Hematogenous Tumor Metastasis: History
View Latest Version
Please note this is an old version of this entry, which may differ significantly from the current revision.
Subjects: Biochemistry & Molecular Biology
Contributor:
Eiji Takahashi
,
Daisuke Yamaguchi
,
Yoshihisa Yamaoka

This work addresses how the migration of cancer cell line MDA-MB-231 cells is regulated. Directional migration of primary cancer cells toward intratumor blood/lymphatic vessels should elevate the probability for intravasation and ultimate hematogenous metastasis. Many presume, on the analogy of chemotaxis by specific chemoattractants, that concentration gradients of energy substrates/metabolites in tumor tissue would be a guiding cue for directional cell migration, whereas strong experimental evidence is scarce at present. In this study, using a novel microfluidic device, we clearly demonstrated that the gradient of extracellular pH is a cue for directional migration of MDA-MB-231 cells in vitro. Much smaller pH gradients compared to those found in Na+/H+ exchanger-driven cell migration were sufficient to guide the cell. This study answers a longstanding and important question regarding the regulation of cancer cell migration and therefore relevant from the standpoint of not only cell physiology but also clinical sciences, particularly in cancer biology.

  • metastasis
  • cell migration
  • pH gradients
  • oxygen gradients
  • MDA-MB-231 cells

Extracellular Gradients of Oxygen Concentration under GCG

We successfully visualized the oxygen concentration gradient under GCG. As shown in Figure 1A, 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 gradient was established under GCG. The oxygen concentration gradient abolished after treatment with a mitochondrial respiratory complex III inhibitor (antimycin A, 2 µM), indicating that the oxygen concentration gradient depends upon mitochondrial respiration (Figure 1B). Magnitude of the oxygen concentration gradient increased in proportion to the number of cells per unit volume (Figure 1C). In our culture conditions, 100% confluence corresponded to the cell density of ~5.5 × 106 cells/mL and, therefore, the largest magnitude of the oxygen concentration gradient attainable in our GCG system was ~6% O2/mm. It is notable that the oxygen concentration at the entrance of the GCG was substantially lower than the air level. This indicates the presence of an oxygen diffusion barrier in the extracellular medium as suggested previously by Metzen et al. [14].
Ijms 21 02565 g001 550
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.

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

As reported elsewhere [12], 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 2A). 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 1C).
Ijms 21 02565 g002 550
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.

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 3 illustrates the trajectory of individual MDA-MB-231 cells for 24 h.
Ijms 21 02565 g003 550
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.
 
Without the GCG, accumulated distances for 24 h in cells migrating into the wound space from the left boundary (L-cells) and from the right boundary (R-cells) were similar (368 ± 94 µm and 358 ± 112 µm, respectively). With the GCG, the accumulated distance was significantly decreased, while values for the L-cells and R-cells were not different from each other (322 ± 118 µm and 295 ± 138 µm, respectively). Without involvement from the GCG, the L-cells and R-cells similarly migrated into the wound space (Figure 3A). Forward migration index (FMI) values were not different between the L-cells and R-cells (Figure 4A).
Ijms 21 02565 g004 550
Figure 4. Forward migration index (FMI) parallel to the diffusion path (FMIx) without (A) and with the GCG in place (B). Without the GCG involved, the FMIx values for the L-cells and R-cells were not different (NS, p = 0.60). In contrast, with the GCG in place, the FMIx 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.
 
In contrast, in the “with GCG” experiments, we demonstrated distinct differences in the direction of cell migration, particularly in the R-cells. With the inclusion of the GCG, the migration of R-cells into the wound space appeared to be considerably hindered (Figure 3B). These cells even migrated in the direction opposite of that toward the wound space, as if they were instead crawling into the crowd of cells. In fact, the FMI value for R-cells was not different from the null (Figure 4B). These data are consistent with that cells under the GCG demonstrate directional migration toward the open-end of the GCG (right side). FMI values perpendicular to the diffusion path (i.e., y-axis) were not different from the null, regardless of GCG involvement. Directional cell movement can be visually confirmed by observing Video S2.
 
We further checked the directional migration of the cells under GCG by counting the numbers of L-cells and R-cells in the wound space. As shown in Figure 5A, without the GCG in place, the numbers of L-cells and R-cells were the same throughout the experiment. In contrast, with the GCG in place, the numbers of R-cells were smaller than that of the L-cells (Figure 5B), reaching statistical significance after five hours. Thus, the numbers of L-cells and R-cells at 24 h were highly different from one another (Figure 5C).
Ijms 21 02565 g005 550
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 (NL and NR, 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.
 
