Hemoglobin Regulates the Migration of Glioma Cells Along Poly(e-caprolactone)-Aligned Nanofibers Alexander D. Roth, Jacob Elmer, David R. Harris, Joseph Huntley, and Andre F. Palmer William G. Lowrie Dept. of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210
Tyler Nelson Dept. of Biomedical Engineering, The Ohio State University, Columbus, OH 43210
Jed K. Johnson Nanofiber Solutions LLC, 1275 Kinnear Road, Columbus, OH 43212
Ruipeng Xue and John J. Lannutti Dept. of Materials Science and Engineering, The Ohio State University, Columbus, OH 43210
Mariano S. Viapiano Dept. of Neurological Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210 DOI 10.1002/btpr.1950 Published online July 24, 2014 in Wiley Online Library (wileyonlinelibrary.com)
Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2)-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(e-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2, Hb did not impact aggregate dispersion. Also at 1% O2, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on C 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214– nanofibers. V 1220, 2014 Keywords: hemoglobin, glioma cells
Introduction Glioblastoma multiforme (GBM) is a form of neurological cancer often associated with a rapid onset of symptoms and a poor prognosis following initial diagnosis.1 The ability of GBM cells to proliferate and invade neural tissue is regulated by multiple growth factors and extracellular matrix (ECM) proteins,2 which are themselves often regulated by oxygen (O2) availability.3 It has been suggested that tumor invasion is triggered by low O2 levels and associated necrosis of tumor tissue.4–6 Therefore, the availability of O2 is critical for the viability of individual cancer cells and overall tumor survival.3 Correspondence concerning this article should be addressed to A. F. Palmer at [email protected]. 1214
Tumor morphology is often characterized by a leaky vasculature containing regions of dense cell mass.7 The centers of these regions possess low O2 tensions, which often leads to cell necrosis in the hypoxic region.7 The cells surrounding the hypoxic regions tend to migrate away from the tumor mass, searching for more nutrients and better O2 availability.6 The use of aligned fibers has been shown to aid in directing cells to migrate along a specific path and presents a suitable substrate for GBM cell migration studies since it mimics the topography of the white matter in the brain.8–10 While natural and synthetic polymers have been used for cell migration studies, poly(e-caprolactone) (PCL) has been shown to be a good scaffold for neural cells and tissues, due to the ability of fibrous PCL to mimic the ECM structure of neural progenitor cells.10,11 Aligned PCL fibers (as opposed to randomly oriented fibers) facilitated the growth and regeneration of nerve C 2014 American Institute of Chemical Engineers V
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gaps.12 Therefore, PCL-aligned fibers present an ideal in vitro system allowing for the effective study of GBM cell migration.9 Previous in vitro experiments showed that hypoxia increased the rate of individual U87 glioma cell migration while aggregate dispersion was relatively constant for cell aggregates.13 However, that study was performed on tissue culture plates with randomly oriented surfaces rather than on aligned fibers. A key limitation to performing extended more realistic GBM cell migration studies is the low solubility of O2 in aqueous cell culture media. Hemoglobin (Hb)-based O2 carriers (HBOCs) have been used to facilitate O2 transport to cells in static tissue culture14–16 due to the ability of HBOCs to store and transport O2. Perfluorocarbons (PFCs) have also been used as O2 carriers due to their ability to dissolve large amounts of O2.17 However, the ability of PFCs to dissolve O2 is a linear function of the dissolved O2 concentration, unlike Hb which exhibits cooperative O2 binding.14 Therefore, HBOCs are better O2 transporters vs. PFCs for the purpose of binding and releasing O2. In this work, it is hypothesized that improved O2 transport via HBOCs to GBM aggregates in vitro will lead to increased cell migration on PCL-aligned fibers. For these experiments, we chose to study bovine Hb (bHb) and human Hb (hHb), for their respective low and high affinities,18 while Lumbricus terrestris (earthworm) Hb (LtHb) was selected for its resistance to oxidation.19–21 Additionally, three O2 levels were used: 19% O2 as an atmospheric control, 5% O2 as a physiological control simulating venous O2 levels in the brain22 and 1% O2 as a condition resembling hypoxia.
