The presence (A).of Rmepresentative data indicating changes in oxygen concentration in the extracellular medium altastasis is an important prognostic factor in the majority of tumor patients. Distant metastasis begins with the intravasation of primary tumor cells into intratumoral blood and lymphatic vessels. Local tumor cell proliferations elevate hydrostatic pressure in the interstitium, which tends to push tumor cells into microvessels. Tumor angiogenesis and lymphangiogenesis further enhance passive intravasation. In addition to passive cell movement, active cell migrations guided by chemokines are also occurring, in which cancer-associated fibroblasts play a role ^[1][2][3].

Considering the oxygen diffusion path as measured by the oxygen sensor foil. The ratio of red and green fluorescence is repanalogy of chemotaxis, where component movement depends on the concentration gradient of specific chemoattractants, it has long been postulated that the gradients of nutrients and metabolic waste might guide tumor cell migration. This prediction is reinforced given the metabolic characteristics peculiar to solid tumors. In solid tumors, the structures of blood vessels are immature and their topological configuration in tissues is generally irregular ^[4]. As a result, thented in pseudo-color convectional and diffusional transports of substances between individual cells and blood are considerably hindered and steep concentration gradients of nutrients and metabolic wastes are established within the tissue ^[4][5]. Alt 10 min aftehough <0.1% of primary tumor cells succeed in intravasation in the local tissue ^[6], directional placing the gap cover glass (GCG), no cell migration guided by these metabolic gradients, if any, could elevate the probability of intravasation and, ultimately, hematogenous metastasis. However, evidence indicating the status of extracellular nutrient/metabolite gradient was demonstrated us as guiding cues for cancer cell migration is scarce at the present time.

Givend the aboverneath. At six hours after placing the GCG, our initial hypothesis is that the metabolic gradients in the tissue might be a cue for guiding the tumor cells to nearby blood vessels. Because acidosis and hypoxia are the hallmark of an invasive hypoxic tumor ^[7][8], awe −6.2% O₂/focused, among num erous cues in tissues ^[9], on H⁺ and oxygen particularly theinear or concentration gradients. Hereafter, we refer to the gradient of pH and oxygen concentration as metabolic gradients.

To test this hypothesis in vitro, wase used a glassware produced ut that we previously devised and call gap cover glass (GCG; Figure 1) ^[10] to apply in proder the GCG. The number of ucing metabolic gradients in cultured monolayer cells. In fact, we demonstrated that gradients of extracellular pH with a magnitude of 0.2–0.3 units/mm are established at three hours after placing the GCG onto confluent monolayer MDA-MB-231 cells ^[11], while gradien the culture dish was 5.2 × 10⁶ts of oxygen concentration under the GCG has not yet been demonstrated. Furthermore, we demonstrated a tendency that MDA-MB-231 cells/ mL. Note that the oigrate toward the opening of GCG; in other words, toward areas of higher pH and/or oxygen concentration at the opening of. One of the serious drawbacks of our approach, however, was that migrating cells under the GCG (distance = 0) was substantially lower than that in the microincubatorfrequently underwent collisions with each other and subsequently changed their direction of migration. Thus, we thought that our tentative conclusions regarding directional migration of MDA-MB-231 cells were far from promising. To overcome the collision artifacts, we refined our technique by combining our GCG with the conventional wound-healing assay for cell migration ^[12].

Figure 1. GCG. (21%A), Three thin glass problates were assembled into the GCG. (B) The GCG wabs gently due placed onto the existence of anmonolayer 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 diffusion barrier inconsumption produces gradients of oxygen concentration in the extracellular medium in the narrow channel. Washout of metabolites such as CO₂/H⁺ to the bulk medium ais suggested by Metzen et aimilarly restricted, and pH gradients are produced in the narrow channel. ^[1]. (BC) EffGCG combinect of mitod with wound-healing assay for cell migration. Gradients of oxygen and H⁺ concentration are also illustrated. In thondrial respiratorye 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⁺ complncex III inhibintration; indicated by the thick arrow).

We demonstor (antimycin A, 2 µM) on the oxygeated with a new improved technique that MDA-MB-231 cells under GCG migrate toward higher pH/O₂ regions concentrationwhile directional migration was abolished after eliminating gradient. Elimination of mitochondris of pH. Thus, relatively small gradients of pH in the extracellular medium compared to those found in Nal⁺/H⁺ exchangerespi-driven cell migration aboli were sufficient to guide MDA-MB-231 cells in vitro.