Effects of cell proliferations on heterogeneous cell migration into the wound space were also evaluated. Without GCG in place, number of the cell in the dashed square on the L-side and that in the dashed square on the R-side (see Figure 3A) similarly increased in 24 h to 111 ± 11% and 109 ± 8% compared to those at time = 0, respectively, representing proliferation of the cell. With the GCG in place, number of the cell in the dashed square on the L-side counted at 24 h increased to 112 ± 14%, while that in the dashed square on the R-side was 98±16%. The difference was not statistically significant (p = 0.055). These results indicate that the difference in the cell proliferation rate in the metabolic gradients had no significant impact on the present wound-healing assays. From these data, we concluded that MDA-MB-231 cells under the GCG demonstrate directional migrations toward the open-end of the GCG.
 
Next, we undertook another series of experiments in which the role of extracellular pH gradients in directional movements of MDA-MB-231 cells was examined. We completed five “L-15” and five “L-15/hepes” experiments, respectively. Figure 6 illustrates the analysis of the directionality of cell migration. With the GCG in place, the magnitude of the FMI for the R-cells was significantly smaller than that for the L-cells in L-15 medium (Figure 6A), while FMI values for the L-cells and R-cells were not different from one another in the L-15/hepes medium (Figure 6B).
Ijms 21 02565 g006 550
Figure 6. Effects of extracellular pH gradients on FMIx in cells underneath GCG. (A) In L-15 medium, the FMIx 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 FMIx 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.
 
In the L-15 medium, the number of cells migrating from the left boundary into the wound space was significantly higher than that from the right boundary (Figure 7A,C). In comparison, in the L-15/hepes medium, such heterogeneity disappeared (Figure 7B,C). These results indicate that the extracellular pH gradient is the dominant cue for the directional migration of MDA-MB-231 cells under our GCG.
Ijms 21 02565 g007 550
Figure 7. The number of cells that migrated into the wound space in 24 h. (A) In L-15 medium, NL and NR were different, consistent with what is observable in Figure 5B. (B) Conversely, in L-15/hepes medium, NL and NR 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.

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 [11]. 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 [15,16,17,18], 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. [19] 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. [20] 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. [16] 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. [18] 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. [17] 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. However, 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 metabolic activity of the cell. More importantly, metabolic activities 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 [5]. Hypoxia-inducible factor 1α (HIF-1α) is an intracellular oxygen sensor that has been reported to affect the intracellular machinery for cell migration [21]. 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 1A) 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 [22]). Thus, it is difficult to attribute the directional cell migration demonstrated in the present study to HIF-1α dependent mechanisms. It should be noted here, however, that the present results do not exclude the possibility that oxygen gradients might direct cell migration because the oxygen concentration can drop to pathophysiological levels in hypoxic tumor tissue in vivo [5,23]. 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. [24] demonstrated using the Dunn chamber that both αVβ3 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. [25] 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 extracellular pH have been demonstrated at the level of the single cell. Martin et al. [26] observed intracellular pH (pHi) 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 pHi 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. [27] determined the pericellular pH on the surface of the plasma membrane (pHem) in polarized MV3 cells, reporting significant gradients of pHem in single cells where front (at focal adhesions)-to-rear pHem 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 pHi and pHem 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 [26,28,29]. Both pHi and pHem gradients may independently affect cell motility through effects on cytoskeletal machinery and cell-matrix interactions, respectively [30]. In fact, a substantial role of NHE1 activities in cell motility has been demonstrated in various cells [28,31,32,33,34].
 
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 [24,25]. Therefore, it is unclear whether the relationship between cell migration and the NHE1-driven pHi and pHem heterogeneities would be directly applicable to the present and other experiments in which the pH of the extracellular bulk medium was manipulated.
 
Although there is a possibility that subcellular heterogeneities in pHi and pHem 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 [25,33,34], without assuming significant intracellular pH gradients. Stock et al. [33] demonstrated that the pH of the extracellular bulk solution significantly affects migratory activities in MV3 cells in vitro. At low extracellular pH values, cell-matrix interactions via integrin α2β1 appear too strong, while 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 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 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 [9,35]. A type of metabolic reprograming known as the Warburg effect in cancer cells and reduced washout of CO2/H+ from the tissue are the known major causes of an acidotic microenvironment [36]. In the process of adaptation to acidosis, cancer cells may acquire a malignant phenotype [37,38]. A low 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 [39], directional cell migration would elevate the probability of intravasation and, ultimately, metastasis. Thus, targeting acidosis in the tumor microenvironment may have therapeutic rationales from the standpoint of control of hematogenous metastasis.
 