Materials and Methods Purification of Hb hHb and bHb were purified from their respective red blood cells according to Palmer et al.23 LtHb was purified according to Elmer et al.19 All purified Hbs were assayed for Hb concentration, methemoglobin (metHb, an oxidized form of Hb that cannot transport O2) level, O2 affinity (P50, partial pressure of O2 (pO2) where the Hb is half saturated with O2) and cooperativity coefficient (n) according to the Hill equation.24 The Hb and metHb concentration were quantified for bHb and hHb using the Winterbourne equation,25 while the concentration of LtHb and met LtHb were quantified using a heme-reduction assay. The saturation of Hb with O2 was measured using a HEMOX analyzer (TCS Scientific Corporation, New Hope, PA) at 37 C. Purified Hbs had metHb levels less than 1%. Hbs were sterile-filtered using a 0.22 lm filter before addition to the cell culture media. Viability and live cell counts Human U251 glioblastoma cells were obtained from American Type Culture Collection (Manassass, VA) and validated by genomic markers at IDEXX RADIL laboratories (St. Louis, MO). Cells were seeded in 6-well plates at 125,000 live cells/mL with 2 mL/well. Cells were grown for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose, 10% (v/v) fetal bovine serum, 50 U/mL penicillin and 50 lg/mL streptomycin, at 19% O2 and 5% CO2. After 24 h, the medium was replaced with 5 mL of fresh medium containing Hb at concentrations of 0, 0.5, 1, or 5 g/L
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and cultured for three days at 37 C in a humidified cell culture incubator at O2 tensions of 1%, 5%, or 19% O2. Cell viability and live cell counts were measured using a trypan blue exclusion assay (n 5). Migration on polystyrene plates C
Wound healing assay inserts were obtained from IbidiV (Martinsried, Germany) and placed in 24-well plates. Cells were seeded on each side of the inserts at 35,000 cells in a final volume of 350 lL, and allowed to attach for 18–24 h at 37 C in 19% O2. After adherence, Hb from different species was added to the wells and the inserts were removed. Cells were cultured for an additional day in different O2 atmospheres. Images of gap closure were taken 0, 8, and 24 hrs after insert removal using an IX-71 microscope under phase contrast (n 5). The total gap area was calculated using the image analysis software ImageJ. Synthesis of PCL-aligned fiber plates PCL nanofiber-coated culture plates (NanoAligned; Nanofiber Solutions, Columbus, OH) were prepared as described previously.10 Sheets of optically-clear polystyrene film (MultiPlastics, Lewis Center OH), were attached to the side of a rotating drum. Nanofibers were deposited by electrospinning, using a syringe positioned perpendicular to the polystyrene film. Alignment was controlled by drum rotational speed and scaffold thickness by the amount of time used to deposit the nanofibers. Films covered with multilayered nanofiber scaffolds were trimmed, attached to bottomless culture plates and sterilized with UV radiation (350 mJ/cm2). Preparation of cell aggregates Hard-agar plates were prepared by applying agarose dissolved in DMEM (1%, w/v) to the bottom of culture plates (100 lL/well in 24-well plates). Agar plates were rehydrated before seeding with 2 mL of culture medium for 1 h at 37 C and U251 cells were seeded at 50,000 cells/well. Cell tracker dyes (Cell-Tracker Green, 5 nM final concentration, and Calcein, 5 nM final concentration) were added to label the cells. Cells were shaken every 4–8 h following seeding and tumor spheroids were recovered after 24–72 h. Cell migration on aligned nanofibers The 24-well plates containing aligned nanofiber scaffolds (NanoAligned Part#2402; Nanofiber Solutions, Columbus, OH) were sterilized with 70% (v/v) ethanol and allowed to air dry. Culture medium was supplemented at 500 lL/well and warmed to 37 C for 30 min. The 3–12 aggregates per well measuring approximately 30–200 lm in diameter were manually seeded on the scaffolds and verified with an Olympus IX-41 microscope. Hb from different species was added to the wells and the dispersion of fluorescent cells was imaged over 72 h using an Olympus IX-71 microscope. Statistical analysis of samples Means and standard deviations were calculated for all samples. One-way or multiparametric analysis of variance was used to determine significant differences between treatments based on O2 level, Hb type, and Hb concentration. A P < 0.05 was used to identify significant differences between treatments.