Results

Extracellular Gradients of Oxygen Concentration under GCG

We shuccessfully visualized the oxygen concentration gradient under GCG. Although the cell shape changed slightly s shown in Figure 2A, immediately after placing the GCG, the oxygen concentration along the oxygen diffusion path was constant up to 1200 µm inside the GCG. Subsequently, the oxygen concentration gradients developed slowly and, at six hours after placement of the GCG, a −6.5% O₂/mm linear gradiespiratory inhibition, the nt was established under GCG. Magnitude of the oxygen concentration gradient increased in proportion to the number of cells were per unit volume (Figure 2B).

Figure 2. (A). Representative data indiable as judgcating changes in oxygen concentration in the extracellular medium along the oxygen diffusion path as measured by the LIVE/DEAD cell imaging kit (R37601, Thermo Fisheoxygen 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 Soxygen concientific)entration gradient was produced under the GCG. The cell densitynumber of MDA-MB-231 cells in the culture dish was 5.52 × 10⁶ cells/mL. (CB) 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.

Effects of Hepes-buffered L-15 on pH and Oxygen Gradients under GCG

As reported elsewhere ^[211], extracellular pH gradients along the diffusion path slowly developed in GCG and reached the steady state in three hours. Gradients of pH with a magnitude of 0.2–0.3 units/mm were consistently demonstrated (Figure 2A). Supplementing hepes into the L-15 medium abolished the pH gradient (Figure 2A3). On the other hand, the addition of hepes in L-15 medium did not affect oxygen concentration gradients under GCG (Figure 2B and the open triangle in Figure 2C).

Cell Migration

We completed five “without GCG” and five “with GCG” experiments, respectively, and representative data associated with these “without GCG” and “with GCG” experiments are shown in Supplementary Video S1 and Video S2, respectively. Figure 34 illustrates the trajectory of individual MDA-MB-231 cells for 24 h.

Withe 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.

Discussion

Directional migration of primary cancer cells toward intratumor blood/lymphatic vessels should elevate the probability for intravasation and ultimate hematogenous metastasis. On the analogy of chemotaxis, many presume that the gradients of nutrients and metabolic waste in the local tissue might guide tumor cells to nearby microvessels. However, at the present time, presence of such metabolic cues still remains an open question.

In the present study, we specifically focused on the gradients of H⁺ and oxygen as candidates for the metabolic cue. To monitor migratory behaviors of the cell in gradients of pH and/or oxygen in vitro, we previously proposed a simple microfluidic glassware, GCG, which is capable of producing gradients of energy substrates and metabolites, including H⁺ and oxygen in monolayer cultured cells ^[310]. Simultaneous changes in H⁺ and oxygen concentrations under the GCG are similar to those found in solid tumors and, therefore, experimentation using the GCG reflects a clinically relevant setting. Unlike the recent microfluidic devices designed for investigating cell migration under oxygen concentration gradients ^[4][5][6][7], the magnitude of the metabolic gradients under our GCG depends on the metabolic activity of cells per unit volume and cannot be easily manipulated; control of the gradient would require a redesign of the GCG or an accurate adjustment of the cell density (Figure 1C). It is also impossible for our GCG to produce concentration gradients of a specific molecule in the extracellular space. Thus, additional experiments are required to specifically pinpoint the molecule-of-importance among various metabolic substrates/metabolites.

Previous in vitro studies demonstrated the possibility that oxygen concentration gradients may act as a guiding cue for cell migration. Mosadegh et al. ^[813] used a unique paper-based 3D cell culture system in which oxygen and nutrient gradients were produced along a stack of eight 40-µm-height cell culture layers. They compared metastatic potential in various subtypes of human alveolar adenocarcinoma A549 cells and demonstrated in one A549 subpopulation that oxygen was the primary chemoattractant in their invasion stack; the cells migrated toward the higher oxygen layers. Neither oxygen concentration in each individual layer nor the magnitude of oxygen concentration gradients along the stack was reported. Lewis et al. ^[914] observed migration of individual sarcoma cells embedded in oxygen-controllable hydrogels in which gradients of oxygen were established. With a 2.5 mm-thick hydrogel (hypoxic gel), the measured oxygen concentrations at the bottom of the hydrogel layer decreased to 0.4%–4% O₂ from air level corresponding to oxygen gradients ranging from 3.4%–5.4% O₂/mm. They found that individual sarcoma cells in the hypoxic gel migrate more quickly, across longer distance, in the direction of increasing oxygen concentration compared to those in the normoxic hydrogel with much smaller oxygen gradient. Chang et al. ^[515] demonstrated using a sophisticated microfluidics platform that under the combination of oxygen and chemokine (SDF-1α) gradients A549 cells migrated toward lower oxygen regions in a 5% O₂/mm oxygen gradient. Sleeboom et al. ^[716] demonstrated that both MDA-MB-231 cells and their stem cell enriched population similarly migrate toward low oxygen levels in a 2% O₂/mm oxygen gradient as determined by cell migration trajectory and the forward migration index. The average local oxygen concentration along the oxygen gradient varied from 1% to 7%, which did not significantly affect the forward migration index of the individual cell. Shih et al. ^[617] proposed a microfluidic device in which oxygen concentration gradients with a magnitude of 18% O₂/mm were established along the 900 µm-wide cell channel using oxygen scavenging chemical reactions. HUVECs located at the boundaries of 300 µm-wide cell regions migrated differently in the oxygen gradient; cells initially located at the boundary with higher oxygen concentration in the gradient migrated slowly compared to those with lower oxygen concentration, resulting in a collective cell migration toward lower oxygen.

