population of TAMsin vivo.A: Single-cell suspensions were prepared from late-stage MacGreen PyMT tumor bearing females and stained for CD115 (external epitope of CSF-1R) (i); F4/80 (ii); CD11b (iii); Gr1 (iv). Shaded gray histograms indicate unstained EGFP+ control.B: Single cell suspension of tumor from late-stage MacGreen PyMT tumor I.V. injected with texas-red conjugated dextran was prepared and analyzed by flow cytometry. Dextran+/EGFP+ (top panel) and dextran−/EGFP+ cells (bottom panel) were separately analyzed for expression of F4/80 and Gr1. Average percentages with SEM of three experiments are indicated. Shown are representative plots.C: Cytospin followed by Wright-Giemsa staining was performed on dextran+/EGFP+ and dextran−/EGFP+ cells sorted by flow cytometry. Dextran+/EGFP+ (top panels) cytospin confirms a pure population of mononuclear phagocytes whereas dextran−/EGFP+ (bottom panels) reveal a heterogeneous population of mononuclear and polymorphonuclear cells. Scale bar = 20 μm.
Figure 2
Figure 2
Localization of TAMs in PyMT Tumors.A: Immunohistochemistry was performed on serial frozen sections of late stage PyMT tumors. Negative irrelevant control antibody (a). F4/80 staining illustrates localization of macrophages in the stroma and diffusely in the tumor islands (b). Gr-1 staining is more sparse and restricted to stroma/peri-vascular areas of the tumor (c). Ly-6G staining is also restricted to stroma/peri-vascular areas of the tumor and appears to overlap with Gr-1 staining as indicated byblack arrows (d). Scale bar = 50 μm.B: Localization of dextran labeling (red) cells within MacGreen tumor.C: Localization of CSF-1R (CD115) cells within MacGreen tumor.D: Localization of F4/80 cells within MacGreen tumor.E: Localization of Gr1 cells within MacGreen tumor.Inset ×2 magnification of DAPI-stained nuclei in merged image.B left panels green (EGFP) channel,middle panels red channel,right panels merged image with DAPI.Arrows indicate cells that are EGFP+, but do not express cell-type specified markers. Scale bars = 10 μm.
Figure 3
Figure 3
TAM gene expression can be robustly assayed by high-density oligoarrays and exhibit a unique expression signature compared with tissue splenic macrophages.A: Schematic of the experimental procedure to compare TAMs versus splenic macrophages.B: Scatter plot. Average log expression of each queried transcript for TAMs compared with splenic macrophages with linear regression line overlaid (r = 0.9698).C: MA-plot. Log ratio of Cy3 expression (TAM) and Cy5 expression (splenic macrophages) (M), plotted against average of the log intensities (A). Symmetry with respect to horizontal line indicates lack of dye bias in array.D: Volcano plot. Log fold change between groups versus significance level as calculated byt-test (−log10[P value]). Horizontal line marksP value = 0.05. Vertical lines mark log fold change less than or greater than 1. With these criteria, 926 genes are called as increased in TAMs compared with splenic macrophages, and 831 genes are called as decreased.E: SAM plot. Significance Analysis of Microarrays with delta value of 1.74, false discovery rate of 0.1. Called genes: 210 increased expression in TAMs; 242 decreased expression; 8 not regulated by fold-change criteria.F: Dendogram and hierarchical clustering. Genes depicted are those called by SAM analysis. Dark shaded boxes indicate low expression; light shaded boxes indicate higher expression. Clustering analysis robustly separate the TAM samples from splenic macrophage samples.
Figure 4
Figure 4
Transcripts mediating immune function and tissue development are differentially regulated in TAMs. Genes selected by SAM subjected to bioinformatics analysis using IPA.A: Most significantly enriched functional groups relating to physiological system development and function.B: Most significantly enriched groups pertaining to molecular and cellular function. Both panels include correspondingP values calculated through IPA using right-tailed Fisher Exact Test.C: Primary gene ontological classification was designated for each differentially regulated gene and a pie-chart was created to visualize enriched gene ontologies in transcripts decreased in abundance (left panel) and increased abundance (right panel) in TAM. Legend on right ascribes classifications. Percentages associated with each wedge represent percent enrichment in regulated gene population.
Figure 5
Figure 5
Quantitative rtPCR validates gene expression array data from numerous ontological clusters. Sixteen genes identified as differentially regulated from arrays validated using qrtPCR using separate biological repeats. All samples were normalized to the housekeeping gene,Pp1a. Data shown indicates relative expression of TAMs (black bars) with respect to splenic macrophages (gray bars), set to one. Gene symbol abbreviations listed in Table 1.
Figure 6
Figure 6
TAMs regulate tumor angiogenesis through multiple mediators.A: Differentially regulated transcripts (P < 0.05) that were ascribed a role in angiogenesis (Gene Ontology/IPA) were plotted on a schematic indicating cellular localization. Log ratio data were overlaid to indicate those genes of increased abundance in TAMs (red) and those of decreased abundance (green). Stars label those results validated by qrtPCR. To determine whether these transcripts can be regulated in macrophages by tumor cells, in vitro cultures were established between primary BMMs and Met-1 cells.B: ELISA for Mip1α (top) and VEGF (middle) is used to compare the validity of thein vitro system to results from isolated TAMs. BMMs were cultured alone and treated with Met-1 cell conditioned media. Shown are representative results from three experiments of collected supernatants at 6, 12, and 24 hours. (bottom) qrtPCR at 12 hours for BMMs treated with Met-1 conditioned media and control BMMs. Results standardized toPp1a housekeeping gene. Relative expression is normalized to that of the control BMM. Abbreviations: ADAMTS 1/8 (ADAM metallopeptidase with thrombospondin type 1 motif, 1/8); ANG (angiogenin); CXCL4 (platelet factor 4 (chemokine (C-X-C motif) ligand 4); ECM1 (extracellular matrix protein 1); FN1 (fibronectin 1); MIP1A (chemokine (CC motif) ligand 3); MMP2 (matrix metallopeptidase 2); NRP2 (neuropilin 2); PTX3 (pentraxin-related gene); SERPINE1 (serpin peptidase inhibitor, clade E, member 1); STAB1 (stabilin 1); SLURP1 (secreted LY6/PLAUR domain containing 1); VEGFA (vascular endothelial growth factor A).
Figure 7
Figure 7
Murine TAM signature overlaps with murine fetal macrophages and human breast cancer.A: Comparison of TAM differentially regulated transcripts (P < 0.05) and top enriched transcripts identified in fetal macrophages indicates that the two macrophage populations are associated (P < 0.001, Fisher’s Exact Test). Overlapping transcripts between both groups and increased in TAMs are in blue and identified in Table 2. Overlapping transcripts decreased in TAMs are in red and identified in Table 3.B: Oncomine-generated box-and-whiskers plots for Spp1/Osteopontin expression considering metastasis status (left) and 5-year survival (right) using vandeVijver_Breast 2002 breast cancer gene expression dataset.P values generated using student’st-test through Oncomine.C: Oncomine-generated heat map from vandeVijver_Breast dataset.Left (S1) are from individuals with no evidence of metastatic disease andright (S2) with metastasis. Top correlated transcripts to Spp1 expression are ranked on the right with associated coefficients. Boxed molecules were also identified as differentially expressed between TAMs and splenic macrophages. All symbols are official gene symbols.
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