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Results Characterization of Hb-O2 binding parameters The O2-Hb equilibrium binding curves for the three types of Hb are shown in Figure 1. The O2-Hb equilibrium curves of LtHb and bHb overlap and show lower O2 affinity compared to hHb. Table 1 lists the P50 and n values for the three types of Hb regressed from the Hill equation and the pO2’s for each Hb as it attains 5 and 95% saturation with O2, respectively.
Dispersion of U251 cell aggregates on aligned nanofibers
Cell viability and live cell counts Viability is defined as the percentage of counted cells that did not uptake the trypan blue dye and were thus assumed to be alive. Live cell counts are the total number of living cells obtained from each well after the individualized culture conditions. Cell viability was not significantly affected by O2 concentrations after three days of culture in the absence of Hb, likely reflecting the ability of glioma cells to maintain glycolytic metabolism even when deprived of atmospheric O2. In addition, at 19% O2 cell viability was essentially unaffected in the presence of all Hbs (Figure 2). However, at lower O2 concentrations, cell viability was significantly reduced by hHb due to the precipitation of heme out of the cell culture medium. Total cell growth was not affected in the presence of Hbs at 19% O2 (Figure 3). In contrast with cell viability, cell growth was significantly reduced at 1% compared to 19% O2, suggesting that O2 is required to increase tumor biomass. At 1 and 5% O2, hHb exerted a strongly negative effect. In contrast, bHb and LtHb did not affect cell growth at 5% O2 and improved growth at 1% O2 compared to control cells cultured without Hb. Cell migration Migration was followed for 24 h after removal of the inserts (Figure 4). The results (Figure 5) show no significant
Figure 1.
differences in cell migration under the majority of the O2 and Hb conditions tested. However, addition of 0.5 g/L bHb at 5% O2 improved cell migration compared to control cells cultured under the same O2 concentration. The opposite effect was observed with LtHb: a significant decrease in migration was observed at 5 g/L in 1% O2, compared to the controls. In contrast, hHb did not appear to impact cell migration.
O2-Hb equilibrium curves of various Hbs used in this study.
The maximum distance between cells originating from the same aggregate dispersed along the fibers was expressed as a ratio of the diameter of the original aggregate plotted vs. time (Figure 6). At most time points, aggregates dispersed more slowly on nanofibers at 1% O2 than at 5% or 19% O2. In contrast, on tissue culture polystyrene migration was completely unaffected by O2 level in the absence of Hb. Migration on nanofibers at 5% O2 vs. 19% O2 was not significantly different. Addition of 0.5 g/L bHb at 5% O2 resulted in a significant increase in cell migration to levels above those of the controls, replicating the effect observed in gap-migration assays. Addition of 5 g/L LtHb at 1% O2 also increased cell dispersion compared against control cells at the same O2 level, in stark contrast with the negative effect of this Hb observed in individual cell migration. However, no improvement was seen when these Hbs, or hHb, were added at 19% O2 (data not shown). Overall, addition of Hbs tended to improve cell migration at low O2 levels compared to the absence of Hb.
Discussion The aggressive behavior of malignant gliomas that leads to tissue necrosis and disseminated invasion has been linked to localized hypoxia26 and underscores the dependence of tumor cells on the available O2 needed to grow and migrate. In addition, migration on 2D vs. 3D substrates—in particular nanofiber-based scaffolds that reproduce the aligned topography of brain structures—has revealed differential expression and activity of genes that constitute key regulators of glioma progression.27 This suggests that major properties of glioma cells, such as its O2 dependence for cell migration are influenced by the topography of the substrate that regulates the overall migratory mechanism. Given their high proliferative rate and constitutive migratory properties, glioblastoma cells represent an excellent model to analyze cellular responses to HBOCs under conditions of O2 deprivation. Unlike PFCs, which bind O2 as a linear function of the pO2,17 Hb binds and releases O2 in a cooperative manner depending on the pO2. This means that cells at low O2 levels supplemented with Hb below 5% O2 saturation are limited in growth by O2 availability. However, cells at high pO2s can function well without the presence of an O2-carrier, while cells existing at low O2 levels with Hb at moderate O2 saturation can function similarly to cells grown at high pO2s
Table 1. Hill Parameters for Various Hbs Hb
P50 (mm Hg)
n
pO2, 5% Saturation (mm Hg)
pO2, 95% Saturation (mm Hg)
Bovine Human Earthworm
23.23 6 0.03 11.78 6 0.01 23.76 6 0.04
2.49 6 0.01 2.62 6 0.01 2.60 6 0.01
7.12 3.83 7.65
75.79 36.24 73.74
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Figure 2.
