Mg-based materials diminish tumor spreading and cancer metastases

Abstract

Cancer metastases are the most common causes of cancer-related deaths. The formation of secondary tumors at different sites in the human body can impair multiple organ function and dramatically decrease the survival of the patients. In this stage, it is difficulty to treat tumor growth and spreading due to arising therapy resistances. Therefore, it is important to prevent cancer metastases and to increase subsequent cancer therapy success. Cancer metastases are conventionally treated with radiation or chemotherapy. However, these treatments elicit lots of side effects, wherefore novel local treatment approaches are currently discussed. Recent studies already showed anticancer activity of specially designed degradable magnesium (Mg) alloys by reducing the cancer cell proliferation. In this work, we investigated the impact of these Mg-based materials on different steps of the metastatic cascade including cancer cell migration, invasion, and cancer-induced angiogenesis. Both, Mg and Mg-6Ag reduced cell migration and invasion of osteosarcoma cells in coculture with fibroblasts. Furthermore, the Mg-based materials used in this study diminished the cancer-induced angiogenesis. Endothelial cells incubated with conditioned media obtained from these Mg and Mg-6Ag showed a reduced cell layer permeability, a reduced proliferation and inhibited cell migration. The tube formation as a last step of angiogenesis was stimulated with the presence of Mg under normoxia and diminished under hypoxia.

Keywords: Angiogenesis; Cancer; Cell invasion; Cell migration; Magnesium degradation; Osteosarcoma.

Graphical abstract

Fig. 1

Scheme of the experimental set-up to investigate the cell invasion.

Fig. 2

Cell migration influenced by Mg and Mg–6Ag under normoxia. Microscopic images of Saos-eGFP (green) and RF Fibroblasts (red) in coculture or monocultures with the initial wound (0 h) and after 48 h. The white dotted lines symbolize the cell fronts. Scale bar is 100 μm. The cell-free areas after 24 h and 48 h were quantified in relation to the initial cell-free area. Relative cell-free areas are shown as the mean ± SD from two experiments with two samples and three randomly chosen positions (n = 12). Statistics: two-way ANOVA (Mg, Mg–6Ag compared to glass control = #; 24 h, 48 h compared to 0 h = *) with Tukey's multiple comparison test. One symbol = p < 0.05; two symbols = p < 0.01; three symbols p < 0.001; four symbols = p < 0.0001.

Fig. 3

Cell invasion influenced by Mg and Mg–6Ag. Cells invaded through an ECM mimetic gel layer from the point of cell seeding (320 μm) to the membrane (0 μm). Fluorescence intensities of representative z-stack (10 μm steps) images are shown. Statistics: ordinary one-way ANOVA with Dunnett's multiple comparison test (n = 12); * = p < 0.01, ** = p < 0.01.

Fig. 4

Visualization of invasive cells. (A) Cells that invaded the ECM mimetic gel layer and crossed the membrane were stained with crystal violet. Red arrows on representative images indicate invaded cells, which were quantified (red numbers). Scale bar is 100 μm. (B) Quantitative analysis of images. Statistics: two-way ANOVA (Mg, Mg–6Ag compared to glass control = *) with Tukey's multiple comparison test (n = 12). * = p < 0.05; *** = p < 0.001; **** = p < 0.0001.

Fig. 5

The impact of Mg-based materials on metastases-associated cytokine release. MMP-2, MMP-9 and TIMP-1 was quantified in the supernatant of migrating cells of the coculture and normalized to the cell numbers. Normalized cytokine concentrations are shown as the mean ± SD from two experiments with two samples in duplicates (n = 12). Statistics: two-way ANOVA (materials, time points) with Tukey's multiple comparison test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Fig. 6

Supernatant Mg and Ag concentration of conditioned media. Saos-eGFP and RF Fibroblasts were seeded as a 1:1 coculture (CC) or monocultures (Saos-eGFP: MCS, RF Fibroblasts: MCF) on Mg, Mg–6Ag or glass (glass control). Furthermore, material without cells served as a mat. control. After one, three and seven days, conditioned medium was harvested, and Mg (in mM) and Ag (orange numbers, in μM) concentration were measured as described previously [12].

Fig. 7

Endothelial cell layer permeability with different conditioned media. HUVEC were incubated with conditioned media from the coculture (CC) or Saos-eGFP (MCS) and RF Fibroblasts (MCF) in monocultures on Mg-based materials or material without cells. Fluorescein-dextran concentrations (in μg/mL; quantified with a standard curve) are shown as the mean ± SD from two experiments with one triplicate (n = 6). Statistics: two-way ANOVA (materials, time points) with Tukey's multiple comparison test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Fig. 8a

Endothelial cell proliferation with different conditioned media. HUVEC were incubated with conditioned media from the coculture (CC) or Saos-eGFP (MCS) and RF Fibroblasts (MCF) in monocultures on Mg-based materials or material without cells. Fluorescence intensities (normalized to day 0) are shown as the mean ± SD from two experiments with six triplicates (n = 12). Statistics: two-way ANOVA (materials, time points; compared to day 0) with Tukey's multiple comparison test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Fig. 8b

Endothelial cell tube formation. Representative pictures of HUVEC capillary structures under normoxia stained with calcein-AM. Scale bar is 100 μm. Images were analyzed with the “Angiogenesis analyzer” from ImageJ and branch numbers are shown as the mean ± SD from two experiments with three samples (n = 6). Statistics: two-way ANOVA (materials, time points) with Tukey's multiple comparison test.

Fig. 9

Endothelial cell migration with different conditioned media. HUVEC were incubated with conditioned media from the coculture (CC) or Saos-eGFP (MCS) and RF Fibroblasts (MCF) in monocultures on Mg-based materials or material without cells. (A) Representative microscopic images of the scratch area in a HUVEC layer within 48 h. The white dotted lines symbolize the cell fronts. Scale bar is 100 μm. (B, C) Quantification of the scratch area in relation to the initial cell-free area under normoxia (B) and hypoxia (C). Relative cell-free areas are shown as the mean ± SD from two experiments with two samples and three chosen positions (n = 12). Statistics: two-way ANOVA (Mg, Mg–6Ag compared to glass control = #; 24 h, 48 h compared to 100% at 0 h = *) with Tukey's multiple comparison test. One symbol = p < 0.05; two symbols = p < 0.01; three symbols p < 0.001; four symbols = p < 0.0001.

Fig. A1

Cancer-induced angiogenesis. Endothelial cells are influenced by VEGF (blue triangle), which results in a leaky cell layer. This ameliorates the release of plasma proteins (red circles).

Fig. A2

Scheme of conditioned media production. Saos-eGFP (green) and healthy fibroblasts (red) were seeded on the different materials (Mg, Mg–6Ag, glass slides) in different culture combinations (monocultures, coculture).

Fig. A3

Migration assay with Mg-based materials. This figure is equivalent to Fig. 2 with time points between 0 h and 72 h. Scale bar is 100 μm.