Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness

Abstract

Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells as well as in hypoxic perinecrotic areas of numerous cancers. Here, we investigated whether ANGPTL4 might affect tumor growth as well as metastasis. Metastatic 3LL cells were therefore xenografted into control mice and mice in which ANGPTL4 was expressed by using in vivo DNA electrotransfer. Whereas primary tumors grew at a similar rate in both groups, 3LL cells metastasized less efficiently to the lungs of mice that expressed ANGPTL4. Fewer 3LL emboli were observed in primary tumors, suggesting that intravasation of 3LL cells was inhibited by ANGPTL4. Furthermore, melanoma B16F0 cells injected into the retro-orbital sinus also metastasized less efficiently in mice expressing ANGPTL4. Although B16F0 cells were observed in lung vessels, they rarely invaded the parenchyma, suggesting that ANGPTL4 affects extravasation. In addition, recombinant B16F0 cells that overexpress ANGPTL4 were generated, showing a lower capacity for in vitro migration, invasion, and adhesion than control cells. Expression of ANGPTL4 induced reorganization of the actin cytoskeleton through inhibition of actin stress fiber formation and vinculin localization at focal contacts. Together, these results show that ANGPTL4, through its action on both vascular and tumor compartments, prevents the metastatic process by inhibiting vascular activity as well as tumor cell motility and invasiveness.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

In vivo angptl4 gene transfer by electrotransfer. Western blot analysis of ANGPTL4-mycHis protein detected by c-myc antibody. (A) Sera from mice electrotransferred with control plasmid (week 2, lane 1) or with ANGPTL4 (week 2, lane 2 and week 6, lane 3), and from mice injected with 1 μg of recombinant ANGPTL4 (lane 4) were analyzed. (B) Assessment of plasma TG from mice injected 30 min before with control buffer or recombinant ANGPTL4 (1, 10, or 50 μg), by enzymatic reaction.

Fig. 2.

ANGPTL4 inhibits 3LL tumor cell intravasation, invasion of lymph nodes, and lung metastases. Two weeks after muscle electroporation, C57BL/6 mice were s.c. implanted with 3LL cells. One, two, three, and four weeks after implantation, tumors, lymph nodes, and lungs were removed and stained by using HES and anti-CD34 immunohistochemistry. Invading tumor cells (emboli) were mainly observed in control mice (intratumor vessels, peritumor vessels, see arrows) (A). The number and the volume of metastatic lymph nodes (B) as well as the number of 3LL micrometastases in lungs (C Left) were quantified in sections from ANGPTL4 mice compared with control mice each week after injection. Typical views of invaded lymph nodes (B Right; see the tumor area surrounded in black in control mouse versus diffuse tumor cells shown by arrows in ANGPTL4 mice) and lung metastases (C Right) are shown.

Fig. 3.

In the B16F0 model, ANGPTL4 inhibits extravasation of tumor cells and macrometastases number. B16F0 cells were injected i.v. into C57BL/6 mice 2 weeks after ANGPTL4 electroporation, and mice were killed either 2 or 3 weeks later. At week 2, tumor islets were observed in lungs by staining sections with HES (A Upper) and anti-CD34 (A Lower), allowing discrimination of C57BL/6 micrometastases implanted in the lung parenchyma from intravascular emboli. Left panel, arrows indicate residual vascular walls associated with tumor cell extravasation in control lung. Right panel, arrows show intact vessels enclosing localized nodules (emboli) in lungs from ANGPTL4 mice (A). Quantification of micrometastases invaded the parenchyma of the lung (B) and localized emboli (C). Typical views of lungs presenting macroscopic metastases at week 3 (D Upper). For each mouse, quantification of total metastatic area (E) and mean surface area per macrometastasis (F) was performed by using scanned images of HES-stained lung sections (D Lower).

Fig. 4.

Inhibition of histamine-induced vascular permeability by ANGPTL4. VP was evaluated by performing a Miles assay induced by histamine in nude mice electrotransferred with ANGPTL4 or control plasmid. Spectrophotometric analysis of vascular leakiness with Evans blue dye was performed. For each group, extravasation of dye after injection of histamine (1 nM, 10 nM) was compared with extravasation of dye after injection of PBS. Mean nanograms of Evans blue per milligram of fresh tissue are represented ± SEM.

Fig. 5.

In vitro characterization of the overexpression of ANGPTL4 in tumor cells. Western blot analysis of ANGPTL4-mycHIS protein secreted by stably transfected B16F0 clones (B16F0/ANGPTL4-Low, -Medium, or -High) compared with control cells (B16F0/Empty vector as negative control and CHO/ANGPTL4 as positive control (16) and detected by c-myc antibody (A). Alteration of tumor cell adhesion, motility, and invasiveness by ANGPTL4. “Adhesion assay”: 5 × 104 stained B16F0 cells expressing ANGPTL4 or control cells were plated on BSA, fibronectin, laminin, or vitronectin for 1 h, and fluorescence was measured (B). “Boyden chamber assay”: 1 × 105 stained cells expressing ANGPTL4 or control cells were seeded in transwells with polycarbonate membrane plates. Migration on a fibronectin coating (C) or invasion through a Matrigel barrier (D) was assessed by measuring fluorescence after 6 h. ANGPTL4 modifies cytoskeleton organization. Cytoskeleton organization of B16F0 ANGPTL4 cells or control cells was evaluated by fluorescence detection of polymerized actin fibers (red) and vinculin (green) (E). Quantification was performed by determining the percentage of cells showing an organized cytoskeleton, i.e., polymerized actin stress fibers (red) and vinculin focal adhesion contacts (green) in control and ANGPTL4 cells (F).