Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis

Abstract

Interplay between various lymphangiogenic factors in promoting lymphangiogenesis and lymphatic metastasis remains poorly understood. Here we show that FGF-2 and VEGF-C, two lymphangiogenic factors, collaboratively promote angiogenesis and lymphangiogenesis in the tumor microenvironment, leading to widespread pulmonary and lymph-node metastases. Coimplantation of dual factors in the mouse cornea resulted in additive angiogenesis and lymphangiogenesis. At the molecular level, we showed that FGFR-1 expressed in lymphatic endothelial cells is a crucial receptor that mediates the FGF-2-induced lymphangiogenesis. Intriguingly, the VEGFR-3-mediated signaling was required for the lymphatic tip cell formation in both FGF-2- and VEGF-C-induced lymphangiogenesis. Consequently, a VEGFR-3-specific neutralizing antibody markedly inhibited FGF-2-induced lymphangiogenesis. Thus, the VEGFR-3-induced lymphatic endothelial cell tip cell formation is a prerequisite for FGF-2-stimulated lymphangiogenesis. In the tumor microenvironment, the reciprocal interplay between FGF-2 and VEGF-C collaboratively stimulated tumor growth, angiogenesis, intratumoral lymphangiogenesis, and metastasis. Thus, intervention and targeting of the FGF-2- and VEGF-C-induced angiogenic and lymphangiogenic synergism could be potentially important approaches for cancer therapy and prevention of metastasis.

Fig. 1.

FGF-2 and VEGF-C collaboratively stimulate hem- and lymphangiogensis. (A) Micropellets containing FGF-2, VEGF-C, or FGF-2 plus VEGF-C together with the slow-release polymer were implanted into micropockets of mouse corneas (n = 5–6 per group). At day 6 after implantation, corneal neovascularization was examined and photographed. Slow-release polymer containing PBS was used as a negative control. P indicates the position of the implanted pellet. (B) Schematic diagram demonstrates the flat-mounted cornea and clock-hours of the circumferential cornea. (C) A representative cornea from each group that were double immunostained with CD31 (red) and LYVE-1 (green), which showed no overlapping signals. (D) Double immunostaining of corneal blood and lymphatic vessels using VEGFR-3 (green) and CD31 (red) specific antibodies. (E) Quantification of corneal CD31⁺ blood vessels at different clock hours (n = 5–6 /group). (F) Quantification of corneal LYVE-1 positive lympghatic vessels at different clockhours (n = 5–6 per group). (G) Quantification of the total CD31⁺ vessels in the entire of circumferential area of each cornea (n = 5–6 per group). (H) Quantification of the total LYVE-1⁺ lymphatic vessels in the entire of circumferential area of each cornea (n = 5–6 per group).

Fig. 2.

In vitro LEC activity and signaling and suppression of hem- and lymphangiogenesis by FGFR-1 blockade. (A) Stimulation of hLEC proliferation by FGF-2, VEGF-C, FGF-2 plus VEGF-C or buffer alone (16 samples per group). (B) Immunoblot analysis of activation of signaling components in hLECs by FGF-2. GDPDH showed the standard level of sample loading. (C) Immunoblot analysis of activation of signaling components in hLECs by VEGF-C. GDPDH showed the standard level of sample loading. (D) Amplification of Fgfr-1 by reverse-transcription PCR using cDNA extracted from LECs. (E) qPCR analysis of Fgfr-1 mRNA expression levels in hLECs stimulated by FGF-2 or VEGF-C. PBS-stimulated cells were used as a control. (F) Quantitative PCR analysis of Vegfr-3 mRNA expression levels in hLECs stimulated by FGF-2 or VEGF-C. PBS-stimulated cells were used as a control. (G) Fgfr-1 specific siRNA substantially inhibited FGF-2–induced LEC proliferation. A scrambled nucleotide sequence was used as a control. (H) Inhibition of FGF-2– or VEGF-C–induced mLEC proliferation by FGFR-1 or VEGFR-3 blockade (six samples per group). (I) Inhibition of FGF-2– or VEGF-C–induced mLEC migration by FGFR-1 or VEGFR-3 blockade (six samples per group). (J) Suppression of FGF-2 plus VEGF-C–induced corneal neovascularization by FGFR-1 blockade. (K) Suppression of FGF-2 plus VEGF-C–induced corneal hemangiogenesis (CD31⁺ blood vessels) and lymphangiogenesis (LYVE-1⁺ lymphatic vessels) by FGFR-1 blockade. P, pellet. (L) Quantification of antiangiogenic activity by FGFR-1 blockade (n = 5–6 per group). (M) Quantification of anti-lymphangiogenic activity by FGFR-1 blockade (n = 5–6 per group).

