Figure 1
Figure 1. Gene expression profile of TEMs, TAMs, MDSCs, PMs, and ECs. (A) Flow cytometry analyses of tumors grown subcutaneously in wild-type FVB mice (n = 4) and made into single-cell suspensions shows that the majority of tumor-infiltrating myeloid cells (gated as Cd11b+ cells, dot plot on the left) are highly enriched in F4/80+ or Cd48+ macrophages (dot plots on the right). One representative experiment is shown. (B) Quantitative PCR analysis of TEMs (n = 4), TAMs (n = 4), and ECs (n = 2) showing the expression level of relevant genes. Results show ΔCT values (mean ± SEM) over endogenous control Gapdh. The lower the ΔCT, the higher the expression level. TEMs and TAMs express Cd45 (pan-hematopoietic–specific marker) and F4/80 (macrophage-specific marker) to similar extent. Vegfr2 (endothelial-specific marker) is expressed robustly only by ECs. TEMs express Tie2 to a significantly higher extent than TAMs (P < .05). (C) Quantitative PCR-based, multigene array analysis of TEMs (n = 3), TAMs (n = 3), and ECs (n = 1) showing the expression level (ΔCT vs β2m) of relevant EC (top panel) and hematopoietic/myeloid (bottom panel) genes. TEMs and TAMs robustly express classic hematopoietic/myeloid genes but not EC genes. (D) Morphology (May-Grünwald-Giemsa staining) of TAMs (top panel) and TEMs (bottom panel) FACS-sorted from N202 tumors grown for 3 weeks in Tie2-GFP transgenic mice (n = 2 independent experiments). Arrows indicate large macrophages containing conspicuous cytoplasmic phagosomes. Scale bar represents 30 μm. Photos are representative of n = 25 photos/sample. (E) One-dot-one-gene representation of the expression profile (280 genes analyzed) of tumor-derived TEMs (n = 3), TAMs (n = 3), and ECs (n = 1), Gr1+Cd11b+ neutrophils/MDSCs (n = 2; isolated from the spleen of tumor bearing mice) and PMs (n = 2), analyzed by quantitative PCR as in panel C. Each one-dot-one-gene plot compares 2 cell types, as indicated. The data show that TEMs are highly related to TAMs (Pearson linear correlation: 0.926) but sharply differ from Gr1+Cd11b+ cells (0.682), PMs (0.701), and ECs (0.240).

Gene expression profile of TEMs, TAMs, MDSCs, PMs, and ECs. (A) Flow cytometry analyses of tumors grown subcutaneously in wild-type FVB mice (n = 4) and made into single-cell suspensions shows that the majority of tumor-infiltrating myeloid cells (gated as Cd11b+ cells, dot plot on the left) are highly enriched in F4/80+ or Cd48+ macrophages (dot plots on the right). One representative experiment is shown. (B) Quantitative PCR analysis of TEMs (n = 4), TAMs (n = 4), and ECs (n = 2) showing the expression level of relevant genes. Results show ΔCT values (mean ± SEM) over endogenous control Gapdh. The lower the ΔCT, the higher the expression level. TEMs and TAMs express Cd45 (pan-hematopoietic–specific marker) and F4/80 (macrophage-specific marker) to similar extent. Vegfr2 (endothelial-specific marker) is expressed robustly only by ECs. TEMs express Tie2 to a significantly higher extent than TAMs (P < .05). (C) Quantitative PCR-based, multigene array analysis of TEMs (n = 3), TAMs (n = 3), and ECs (n = 1) showing the expression level (ΔCT vs β2m) of relevant EC (top panel) and hematopoietic/myeloid (bottom panel) genes. TEMs and TAMs robustly express classic hematopoietic/myeloid genes but not EC genes. (D) Morphology (May-Grünwald-Giemsa staining) of TAMs (top panel) and TEMs (bottom panel) FACS-sorted from N202 tumors grown for 3 weeks in Tie2-GFP transgenic mice (n = 2 independent experiments). Arrows indicate large macrophages containing conspicuous cytoplasmic phagosomes. Scale bar represents 30 μm. Photos are representative of n = 25 photos/sample. (E) One-dot-one-gene representation of the expression profile (280 genes analyzed) of tumor-derived TEMs (n = 3), TAMs (n = 3), and ECs (n = 1), Gr1+Cd11b+ neutrophils/MDSCs (n = 2; isolated from the spleen of tumor bearing mice) and PMs (n = 2), analyzed by quantitative PCR as in panel C. Each one-dot-one-gene plot compares 2 cell types, as indicated. The data show that TEMs are highly related to TAMs (Pearson linear correlation: 0.926) but sharply differ from Gr1+Cd11b+ cells (0.682), PMs (0.701), and ECs (0.240).

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