A Relatively Small Gradient of Extracellular pH Directs Migration of MDA-MB-231 Cells In Vitro

Abstract

Hematogenous tumor metastasis begins with the invasion and spread of primary tumor cells in the local tissue leading to intravasation. We hypothesized that tumor cells might actively migrate toward intratumor vessels with the extracellular metabolic gradient acting as a guiding cue. Here, we determined in vitro whether the extracellular gradient of pH can act as a cue for directional migration in MDA-MB-231 cells. Cell migration was determined by the wound-healing assay under gradients of extracellular pH (~0.2 units/mm) and oxygen concentration (~6% O₂/mm) that were produced by a microfluidic device, gap cover glass (GCG). Without GCG, the migration of cells was spatially homogeneous; the same number of cells migrated to the rectangular wound space from the left and right boundaries. In contrast, when GCG generated pH/O₂ gradients across the wound space, the number of cells migrating to the wound space from the boundary with higher pH/O₂ values was considerably decreased, indicating a preferential movement of cells toward the region of higher pH/O₂ in the gradient. The addition of hepes in the extracellular medium abolished both the extracellular pH gradient and the directional cell migration under GCG. We conclude that relatively small gradients of pH in the extracellular medium compared to those found in Na⁺/H⁺ exchanger-driven cell migration were sufficient to guide MDA-MB-231 cells. The directional cell migration as guided by the metabolic gradient could effectively elevate the probability of intravasation and, ultimately, hematogenous metastasis.

Keywords: MDA-MB-231 cells; cell migration; metastasis; oxygen gradient; pH gradient.

Figure 1

(A). Representative data indicating changes in oxygen concentration in the extracellular medium along the oxygen diffusion path as measured by the oxygen sensor foil. The ratio of red and green fluorescence is represented in pseudo-color. At 10 min after placing the gap cover glass (GCG), no gradient was demonstrated underneath. At six hours after placing the GCG, a −6.2% O₂/mm linear oxygen concentration gradient was produced under the GCG. The number of MDA-MB-231 cells in the culture dish was 5.2 × 10⁶ cells/mL. Note that the oxygen concentration at the opening of the GCG (distance = 0) was substantially lower than that in the microincubator (21%), probably due to the existence of an oxygen diffusion barrier in the bulk medium as suggested by Metzen et al. [14]. (B) Effect of mitochondrial respiratory complex III inhibitor (antimycin A, 2 µM) on the oxygen concentration gradient. Elimination of mitochondrial respiration abolished the oxygen concentration gradient under GCG. Although the cell shape changed slightly six hours after respiratory inhibition, the cells were viable as judged by the LIVE/DEAD cell imaging kit (R37601, Thermo Fisher Scientific). The cell density was 5.5 × 10⁶ cells/mL. (C) Relationship between the magnitude of the oxygen concentration gradient and cell density. The linear relationship indicates that the oxygen concentration gradient depends upon oxygen consumption of the cell per unit volume. The open triangle represents the slope in which L-15 medium was buffered with 15 mM hepes.

Figure 2

Gradients of extracellular pH and oxygen concentration under GCG in hepes (15 mM)-buffered L-15 medium. (A) Representative pH data in L-15/hepes medium. Average pH gradients in L-15 medium are also shown, where the range of ±standard deviation (SD) is represented in gray (n = 4). Data were collected three hours after placing the GCG. (B) Representative oxygen gradients in L-15/hepes medium. Cell density-corrected oxygen gradients represented as the open triangle in Figure 1C were comparable to those in L-15 medium. Data were collected six hours after placing the GCG.

Figure 3

Trajectories of MDA-MB-231 cells measured for 24 h. The oxygen concentration in the microincubator was 21% (room air). Values presented in the graphs are in micrometers. (A) Without the GCG placed, cells migrated into the wound space equally from the right (R) and left (L) boundaries of the wound space. (B) With the GCG placed, the migration of the cells initially located at the right boundary (R) into the wound space was hindered and they even appeared to migrate in a direction opposite to that toward the wound space as if they were crawling into the crowd of cells. Trajectories of the 50 cells are superimposed. To assess the rate of cell proliferation under GCG, 300 × 500 µm rectangular regions of interest (ROIs) were defined close to but outside the wound space (dashed squares) and the number of cells in the respective ROI was counted and compared at time = 0 and time = 24 h.

Figure 4

Forward migration index (FMI) parallel to the diffusion path (FMI_x) without (A) and with the GCG in place (B). Without the GCG involved, the FMI_x values for the L-cells and R-cells were not different (NS, p = 0.60). In contrast, with the GCG in place, the FMI_x value for the R-cells was significantly smaller compared to that of the L-cells. Data were accumulated from five independent experiments in which 10 cells were sampled in each experiment. Error bars represent the SD. *, p < 0.05, as judged by Student’s t-test.

Figure 5

The number of cells that migrated into the wound space in 24 h. (A) Without the GCG, the numbers of cells migrating into the wound space from the left boundary and the right boundary were the same. (B) With the GCG, the numbers of cells migrating into the wound space from the left boundary and the right boundary were different, with a statistically significant difference demonstrated after five hours. (C) The ratio of the numbers of cells migrating into the wound space from the left boundary and the right boundary determined at 24 h (N_L and N_R, respectively). The directional migration of MDA-MB-231 cells was clearly demonstrated with involvement of the GCG. Error bars represent the SD. Data were accumulated from five independent experiments. *, p < 0.05, as judged by Student’s t-test.

Figure 6

Effects of extracellular pH gradients on FMI_x in cells underneath GCG. (A) In L-15 medium, the FMI_x for the L-cells was significantly higher than that for the R-cells. This result is consistent with that in Figure 4B. (B) In L-15/hepes medium in which extracellular pH gradients disappeared, the FMI_x values for the L-cells and R-cells were not different (p = 0.20), indicating that the directionality in cell migration also disappeared. Data were accumulated from five independent experiments in which 10 cells were sampled in each experiment. Error bars represent the SD. *, p < 0.05, as judged by Student’s t-test.

Figure 7

The number of cells that migrated into the wound space in 24 h. (A) In L-15 medium, N_L and N_R were different, consistent with what is observable in Figure 5B. (B) Conversely, in L-15/hepes medium, N_L and N_R were the same. (C) The heterogeneity in cell migration ultimately disappeared in L-15/hepes medium. Error bars represent the SD. Data were accumulated from five independent experiments. *, p < 0.05, as judged by Student’s t-test.

Figure 8

GCG. (A) Three thin glass plates were assembled into the GCG. (B) The GCG was gently placed onto the monolayer of MDA-MB-231 cells. The diffusional supply of energy substrates such as oxygen to cells in the narrow channel of the GCG is restricted. Thus, cellular oxygen consumption produces gradients of oxygen concentration in the extracellular medium in the narrow channel. Washout of metabolites such as CO₂/H⁺ to the bulk medium is similarly restricted, and pH gradients are produced in the narrow channel. (C) GCG combined with wound-healing assay for cell migration. Gradients of oxygen and H⁺ concentration are also illustrated. In the present study, we demonstrated a vectorial movement of MDA-MB-231 cells toward the open-end of GCG (i.e., higher oxygen concentration or lower H⁺ concentration; indicated by the thick arrow).