Targeting VEGFR-3/-2 signaling pathways with AD0157: a potential strategy against tumor-associated lymphangiogenesis and lymphatic metastases

Abstract

Background: Lymphatic metastasis is one of the leading causes of death in patients with different types of cancer and is the main prognostic factor for the disease survival. The formation of new lymphatic vessels (lymphangiogenesis) in primary tumors facilitates tumor cell dissemination to regional lymph nodes and correlates with distant metastases. Lymphangiogenesis has thus emerged as a suitable therapeutic target to block metastases, but no anti-lymphangiogenic compounds have been approved for clinical use to date. Therefore, new or improved therapies blocking lymphatic metastases are urgently required.

Methods: We established murine breast tumors to assess the effect of AD0157 on tumor growth, lymphangiogenesis, and lymphatic dissemination. Then, a battery of in vivo (mouse corneal neovascularization and ear sponges), ex vivo (mouse lymphatic rings and rat mesentery explants), and in vitro (proliferation, tubulogenesis, wound-healing, Boyden chambers, and spheroids) assays was used to give insight into the lymphangiogenic steps affected by AD0157. Finally, we investigated the molecular pathways controlled by this drug.

Results: AD0157 was found to inhibit the growth of human breast cancer xenografts in mice, to strongly reduce tumor-associated lymphangiogenesis and to block metastatic dissemination to both lymph nodes and distant organs. The high anti-lymphangiogenic potency of AD0157 was further supported by its inhibitory activity at low micromolar range in two in vivo pathological models and in two ex vivo assays. In addition, AD0157 inhibited lymphatic endothelial cell proliferation, migration and invasion, cellular sprouting, and tube formation. Mechanistically, this compound induced apoptosis in lymphatic endothelial cells and decreased VEGFR-3/-2, ERK1/2, and Akt phosphorylations.

Conclusions: These findings demonstrate the suitability of AD0157 to suppress tumor-associated lymphangiogenesis. Beyond discovering a new potent anti-lymphangiogenic drug that is worth considering in future clinical settings, our study supports the interest of designing anti-lymphangiogenic therapies to avoid distant metastatic processes.

Keywords: AD0157; Lymph nodes; Lymphangiogenesis; Lymphatic metastasis; VEGFR-3/-2.

Fig. 1

AD0157 reduces tumor growth and lymphangiogenesis in orthotopic mammary xenografts. a Evolution of tumor volumes in NOD/SCID mice implanted with MDA-MB-231/Luc+ cells and treated daily with vehicle (saline solution) or AD0157 (1.5 or 3 mg/kg) for 25 days, as indicated on top of the graph. Dashed line indicates the tumor volume (50 mm³) reached by tumors before drug treatment. Crosswise lines indicate the starting point for the statistical differences shown at right. b Mouse body weights before and during treatments. ns denotes no statistical differences between groups. c Representative in vivo bioluminescent signals of mice at the end of the treatment. d Quantification of tumor bioluminescent radiance. e Tumor weights at the end of the experiment. f Representative pictures of tumors harvested from mice treated with vehicle (control) or AD0157 (1.5 and 3 mg/kg). Each square represents a surface of 2.25 cm². g Representative histological sections showing human ki-67 (proliferation marker, in brown) and LYVE-1 (lymphatic vessel marker, in dark pink) stainings in mammary tumors. Scale bars represent 100 μm. h Quantification of human ki-67⁺ cell and lymphatic densities in histological sections of mammary tumors. Data are presented as mean ± s.e.m. One-way ANOVA significance test, followed by the Bonferroni test, was used to compare the tumor growth in the different conditions. The Wilcoxon-Mann-Whitney was applied for the rest of statistical tests. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 12 mice/group)

Fig. 2

AD0157 reduces tumor colonization and lymphatic density in draining LNs. a Representative pictures of ex vivo bioluminescent signals recorded in draining LNs at mice sacrifice. The percentage of positive LNs for tumor cells is indicated. b Quantification of bioluminescent radiance in LNs. c Immunostainings of human Ki-67 (proliferative cells in brown, left panels) and LYVE-1 (lymphatic vessels in dark pink, right panels) in whole LN histological sections. Panels at high magnification illustrate the region delineated by the square in the complete LNs. Scale bars represent 250 μm. d Human ki-67⁺ cell density (surface occupied by tumor cells divided by the total LN surface), lymphatic density (surface occupied by lymphatic vessels divided by the total LN surface), and spatial distributions of tumor cells and lymphatic vessels in LNs. The spatial distribution is calculated from the LN edge, where the distance is equal to 0

Fig. 3

AD0157 prevents distant metastases. a Representative ex vivo bioluminescent signals of distant organs (brain, lungs, liver, intestines, kidneys, spleen, and uterus). b Immunohistochemical images visualizing micrometastases by human Ki-67 detection (in brown). Scale bars represent 250 μm. c Number and percentage of organs colonized by metastatic cells. Data are presented as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between vehicle (control) and AD0157 treatment. Chi-square with Yates’ correction test was used to analyze the metastasis incidence. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 12 mice/group)

