Pigment epithelium-derived factor stimulates tumor macrophage recruitment and is downregulated by the prostate tumor microenvironment

Abstract

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis but whether it has additional effects on the tumor microenvironment is largely unexplored. We show that overexpression of PEDF in orthotopic MatLyLu rat prostate tumors increased tumor macrophage recruitment. The fraction of macrophages expressing inducible nitric oxide synthase, a marker of cytotoxic M1 macrophages, was increased, suggesting that PEDF could enhance antitumor immunity. In addition, PEDF overexpression reduced vascular growth both in the tumor and in the surrounding normal tissue, slowed tumor growth, and decreased lymph node metastasis. Contrary, extratumoral lymphangiogenesis was increased. PEDF expression is, for reasons unknown, often decreased or lost during prostate tumor progression. When AT-1 rat prostate tumor cells, expressing high levels of PEDF messenger RNA (mRNA) and protein, were injected into the prostate, PEDF is markedly downregulated, suggesting that factors in the microenvironment suppressed its expression. One such factor could be macrophage-derived tumor necrosis factor alpha (TNFalpha). A fraction of the accumulating macrophages expressed TNFalpha, and TNFalpha treatment downregulated the expression of PEDF protein and mRNA in prostate AT-1 tumor cells in vitro and in the rat ventral prostate in vivo. PEDF apparently has multiple effects in prostate tumors: it suppresses angiogenesis and metastasis, but it also causes macrophage accumulation. Accumulating macrophages may inhibit tumor growth, but they may also suppress PEDF and enhance lymph angiogenesis and, in this way, eventually enhance tumor growth.

Figure 1

(A) Western blot analysis of PEDF in PEDF-transfected (MatLyLu-PEDF) and vector-transfected MatLyLu (MatLyLu-CON) cells in vitro. (B) PEDF purified from MatLyLu-PEDF medium was tested for its ability to inhibit migration of HUVECs toward angiogenic FGF2. Results are presented as percentage maximum migration induced by the positive control (FGF2) after the background migration of endothelial cells was subtracted. (C) Cell viability was measured in MatLyLu-PEDF and control cells after 72 hours using an MTT assay. (D) Sections from orthotopic MatLyLu-PEDF and MatLyLu-CON tumors immunostained with an antibody against human PEDF (Chemicon).

Figure 2

(A) Sections from MatLyLu-PEDF tumors and controls at day 7 showing consecutive areas stained for CD68 and iNOS-positive macrophages. (B) Volume densities (%) of CD68-positive macrophages and (C) iNOS-positive macrophages in the nonmalignant extratumoral tissue and intratumorally in MatLyLu-PEDF tumors and controls at day 7. (D) Percentage of iNOS-positive macrophages (iNOS/CD68) in the extratumoral and intratumoral tissues of MatLyLu-PEDF tumors and controls at day 7. Values are expressed as means ± SD. *P < .05, **P < .01.

Figure 3

(A) Western blot analysis of PEDF in conditioned medium of AT-1 cells in vitro and in tumor lysates from orthotopic AT-1 tumors at day 10. (B) Relative PEDF mRNA levels in AT-1 cells in vitro (n = 3 different batches) and in orthotopic AT-1 tumors in vivo at day 10 (n = 5). (C) Sections from an AT-1 tumor at day 10 showing consecutive areas stained for macrophages (CD68) and TNFα. (D) Relative PEDF mRNA expression in AT-1 cells stimulated with indicated concentrations of TNFα for 18 hours in vitro. (E) Western blot analysis of PEDF in conditioned medium of AT-1 cells stimulated with indicated concentrations of TNFα for 24 hours. (F) Relative PEDF mRNA levels quantified from ventral prostate of Copenhagen rats injected with control (PBS, n = 5) or recombinant rat TNFα (1 µg, n = 5). Values are means ± SD. *P < .05, **P < .01.