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Cell viability of U251 cells after three days of culture exposed to different O2 and Hb conditions. Cells were cultured at 19% O2 (A), 5% O2 (B), or 1% O2 (C). Asterisks represent statistically significant differences against control cells cultured without Hb.
Figure 3. Total growth of U251 cells after three days of culture exposed to the different concentrations of O2 and Hb shown. Cells were cultured at 19% O2 (A), 5% O2 (B), or 1% O2 (C). Asterisks represent statistically significant differences against control cells cultured without Hb. The color of the asterisk corresponds to the type of Hb.
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Figure 4.
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Wound healing assay for cells cultured in 5 g/L hHb and 1% O2. Images show gap closure at 0 hr (A), 8hr (B), and 24 hr (C) after insert removal. Some Hb precipitate is visible in C.
Figure 5. Percentage of gap area remaining after 8 h (A, C, and E) and 24 h of incubation(B, D, and F). Samples were cultured at 19% O2 (A and B), 5% O2(C and D), or 1% O2 (E and F). Asterisks indicate significant differences between each condition and control cells with no Hb at the corresponding O2 level. The color of the asterisk corresponds to the type of Hb.
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Figure 6.
Dispersion of U251 cells from aggregates seeded on PCL-aligned nanofibers. Cell dispersion was measured at different O2 levels, in presence or absence of bHb and LtHb. Asterisks indicate significant differences between each treatment and control cells with no Hb at the corresponding O2 level.
because of improved O2 transport from Hb that can supply the cells with enough O2. Previous studies with glioma cells have suggested that cell migration and invasion are increased under hypoxia,26,29 although these effects were moderate and only observed following short-term exposures to hypoxia. Under the same O2 levels, we were unable to detect significant increases in cell migration on tissue culture polystyrene while experimental results suggested an effective decrease in cell migration out of spheroids migrating onto nanofiber scaffolds.12 While prolonged O2 depletion in our models did not affect cell viability, which is predominantly dependent on aerobic glycolysis, it is likely that decreased O2 availability affected cell metabolism and O2-dependent genes involved in the regulation of cell motility. This effect was more noticeable on nanofiber scaffolds, where 1% O2 significantly decreased cell migration from aggregates in a manner that was rescued when O2 availability was increased by adding bHb or LtHb. Given the expected saturation of bHb at 5% O2, the improved migration on nanofibers after addition of 0.5 g/L bHb likely indicates an improvement in the availability of dissolved O2, since aggregate dispersion was only improved in the steep region of the Hb-O2 equilibrium curve. The results indicate that precipitation of hHb out of the cell culture media can reduce long term in vivo cell growth and viability. Additionally, while single cell migration was not impacted, aggregate dispersion was impacted by the presence of Hb. Most of the Hbs are almost fully saturated withO2 at 19% O2 based on the Hill equation parameters (Table 1). Furthermore, the O2 solubility in aqueous media is fairly high at 19% O2. Thus, the ability of Hb to carry O2 is impeded by the fact that the cells are already receiving sufficient O2, and the fact that O2 is tightly bound to Hb and is not being released from it. At 5% O2, hHb is mostly fully saturated with O2, while bHb and LtHb are approximately 75–80% saturated with O2. At 1% O2, bHb and LtHb are not effectively binding O2 (under 5% saturation), while hHb exists in the steep region of the Hb-O2 equilibrium curve. 5% O2 was the closest to the physiologically equivalent O2 level that glioma cells experienced in this study,22 while