Fig. 4

AD0157 impairs corneal neovascularization in mice. a Representative flat-mounted cauterized corneas harvested from control and AD0157-treated mice at day 9 post-injury. Lymphatic (LYVE-1 positive) and blood (CD31 positive) vessels appear in green and in red, respectively, at low (left panels) and high (middle panels) magnifications. Scale bars represent 1000 and 500 μm, respectively. Panels at right correspond to representative pictures of filopodia-like structures (white arrowheads) displayed by migrating LECs. Scale bars represent 10 μm. b Computerized measurements of different parameters: lymphatic area density (area covered by neoformed lymphatic vessels), length density (cumulative length of vessels), branching density (number of bifurcations), end-point density (number of sprout tips), spatial lymphatic distribution (a grid was applied on each cornea picture to establish the distribution curves of capillaries around the limbal vessels; N_i corresponds to the number of vessel intersections with the grid versus the distance to the limbus), number and length of filopodia-like structures in a total length of 25 μm at the end of the lymphatic vessel, and blood area density (area covered by neoformed blood vessels). Results were divided by the total cornea area to obtain densities. Values are expressed as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between control and AD0157 treatments.*p < 0.05, **p < 0.01, ***p < 0.001 (n = 12 mice/group)

Fig. 5

AD0157 inhibits in vivo VEGF-C-induced lymphangiogenesis and blocks the lymphangiogenic process ex vivo. a Representative pictures of gelatin sponges soaked with either rhVEGF-C (1 μg/mL) alone (positive control) or rhVEGF-C + AD0157 at different concentrations and implanted between the mouse ear skin layers. Lymphatic and blood vasculatures were examined by LYVE-1 (green) and CD31 (red) immunostainings, respectively. Dapi staining was used to detect cell nuclei (blue). Scale bars represent 1500 and 500 μm on higher magnification. A total of 10 mice were analyzed in each experimental condition. b The graphs represent the computerized quantification of the lymphatic density (left panel), the normalized frequency of lymphatic vessels from the sponge edge to its center (middle panel), and the blood density (right panel). Arrows indicate the maximal distance of LEC migration (Lmax). c Ex vivo lymphatic sprouting from mouse thoracic duct explants embedded in a type I collagen gel and cultured in the absence (control) or presence of AD0157 for 7 days. Scale bars represent 500 μm. d Graphs represent the number of microvessel intersections (N_i) quantified on binarized images using a grid of concentric rings (left graph) and the LEC density at a distance (d) = 0.25 mm from the ring border (right graph). A total of 10 lymphatic rings collected from five different mice were analyzed in each experimental condition. e Microvessel outgrowth in rat mesenteric windows incubated in medium containing either rhVEGF-C (200 ng/mL) alone or in combination with AD0157, ranging from 0.5 to 2.5 μM, for 5 days. Scale bars represent 200 μm. f Lymphatic density and number of branchings in rat mesenteric windows exposed or not to AD0157. A total of 10 mesenteric windows harvested from five different rats were evaluated in each experimental condition. Values are expressed as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between control and AD0157 treatment. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

AD0157 suppresses lymphangiogenesis in vitro by interfering with LEC tubulogenesis, migration, invasion, and sprouting. a Tube-like structures formed by LECs in a collagen matrix, in the absence (control) or presence of different AD0157 concentrations. Scale bars represent 200 μm. b Computerized quantification of the tube area density and the number of branchings (common point between 2 or more tubes). c Pictures showing the area recovered by LECs after 48 h of wounded LEC monolayers, in the absence or presence of different AD0157 doses. Dashed lines in pictures indicate the initial (time 0) wound edges. Scale bars represent 100 μm. d Percentage of the initial cell-free area recovered by endothelial cells. e Representative images of the invaded LECs across a Transwell chamber coated with 0.2% gelatin, after 48 h of treatment. f Percentage of invaded cells. g LEC sprouting from spheroids embedded in a collagen-methyl cellulose gel under different experimental conditions. Scale bars represent 100 μm. h Computerized quantifications of the convex envelope area (minimal convex polygon area containing the spheroid core and all sprouting cells), the migrated cell area (area containing migrating cells), the LEC density (number of pixels belonging to cells that intersect the circle “i”, N_i, normalized by the corresponding perimeter) at different distances from the spheroid core and values of the LEC density at the specific distance of 0.13 mm from the spheroid centre, in control and AD0157-treated spheroids. Arrows indicate the maximal length (Lmax). Values are expressed as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between control and AD0157 treatment. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5 independent tests)

Fig. 7

AD0157 induces apoptosis in LECs. Analysis of apoptosis in LECs treated or not with AD0157 for 14 h. a Chromatin condensation revealed by Hoechst 33258 staining. Scale bars represent 100 μm. b Percentages of cells with condensed chromatin (total cells were counted by using bright field). c Cell cycle distribution in LECs stained with propidium iodide and analyzed by flow cytometry. d Percentages of subG1 (apoptotic cells with invariable DNA content), G1 (cells in interphase before cell division), and S/G2/M (DNA replication and mitosis) LEC subpopulations. e LEC Caspase-3/-7 activity. Values are expressed as mean ± s.e.m., and the Wilcoxon-Mann-Whitney significance tests were used to compare the differences between control and AD0157 treatment. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 5 independent tests)

Fig. 8

AD0157 blocks VEGFR-3 and VEGFR-2 signaling cascades in LECs. a Values of VEGFR-3 and VEGFR-2 mRNA expression levels in LECs incubated with different AD0157 concentrations. b Representative Western-blots showing the effect of AD0157 on VEGFR-3, VEGFR-2, ERK1/2, and Akt phosphorylations after stimulation with rhVEGF-C (400 ng/mL), rhVEGF-C156S (500 ng/mL), or rhVEGF-A (100 ng/m). c Quantification by densitometry of Western blots. Results are expressed as the percentage of phosphorylated proteins/total protein ± s.e.m. of five Western blots. The Mann-Whitney-Wilcoxon test was used to determine if the differences among control (stimulated LECs without AD0157 treatment) and AD0157-treated and stimulated LECs were statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001 versus control