Fig. 3.

Anti-VEGFR-3 blocks FGF-2–induced lymphangiogenesis and lymphatic tip formation. (A) FGF-2–, VEGF-C–, or FGF-2 plus VEGF-C–induced corneal neovascularization was treated with or without VEGFR-3 blockade. (B) FGF-2–, VEGF-C–, or FGF-2 plus VEGF-C–induced corneas in response to anti–VEGFR-3 treatment were double immunostained with CD31 (red) and LYVE-1 (green). VEGFR-3 blockade almost completely suppressed FGF-2–, VEGF-C–, or FGF-2 plus VEGF-C–induced corneal lymphangiogenesis but not hemangiogenesis. (C) Suppression of FGF-2–, VEGF-C–, or FGF-2 plus VEGF-C–induced tip formation of lymphatic vessels by VEGFR-3 blockade. (D) Quantification of LYVE-1⁺ lymphatic vessels in various groups treated with or without VEGFR-3 blockade (n = 4–6 per group). (E) Quantification of lymphatic tips in various groups treated with or without VEGFR-3 blockade (n = 4–6 per group). (F) Quantification of CD31⁺ blood vessels in various groups treated with or without VEGFR-3 blockade (n = 4–6 per group).

Fig. 4.

FGF-2 and VEGF-C collaboratively promote tumor angiogenesis and hematogenous metastasis. (A) Growth rates of vector, FGF-2–, VEGF-C–, and FGF-2 plus VEGF-C–expressing mouse fibrosarcomas. (B) Detection of tumor microvessels in various tumor groups by CD31 immunostaining. (C) Quantification of CD31⁺ vessel density in various tumor groups (n = 6 per group). (D) Lung morphology of a representative mouse from each tumor-bearing group (n = 6 per group). Arrowheads point to lung surface metastatic nodules. (E) Quantification of percentage of tumor-bearing mice that had lung metastases (n = 6 per group). (F) Histological examination of pulmonary metastases in mice bearing various tumors. Dashed lines encircle metastatic tumors that express EGFP (green). T, tumor.

Fig. 5.

FGF-2 and VEGF-C collaboratively promotes tumor lymphangiogenesis and lymphatic metastasis. (A) Detection of peritumoral and intratumoral lymphatic vessels (PTL and ITL) in various tumor groups by LYVE-1 immunostaining. Dashed line defines the rim between the tumor and peritumoral regions. Arrowheads indicate the tumoral lymphatics. (B) Quantification of intratumoral LYVE-1⁺ lymphatic vessel density in various tumor groups (n = 6 per group). (C) Sentinel lymph node morphology of various tumor-bearing groups (n = 6 per group). (D) Quantification of percentage of tumor-bearing mice that had sentinel lymph-node metastases (n = 6 per group). (E) Average weight of sentinel lymph nodes in various tumor-bearing mice (n = 6 /group). (F) Average volume of sentinel lymph nodes in various tumor-bearing mice (n = 6 per group). (G) Sentinel lymph node histology showed the presence of lymphatic metastases in FGF-2, VEGF-C, and FGF-2 plus VEGF-C tumor-mice. Dashed lines encircle metastatic tumors that express EGFP (green). T, tumor. (H) Schematic diagram of molecular mechanisms by which FGF-2 and VEGF-C collaboratively induce hem-/lymphangiogenesis and metastasis. VEGF-C activates VEGFR-3 receptor on LECs, leading to LEC tip formation, proliferation, and migration. FGF-2 via activation of FGFR-1 stimulates LEC proliferation and migration. However, VEGFR-3–triggered tip cell formation is a prerequisite for FGF-2–induced lymphangiogenesis. Blockade of the VEGFR-3 signaling system completely inhibits FGF-2–induced lymphangiogenesis. Lymhangiogenic interplay between FGF-2 and VEGF-C in the tumor environment promoted lymphatic metastasis. On blood vessel endothelial cells (BVECs), VEGF-C binds to VEGFR-2 and induced hem-angiogenic signals. FGF-2 directly induces hemangiogenesis by activation of FGFR-1 to -4 expressed on BVECs. The FGF-2 plus VEGF-C–induced high numbers of disorganized tumor blood vessels facilitate hematogenous metastasis.