Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange

Abstract

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell-matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin alpha2beta1. Cells reached their maximum motility at pHe approximately 7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell-matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin alpha2beta1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell-matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

Figure 1. Migratory speed, translocation and trajectories of MV3 cells depend on pH_e

Cells were either seeded on collagen matrices (2D: A, B and E) or incorporated in collagen lattices (3D: C and D; Supplemental material video 1; video 2; video 3) and recorded for 5 h. Migratory speed (A and C) and translocation within 5 h (B and D) under control conditions (▪), after the application of EIPA (○; video 4) or HOE642 (▵; video 5) and upon treatment with rhodocetin (grey diamonds). n varied between 30 (2D) and 10–20 (3D) cells taken from three to five different trials. E, trajectories of MV3 cells migrating on collagen (2D) at different pH_e values. Trajectories obtained under the same experimental conditions were standardized. They all begin at the same starting post represented by the centre of a circle. The radii of the circles represent the mean distances covered within 5 h. n = 30 cells each, taken from three to five different trials.

Figure 2. HOE642 affects the migratory speed (v), translocation and morphology (SI) of MV3 cells in a dose-dependent manner

pH_e was 7.0 throughout. n = 20–30 cells from three to five different trials in each case.

Figure 3. Morphology of MV3 cells

A and B, cells seeded on collagen matrices (A) or incorporated into collagen lattices (B) after a 3 h exposure to different pH_e values (a–c; Supplemental material videos 1–3) or 3 h after the application of 10 μmol l⁻¹ EIPA (d; video 4) or 10 μmol l⁻¹ HOE642 (e; video 5) at pH_e 7.2. C, cells seeded on collagen matrices were exposed to sodium propionate (a) or to sodium propionate plus HOE642 (b) or treated with rhodocetin (c). The arrow heads in Ca indicate shedding of cell processes.

Figure 4. Structural index (A) and formation of lamellipodia (B) in MV3 cells depend on pH_e and NHE activity

Cells were seeded on collagen and incubated in RPMI⁻ media of different pH_e in the presence (○) or absence (▪) of 10 μmol l⁻¹ EIPA or in the presence of 5 μg ml⁻¹ rhodocetin (grey diamonds). The parameters were determined at t = 60 after seeding. The results shown in A and B were obtained from the same cells. Each data point represents the mean ±s.e.m. obtained from n = 50 cells from five different experiments (5 cells per area (2 × 10⁵μm²), 2 areas per experiment).

Figure 5. Cytosolic pH (pH_i) of MV3 cells depends on the external pH (pH_e) or NHE activity

pH_i was measured after a 3 h exposure to different pH_e values (▪) or 3 h after the addition of 10 μmol l⁻¹ EIPA (at pH_e 6.8 and 7.2; ○) and 10 μmol l⁻¹ HOE642 (at pH_e 7.2; ▵). n varied between 21 and 106 cells from three to six trials.

Figure 6. Adhesion of MV3 cells to a collagen matrix depends on pH_e and NHE activity

The adhesiveness is represented by the number of cells that remained stuck to the collagen matrix after washing with PBS 60 min after seeding. Cells were treated with rhodocetin (grey diamonds, n = 3 trials, four areas per trial) or exposed to solutions of various pH_e with (○, n = 4 trials, two areas per trial) or without (▪, n = 6 trials, two areas per trial) 10 μmol l⁻¹ EIPA.

Figure 7. Cells exposed to low pH_e values (pH 6.8) pull on the matrix when they retract their lamellipodia

This is shown by the displacement of microbeads incorporated in the collagen matrix. A, bead 1 moved 1.1 μm within 30 min, beads 2 and 3 were displaced by 1.3 and 1.9 μm, respectively. At high pH_e (pH 7.5) cells did not exert traction on the matrix. The labelled bead moved 0.2 μm. Scale bar = 10 μm. B, statistics of bead displacement at pH_e 6.8 (n = 12 beads from four different trials, three beads per cell) and at pH_e 7.5 (n = 15 beads from five different trials, three beads per cell).

Figure 8. In MV3 cells the expression of integrin β1 does not depend on pH_e

A, Western blot of human integrin β1. Equal amounts of protein (2 μg) were loaded. B, the 110 kDa (precursor protein, open bars) as well as the 130 kDa bands (mature protein, filled bars) did not differ depending on pH_e (mean ±s.e.m.; n = 5 blots).

Figure 9. Comparison of the distributions of vinculin (A–C) and integrin β1 (D–F) in MV3 cells at different pH_e

Cells were seeded on collagen at different pH_e values and then fixed 60 min after seeding. pH_e varied between pH 6.8 and pH 7.5. Scale bar: 50 μm.

Figure 10. Hypothetical model of the integrin–collagen interaction

Integrins and NHE1 are colocalized. Protons stabilize the collagen–integrin (α2β1 dimer) bond. A, acidosis, a surplus of protons or a lively NHE activity, leads to tight adhesion which generates a dendritic cell shape. B, alkalosis, a lack of protons or an inhibited NHE activity, prevents adhesion and cells become spherical.