CCL2 as an important mediator of prostate cancer growth in vivo through the regulation of macrophage infiltration

Abstract

The ability of CCL2 to influence prostate cancer tumorigenesis and metastasis may occur through two distinct mechanisms: 1) a direct effect on tumor cell growth and function, and 2) an indirect effect on the tumor microenvironment by the regulation of macrophage mobilization and infiltration into the tumor bed. We have previously demonstrated that CCL2 exerts a direct effect on prostate cancer epithelial cells by the regulation of their growth, invasion, and migration, resulting in enhanced tumorigenesis and metastasis. Here we describe an indirect effect of CCL2 on prostate cancer growth and metastasis by regulating monocyte/macrophage infiltration into the tumor microenvironment and by stimulating a phenotypic change within these immune cells to promote tumor growth (tumor-associated macrophages). VCaP prostate cancer cells were subcutaneously injected in male SCID mice and monitored for tumor volume, CD68(+) macrophage infiltration, and microvascular density. Systemic administration of anti-CCL2 neutralizing antibodies (CNTO888 and C1142) significantly retarded tumor growth and attenuated CD68(+) macrophage infiltration, which was accompanied by a significant decrease in microvascular density. These data suggest that CCL2 contributes to prostate cancer growth through the regulation of macrophage infiltration and enhanced angiogenesis within the tumor.

Figure 1

Inhibition of tumor growth by neutralization of CCL2. Tumor volume was monitored weekly by caliper measurements in mice receiving C1142 versus C1322 (A), and huIgG versus CNTO888 (B). Antibody treatment was initiated on day 28 (Tx) and administered at 2 mg/kg, i.p., twice weekly, for the duration of the experiment. Immunohistochemical analysis of xenograft tumors displayed normal prostate adenocarcinoma morphology by hematoxylin-eosin staining in mice treated with C1322 (C), C1142 (D), huIgG (G), or CNTO 888 (H). Neovascularization for each individual tumor (n = 5 per group; four fields per tumor) was visualized by immunohistochemical staining of CD31, and representative sections are presented (E, F, I, and J). (K) The ability of CCL2 inhibition to reduce blood vessel formation was analyzed using an in vitro tube formation assay. Tube formation using HDMVECs was induced by VCaP CM, and the number of sprouts per x 100 objective field is displayed. The graph represents the mean of three independent experiments, each performed in triplicate (mean ± SD; *P < .01).

Figure 2

Inhibition of mouse CCL2 reduces proliferation in VCaP xenografts. Apoptosis was visualized by immunohistochemical staining with ApopTag (A–D, H–K). Proliferation was visualized by immunohistochemical staining for Ki67 (E–F, L–M).

Figure 3

Inhibition of mouse CCL2 attenuates Akt and MAPK p44/p42 activity. Tumor specimens were collected and stained with anti-phospho Akt (A and B; E and F) or anti-phospho p44/p42 (C and D; G and H) to visualize the intratumoral activity of these signaling pathways.

Figure 4

CCL2 stimulates Akt and p70S6 kinase activation in VCaP cells. CCL2 induces Akt phosphorylation (A) and p70S6 kinase phosphorylation (B) in a dose-dependent fashion. (C) VCaP cells (8 x 10⁵ cells/well) were treated with hrCCL2 (100 ng/ml) for 24 to 72 hours in the presence or in the absence of CNTO888 (30 µg/ml). The proliferation of VCaP cells was assessed by WST-1 assay (□ control; ◆ 30 µg/ml CNTO888; ▴ 100 ng/ml hrCCL2; ▾ 100 ng/ml hrCCL2 + 30 µg/ml CNTO888).

Figure 5

Inhibition of CCL2 reduced the number of infiltrating macrophages in VCaP xenografts. Macrophage infiltration was visualized by immunohistochemical staining for CD68 (A–D). (E) The number of infiltrating macrophages was determined by counting the total number of macrophages in four independent fields per section (n = 3 tumors per group were analyzed for CD68). The data are presented as mean ± SD (*P < .01).

Figure 6

Inhibition of tumor-derived CCL2 reduces the number of migrating U937 monocytes. U937 cell migration was measured in response to VCaP CM. The total number of migrating U937 cells was calculated by manually counting four independent fields (migration assays were performed in triplicate). The data are presented as mean ± SD (*P < .001).