Increased melanoma growth and metastasis spreading in mice overexpressing placenta growth factor

Abstract

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination.

Figure 1-6914

Melanoma cells grow at a higher rate in K14-PlGF transgenic mice. A: Tumor volume was measured at different time points after melanoma cell injection in WT and transgenic mice (K14-PlGF). Data were obtained from nine mice per group and were expressed as mean ± SEM. Mann-Whitney’s test was used: *P < 0.001 versus WT. B: Photomicrographs of B16-BL6 melanoma implants of WT and transgenic mice (K14-PlGF) 25 days after tumor injection. An increased number of vessels extending for a large area around the tumor are visible in the transgenic animals.

Figure 2-6914

PlGF does not directly promote melanoma cell proliferation. A: Reverse transcription and real-time PCR was performed on total RNA extracted from B16-BL6 cells treated or not with rmPlGF for 3 hours. Results are expressed as relative fold induction of the VEGFR-1 mRNA in treated (+PlGF) versus untreated cells (C). B: Western blot analysis of cell extracts from B16-BL6 cells, treated or not with rmPlGF for 4, 12, and 24 hours was done by using a polyclonal antibody against the C-terminus of VEGFR-1. Bar and arrow highlight the VEGFR-1-related signal. HUVECs and M14 cells were used as positive and negative controls, respectively. Molecular weight marker is reported in kd. C: B16-BL6 proliferation assay. Cells were incubated with or without the indicated concentrations of rmPlGF in the presence or absence of 10 ng/ml of rmVEGF. HUVECs were used as a positive control in the same proliferation assay. Bars represent the mean of eight replicates ± SEM of a representative experiment. *P < 0.05 and **P < 0.001 versus untreated cells, unpaired Student’s t-test.

Figure 3-6914

Tumors grown in K14-PlGF transgenic and WT mice display a similar percentage of necrosis. A: H&E staining of melanoma cell implant in the transgenic mice. B: Higher magnification of the tumor mass showing the intracytoplasmic accumulation of melanin pigment (asterisks) and mitotic figures (arrowheads). C and D: Quantitative analysis of the necrosis expressed as a percentage of tumor area (C) and of the tumor residual area expressed in mm² (D). Data were obtained from nine mice per group. Results were expressed as mean ± SEM, *P < 0.05 versus WT, Mann-Whitney’s test. Original magnifications: ×20 (A); ×400 (B).

Figure 4-6914

Tumor vessel area is increased in K14-PlGF transgenic with respect to WT mice. Quantitative analysis was performed by immunohistochemical staining of PECAM/CD31- and SMA-expressing vessels. Ten fields per animal were analyzed for each parameter (nine mice per group). Results are expressed as mean ± SEM. Mann-Whitney’s test was used: *P < 0.05 and **P < 0.001 versus WT.

Figure 5-6914

HSC levels are higher in the serum of K14-PlGF transgenic mice. A: Methylcellulose-based colony assay of peripheral blood mononuclear cells derived from periphery blood of WT and transgenic (K14-PlGF) mice (10 mice per group). Mann-Whitney’s test was used and resulted in a significant difference: P < 0.001 transgenic versus WT mice. B: Immunohistochemistry for SDF-1α on tumor sections obtained from inoculated animals (nine mice per group). An SDF-1α-specific staining is detected in clusters of melanoma cells inside the tumor mass. Original magnification, ×400.

Figure 6-6914

K14-PlGF mice show incremented metastasis spreading. A: H&E staining of lung metastatic nodules of WT and transgenic mice (K14-PlGF) 25 days after melanoma cell injection. Original magnifications, ×20. B: Number and area of metastases were evaluated by computer-assisted imaging analysis of lung sections in transgenic and WT animals. Data were obtained from nine mice per group. Metastasis area was expressed as percentage of the lung surface. Data were expressed as mean ± SEM, *P < 0.05 versus WT, Mann-Whitney’s test. C: Quantitative analysis of lung vessels in transgenic and WT animals (eight mice per group) by immunohistochemical staining of PECAM/CD31. Ten fields per animal were analyzed. Results were expressed as mean ± SEM, *P < 0.05 versus WT, Mann-Whitney’s test.

Figure 7-6914

PlGF induces melanoma cell invasion in vitro. A: B16-BL6 cell capability to respond to rmPlGF was tested in an in vitro invasion assay using graded concentrations of the growth factor. **P < 0.001 versus basal invasion, two-tailed Student’s t-test. B: B16-BL6 cell invasion of the extracellular matrix in response to 50 ng/ml of rmPlGF was evaluated in the presence of 10 μg/ml of anti-PlGF blocking monoclonal antibody or the same concentration of an unrelated antibody. **P < 0.001 versus no Ab, two-tailed Student’s t-test. C: Treatment with an anti-PlGF blocking monoclonal antibody did not affect cell invasion stimulated by another chemotactic stimulus, ie, the conditioned medium of human fibroblasts (FBCM). D: Treatment with an anti-mouse VEGFR-1 blocking antibody inhibited PlGF stimulation, whereas anti-mouse VEGFR-2 blocking antibody had a minor but significant effect on PlGF-induced cell invasion. *P < 0.05 and **P < 0.001 versus no antibody, two-tailed Student’s t-test. Results represent invasion index or percentage of invasion ± SEM.

Figure 8-6914

PlGF activates MMP-2 and MMP-9 in a dose-dependent manner. A: Reverse transcription and real-time PCR on total RNA extracted from B16-BL6 cells treated with rmPlGF for 3 hours. Amplifications were performed with oligonucleotides specific for the indicated metalloproteinases. Results are expressed as relative fold mRNA induction over that of the untreated cells considered as 1. B: Gelatin zymography of conditioned medium from B16-BL6 cells stimulated with the indicated concentration of rmPlGF. C and D: Densitometric analysis of gelatin zymography of MMP-9 and MMP-2, respectively. Data are expressed as means ± SEM, *P < 0.02 and **P < 0.001 versus WT, Mann-Whitney’s test.