Bone marrow-derived mesenchymal stem cells drive lymphangiogenesis

Abstract

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.

Figure 1. BM-MSC enhance tumor growth and stimulate lymphangiogenesis in vivo.

(A) In vivo bioluminescent signal of tumors developed following injection of 5×10⁴ LLC-Luc cells alone (n = 30) or mixed with 2,5×10⁵ MSC (LLC+MSC) at day 21 (n = 21). The graph corresponds to the quantification of luciferase activity revealing a strong increase of the signal in the LLC+MSC group. (B) BM-MSC enhance lymphatic vessel density in tumors. Sections of tumors induced by injection of LLC-Luc alone (LLC) or with MSC (LLC+MSC) were immunostained with an anti-podoplanin antibody. A computer-assisted quantification of lymphatic vessel density in LLC-Luc tumors (LLC) or in LLC-Luc tumors mixed with BM-MSC (LLC+MSC) is provided on the right. Bar: 100 µm (C) BM-MSC enhance in vivo lymphatic vessel recruitment in sponge implanted in mice ear. Sponges soaked with control medium (CTR; n = 8) or with MSC conditioned medium (MSC CM; n = 7) were implanted in mice ear between skin and cartilage. Lymphatic vessels were identified by LYVE-1 immunolabeling (green) and nuclei were evidenced with Dapi (blue). Bars: 5 mm and 1,5 mm on magnification. * P<0.05. (D). The graphs correspond to LYVE-1 positive lymphatic vessel quantification expressed as (1) the number of vessels plotted as a function of distance to the sponge edge (top graph), and (2) the number of vessels at a distance of 0.3 mm from the edge of the sponge (bottom graph). * P<0.05.

Figure 2. BM-MSC stimulate lymphangiogenesis in vitro.

(A) Lymphatic rings were cultured during 5 days alone (CTR) or in presence of BM-MSC spheroids (+MSC spheroids), and during 10 days with control medium (CTR2) or with MSC conditioned medium (MSC CM) prepared as described in material and methods section. For quantification, a grid corresponding to successive increments at fixed intervals of explant boundary was used on binarized images and the number of microvessel–grid intersections (N_i) was quantified on binarized images. Quantification was performed at a distance of 0.5 mm and results are expressed as the number of intersections (N_i) plotted as a function of distance (mm) to the lymphatic ring. Bar: 500 µm. * P<0.05. (B, C) MSC conditioned medium significantly stimulates the proliferation and migration of LEC in vitro as compared to control medium. (B) Proliferation rate was measured by a WST-1 and BrdU incorporation assays. ** P<0.01, *** P<0.001. (C) Migration was measured in a Boyden chamber assay. *** P<0.001.

Figure 3. VEGF-A secreted by BM-MSC activate LEC.

(A) Western blot analyses of VEGF-A and VEGF-C production on serum-free EBM-2 (CTR) and MSC conditioned medium (MSC CM). (B) VEGFR-2 (top) and VEGFR-3 (bottom) proteins were detected following a phosphorylated tyrosine-containing protein (pY) immunoprecipation (IP) of LEC lysates after cell stimulation with control medium (CTR) or with MSC conditioned medium (MSC CM). Cells treated with VEGF-A (10 ng/ml) or VEGF-C (400 ng/ml) were used as negative and positive controls, respectively. GAPDH western blot was performed on the flowthrough of each sample.

Figure 4. VEGF-A is an important factor implicated in LEC stimulation by MSC conditioned medium.

(A, B) The trapping of VEGF-A by the addition of soluble receptors-1 and -2 decreased MSC conditioned medium-induced LEC proliferation, measured by WST-1 assay (A) and migration in a Boyden chamber assay (B). *** P<0.001. (C) Specific inhibition of VEGFR-2 with ZM 323881 10 nM decreased MSC conditioned medium-induce LEC proliferation measured by WST-1 assay. *** P<0.001. (D) Phosphorylation of VEGFR-2 and ERK1/2 analyzed by western blotting on LEC treated or not with soluble VEGF receptors or ZM 323881 10 nM.