Results of these in vitro studies appear conflicting in terms of the direction of cell migration in oxygen gradients. HowIndevered, effects of oxygen gradient on the cell migration should differ according to cell type, culture conditions (2D or 3D, culture media) and spatial profiles of oxygen concentration, including the magnitude and the local oxygen concentration at which cell is exposed. Furthermore, in most microfluidic devices, profile of oxygen concentration gradients may, more or less, change after loading cells in the device due to metametabolic activity of the cell. More importantly, metabolic activitieies of the cell may also change spatial distribution of nutrients and metabolites that may affect the cellular migratory behavior in addition to the effect of the oxygen concentration gradient.

Initially, we hypothesized that the extracellular gradients of oxygen might be a cue for MDA-MB-231 cells to migrate directionally because steep gradients of oxygen concentration (~10 mmHg/100 µm) have been demonstrated in vivo ^[105]. Hypoxia-inducible factor 1α (HIF-1α) is an intracellular oxygen sensor that has been reported to affect the intracellular machinery for cell migration ^[1118]. Therefore, it is likely that the HIF-1α pathway plays a role in directing cell migration in the steep gradient of oxygen concentration. However, in the present setting, the direct measurement of the oxygen concentration under the GCG achieved in the present study (Figure 12A) revealed oxygen gradients with relatively small magnitudes such that the oxygen concentration recorded at ~400 µm inside the GCG was much higher (~14%) than the oxygen level at which HIF-1α is responsive (5% or lower ^[1219]). Thus, it is difficult to attribute the directional cell migration demonstrated in the present study specifically to HIF-1α dependent mechanisms. It should be noted here, however, that the present results Although we do not exclude the possibility that oxygen gradients might direct cell migration because the oxygen concentration can drop to pathophysiological levels in hypoxic in hypoxic microenvironment in tumor tissues in vivo ^[10][13]. Nevertheless, directional cell migration at unphysiologically high oxygen concentrations demonstrated in the present study prompted us to seek another possible cue for directional cell migration.

A few studies to date have addressed directional cell migration under gradients of extracellular pH in vitro. Paradise et al. ^[1420] demonstrated using the Dunn chamber that both α_Vβ₃ CHO-B2 cells and primary microvascular endothelial cells preferentially migrate toward acid in an extracellular pH gradient. In their research, the Dunn chamber produced a pH gradient of 6.0 to 7.5 over 1 mm. Elsewhere, Jagielska et al. ^[1521] determined that oligodendrocyte precursor cells migrate toward areas of acidic extracellular pH produced by the Zigmond chamber. Here the pH gradient was set to 6.0 to 7.0 over a distance of 1 mm.

In the current study, we found that, first, a gradient of pH was in fact established in the extracellular medium under the GCG (0.2–0.3 units/mm); second, MDA-MB-231 cells under the GCG preferentially migrated toward the open-end of the GCG (i.e., higher pH/O₂ regions); and third, such findings of directional cell migration completely disappeared when the extracellular pH gradient was abolished. Albeit, while gradients of various metabolic substances should exist under the GCG, these results strongly indicate that extracellular pH gradient is the predominant cue for the migration of MDA-MB-231 cells under the GCG.