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.
 
 
  • Enokida, Y.; Tsuruno, Y.; Okubo, K.; Yamaoka, Y.; Takahashi, E. Directional migration of MDA-MB-231 cells under O2/pH gradients. Adv. Exp. Med. Biol. 2017, 977, 169–174. [Google Scholar] [PubMed]
  • Tsuruno, Y.; Okubo, K.; Fujiwara, T.; Yamaoka, Y.; Takahashi, E. An in vitro model for determining tumor cell migration under metabolic gradients. Adv. Exp. Med. Biol. 2018, 1072, 201–205. [Google Scholar] [PubMed]
  • Metzen, E.; Wolff, M.; Fandrey, J.; Jelkmann, W. Pericellular PO2 and O2 consumption in monolayer cell cultures. Respir Physiol 1995, 100, 101–106. [Google Scholar] [CrossRef]
  • Acosta, M.A.; Jiang, X.; Huang, P.-K.; Cutler, K.B.; Grant, C.S.; Walker, G.M.; Gamcsik, M.P. A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia. Biomicrofluidics 2014, 8, 054117. [Google Scholar] [CrossRef] [PubMed]
  • Chang, C.-W.; Cheng, Y.-J.; Tu, M.; Chen, Y.-H.; Peng, C.-C.; Liao, W.-H.; Tung, Y.-C. A polydimethylsiloxane-polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies. Lab Chip 2014, 14, 3762–3772. [Google Scholar] [CrossRef] [PubMed]
  • Shih, H.-C.; Lee, T.-A.; Wu, H.-M.; Ko, P.-L.; Liao, W.-H.; Tung, Y.-C. Microfluidic collective cell migration assay for study of endothelial cell proliferation and migration under combinations of oxygen gradients, tensions, and drug treatments. Sci. Rep. 2019, 9, 8234. [Google Scholar] [CrossRef]
  • Sleeboom, J.J.F.; Den Toonder, J.M.J.; Sahlgren, C.M. MDA-MB-231 breast cancer cells and their CSC population migrate towards low oxygen in a microfluidic gradient device. Int. J. Mol. Sci. 2018, 19, 3047. [Google Scholar] [CrossRef]
  • Mosadegh, B.; Lockett, M.R.; Minn, K.T.; Simon, K.A.; Gilbert, K.; Hillier, S.; Newsome, D.; Li, H.; Hall, A.B.; Boucher, D.M.; et al. A paper-based invasion assay: Assessing chemotaxis of cancer cells in gradients of oxygen. Biomaterials 2015, 52, 262–271. [Google Scholar] [CrossRef]
  • Lewis, D.M.; Park, K.M.; Tang, V.; Xu, Y.; Pak, K.; Eisinger-Mathason, T.S.K.; Simon, M.C.; Gerecht, S. Intratumoral oxygen gradients mediate sarcoma cell invasion. Proc. Natl. Acad. Sci. USA 2016, 113, 9292–9297. [Google Scholar] [CrossRef]
  • Gilkes, D.M.; Xiang, L.; Lee, S.J.; Chaturvedi, P.; Hubbi, M.E.; Wirtz, D.; Semenza, G.L. Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells. Proc. Natl. Acad. Sci. USA 2014, 111, E384–E393. [Google Scholar] [CrossRef] [PubMed]
  • Jiang, B.H.; Semenza, G.L.; Bauer, C.; Marti, H.H. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am. J. Physiol. 1996, 271, C1172–C1180. [Google Scholar] [CrossRef] [PubMed]
  • McKeown, S.R. Defining normoxia, physoxia and hypoxia in tumours-implications for treatment response. Br. J. Radiol. 2014, 87, 20130676. [Google Scholar] [CrossRef] [PubMed]
  • Paradise, R.K.; Whitfield, M.J.; Lauffenburger, D.A.; Van Vliet, K.J. Directional cell migration in an extracellular pH gradient: A model study with an engineered cell line and primary microvascular endothelial cells. Exp. Cell Res. 2013, 319, 487–497. [Google Scholar] [CrossRef]
  • Jagielska, A.; Wilhite, K.D.; Van Vliet, K.J. Extracellular acidic pH inhibits oligodendrocyte precursor viability, migration, and differentiation. PLoS ONE 2013, 8, e76048. [Google Scholar] [CrossRef]
  • Martin, C.; Pedersen, S.F.; Schwab, A.; Stock, C. Intracellular pH gradients in migrating cells. Am. J. Physiol. Cell Physiol. 2011, 300, C490–C495. [Google Scholar] [CrossRef]
  • Ludwig, F.T.; Schwab, A.; Stock, C. The Na+/H+-exchanger (NHE1) generates pH nanodomains at focal adhesions. J. Cell. Physiol. 2013, 228, 1351–1358. [Google Scholar] [CrossRef]
  • Denker, S.P.; Barber, D.L. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1. J. Cell Biol. 2002, 159, 1087–1096. [Google Scholar] [CrossRef]
  • Jensen, H.H.; Pedersen, G.A.; Morgen, J.J.; Parsons, M.; Pedersen, S.F.; Nejsum, L.N. The Na+/H+ exchanger NHE1 localizes as clusters to cryptic lamellipodia and accelerates collective epithelial cell migration. J. Physiol. 2019, 597, 849–867. [Google Scholar] [CrossRef]
  • Stock, C.; Pedersen, S.F. Roles of pH and the Na+/H+ exchanger NHE1 in cancer: From cell biology and animal models to an emerging translational perspective? Semin. Cancer Biol. 2017, 43, 5–16. [Google Scholar] [CrossRef]
  • Klein, M.; Seeger, P.; Schuricht, B.; Alper, S.L.; Schwab, A. Polarization of Na+/H+ and Cl−/HCO3− exchangers in migrating renal epithelial cells. J. Gen. Physiol. 2000, 115, 599–608. [Google Scholar] [CrossRef]
  • Schwab, A.; Fabian, A.; Hanley, P.J.; Stock, C. Role of ion channels and transporters in cell migration. Physiol. Rev. 2012, 92, 1865–1913. [Google Scholar] [CrossRef] [PubMed]
  • Stock, C.; Gassner, B.; Hauck, C.R.; Arnold, H.; Mally, S.; Eble, J.A.; Dieterich, P.; Schwab, A. Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange. J. Physiol. 2005, 567, 225–238. [Google Scholar] [CrossRef] [PubMed]
  • Stüwe, L.; Müller, M.; Fabian, A.; Waning, J.; Mally, S.; Noël, J.; Schwab, A.; Stock, C. pH dependence of melanoma cell migration: Protons extruded by NHE1 dominate protons of the bulk solution. J. Physiol. 2007, 585, 351–360. [Google Scholar] [CrossRef] [PubMed]
  • Corbet, C.; Feron, O. Tumour acidosis: From the passenger to the driver’s seat. Nat. Rev. Cancer 2017, 17, 577–593. [Google Scholar] [CrossRef] [PubMed]
  • Pillai, S.R.; Damaghi, M.; Marunaka, Y.; Spugnini, E.P.; Fais, S.; Gillies, R.J. Causes, consequences, and therapy of tumors acidosis. Cancer Metastasis Rev. 2019, 38, 205–222. [Google Scholar] [CrossRef]
  • Estrella, V.; Chen, T.; Lloyd, M.; Wojtkowiak, J.; Cornnell, H.H.; Ibrahim-Hashim, A.; Bailey, K.; Balagurunathan, Y.; Rothberg, J.M.; Sloane, B.F.; et al. Acidity generated by the tumor microenvironment drives local invasion. Cancer Res. 2013, 73, 1524–1535. [Google Scholar] [CrossRef]
  • White, K.A.; Grillo-Hill, B.K.; Barber, D.L. Cancer cell behaviors mediated by dysregulated pH dynamics at a glance. J. Cell. Sci. 2017, 130, 663–669. [Google Scholar] [CrossRef]
  • Fukumura, D.; Xu, L.; Chen, Y.; Gohongi, T.; Seed, B.; Jain, R.K. Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo. Cancer Res. 2001, 61, 6020–6024. [Google Scholar]

This entry is adapted from the peer-reviewed paper 10.3390/ijms21072565

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
This entry is offline, you can click here to edit this entry!
Video Production Service
Feedback