Among migrating cells, gradients of intracellular and pextraricellular pH have been demonstrated at the level of the single cell. Martin et al. ^[1622] observed intracellular pH (pH_i) gradients within single melanoma cells incubated in hepes-buffered Ringer solution at a pH of 7.0 and reported that the mean front-to-rear pH_i gradients measured ~0.15 units over a 20-µm distance in migrating human (MV3) and murine (B16V) melanoma cells, where the front (leading edge) was more alkaline. Using a combination of pH-sensitive fluorescent dye and total internal reflection microscopy, Ludwig et al. ^[1723] determined the pericellular pH on the surface of the plasma membrane (pH_em) in polarized MV3 cells, reporting significant gradients of pH_em in single cells where front (at focal adhesions)-to-rear pH_em gradients were ~0.2 units (the cell front was more acidic), indicating the existence of nano-domains with distinct pH values on the surface of the plasma membrane. These subcellular heterogeneities in pH_i mand pH_em y arise from the heterogeneous distribution of the Na⁺/H⁺ exchanger isoform 1 (NHE1), a major plasma membrane protein that extrude protons from cytosol, where NHE1 accumulates in the leading edge of migrating cells ^{[1622][1824][1925]}. Both pH_iintracellular and pericellular pH_em gradients may independently affect cell motility through effects on cytoskeletal machinery and cell-matrix interactions, respectively ^[2026]. In fact, a substantial role of NHE1 activities in cell motility has been demonstrated in various cells ^{[18][21][22][23][24]}.

In the present experiment, we imposed 0.2–0.3 units/mm gradients of pH in the extracellular medium that correspond to a gradient of 0.02 units per single MDA-MB-231 cell. The value is far smaller, if compared at the single-cell level, than the NHE1-driven gradients of pH. This is also true in previous studies in which microfluidic devices produced ~1-unit/mm gradients of pH in the extracellular medium ^[1420][1521]. Therefore, it is unclear whether the relationship between cell migration and the NHE1-driven pH_i and pH_em heterogeneities would be directly applicable to the present and other experiments in which the pH of the extrtracellular bulk medium was manipulated.

Although the/pere is a possibility that subcellular heterogeneities in pH_i and pH_em might endow migrating cells with directionality, we are reluctant to conclude that relatively small gradients of extracellular pH at the single-cell level could be a consistent cue for the migration behavior demonstrated in the present study. Instead, we propose a different model of directional cell migration in which stochastic cell movement is modified by macroscopic (spanning a few hundred microns) gradients in the extracellular pH. This model is based on the dependence of cell migration activity on extracellular pH ^[14][15][24], without assuming significant intracellular pH gradients. Stock et al. ^[23] demonstrated that the pH of the extracellular ould be directly applicabulk solution significantly affects migratory activities in MV3 cells in vitro. At low extracellular pH values, cell-matrix interactions via integrin α₂β₁ appear too strong, we to thile they are too weak at high extracellular pH values, both of which hinder migratory activity. Thus, cell migration was most optimally facilitated at an extracellular pH of ~7.0. Based on this bell-shaped extracellular-pH migration-velocity relationship, it is predicted that cells initially located in regions with extracellular pH values of ~7.0 would vigorously move toward either lower or higher pH regions (random directions). As cells happen to migrate into lower or higher pH regions, the migration velocity gradually decreases and, finally, cells would become trapped in the lowest or highest pH regions, respectively. Thus, a degree of heterogeneity in cell distribution across tpresent and other experiments in which the pH range would be established. It is predictable from this model that the direction of migration (toward the lower or higher pH regions) depends upon the initial eof the extracellular pH, specifically, whether it is lower or higher than the pH at which cell migration is most optimally facilitated.

In association with hypoxia, acidosis is another metabolic hallmark of solid tumors ^[25][26]. A type of metabolic reprograming known as the Warburg effect in cancer cells and reduced washout of CO₂/H⁺ fro m the tissue are the known major causes of an acidotic microenvironment ^[27]. In the process of adaptation to acidosis, cancer cells may acqdiuire a malignant phenotype ^[28][29]. A lo w pH in the tumor microenvironment in vivo reflects the presence of steep gradients of pH between cells and the blood. If the present results are applicable to in vivo conditions, acidotic tumor cells might preferentially migrate toward more alkaline intratumor vessels. With the concomitant acidotic induction of vascular endothelial growth factor and angiogenesis ^[30], directional cell migration would elevate the s maniprobability of intravasation and, ultimately, metastasis. Thus, targeting acidosis in the tumor microenvironment may have therapeutic rationales from the standpoint of control of hematogenous metastasisated.

In summary, the use of novel microfluidic devise GCG produced gradients of pH and oxygen concentration in the extracellular medium in monolayer MDA-MB-231 cells. We clearly demonstrated heterogeneous migration of the cells into the wound space in such a way that cells preferentially migrated in the direction of higher pH/oxygen concentration. Elimination of pH gradients also abolished the directional cell movement under the GCG thus indicating a possibility that extracellular pH gradients are the dominant guiding cue for migration of MDA-MB-231 in the present setting. Because, under GCG, extracellular oxygen concentration remained at unphysiologically high ranges despite the presence of significant gradients, the effect of oxygen concentration gradients on directional migration is still remain to be determined.