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at 1% O2, the amount of dissolved O2 in cell culture media is less than what glioma cells are exposed to in vivo. While viability levels and individual cell migration are similar between all O2 levels, dispersion on aligned fibers and cell growth is decreased at 1% O2, which is more apparent after 24 h in cultured media. Without Hb, 5% O2 and 19% O2 did not vary significantly when comparing viability, cell growth, aligned fiber dispersion, and individual cell migration. Significant differences occurred for aligned fiber migration at 5% O2 in the presence of bHb (at all concentrations), indicating that the O2 binding properties and the type of Hb in question does favorably impact U251 migration on aligned fibers. While improvements in migration from LtHb supplementation were not significant at 5% O2, the average displacement and Feret diameter measurements were greater for samples in the presence of LtHb than without Hb. The reduced cell growth also correlates with a decrease in aggregate dispersion, suggesting that the mechanisms for tumor growth and migration are linked to each other and O2 availability. The presence of hHb at 1% O2 corresponds with hHb being in the steep region of the Hb-O2 equilibrium curve, indicating its ability to transport O2. While hHb should function as an effective HBOC, the formation of precipitate in cell culture media supplemented with hHb dramatically decreased cell growth and viability. The reduced viability of cells cultured in the presence of hHb under1% O2 and 5% O2 suggests the production of reactive O2 species, which can kill cells. However, aggregate dispersion on aligned fibers and the wound healing assay results were not affected by the presence of hHb. It is possible that despite significantly reduced viability and decreased cell growth, another mechanism is responsible for cell migration in the presence of hHb. The addition of LtHb at 1% O2 improved cell viability, growth, and aggregate dispersion but decreased individual cell migration. However, the total number of live cells and aggregate dispersion was still less than that observed at 5 or 19% O2 without Hb. The improvements observed in aligned fiber migration and cell growth at 1% O2 in the presence of LtHb could be attributed to a function of LtHb not related to O2 transport, but still dependent on O2. In sharp contrast, the results from wound healing assays suggest that O2 level has little or no effect on individual cell migration. Additionally, these results show that individual cell migration on polystyrene plates does not correlate with cell viability. In this work, cell viability and growth were improved by supplementation of bHb and LtHb in the cell culture medium at 1% O2. hHb reduced cell viability and growth of individual cells, and was ineffective as an HBOC in vitro. Individual cell migration was not dependent on Hb concentration, Hb type, presence of Hb, or O2 levels for 1–19% O2. However, dispersion of individual cells away from cell aggregates on nanofibers was dependent on the O2 level with regards to the presence or absence of Hb and its location on the Hb-O2 equilibrium curve. On an aligned fiber platform, aggregate dispersion increased at higher O2 availability, which is in direct contrast with previous migration studies performed on tissue culture plates.
Acknowledgments This work was partly supported by research grants from the National Science Foundation under Grant Nos. CBET1033991, EEC-0425626, and CMMI-0928315. We would
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like to thank Nanofiber Solutions LLC for the donation of “NanoAligned” fiber plates and Dr. Jessica Winter in the William G. Lowrie Department of Chemical and Biomolecular Engineering at the OSU for use of her Confocal Microscope.
15.
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1. Michotte A, Neyns B, Chaskis C, Sadones J, In‘t Veld P. Neuropathological and molecular aspects of low-grade and high-grade gliomas. Acta Neurol Belg. 2004;104:1482153. 2. Bellail AC, Hunter SB, Brat DJ, Tan C, Van Meir EG. Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion. Int J Biochem Cell Biol. 2004;36: 104621069. 3. Vaupel P, Harrison L. Tumor hypoxia: causative factors, compensatory mechanisms, and cellular response. Oncologist. 2004; 9:429. 4. Pennacchietti S, Michielli P, Galluzo M, Mazzone M, Giordano S, Comoglio PM. Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. Cancer Cell. 2003;3:3472361. 5. Rakic P. Principles of neural cell migration. Experientia. 1990; 46:8822891. 6. Brat DJ, Castellano-Sanchez AA. Hunter SB, Pecot M, Cohen C, Hammond EH, Devi SN, Kaur B, Van Meir EG. Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population. Cancer Res. 2004;64: 9202927. 7. Baban DF, Seymour LW. Control of tumor vascular permeability. Adv Drug Deliv Rev. 1998;34:1092119. 8. Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, NasrEsfahani M, Ramakrishna S. Electrospun poly(epsilon-caprolactone) gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials. 2008;29:453224539. 9. Johnson J, Nowicki MO, Lee CH, Chiocca EA, Viapiano MS, Lawler SE, Lannutti JJ. Quantitative analysis of complex glioma cell migration on electrospun polycaprolactone using time-lapse microscopy. Tissue Eng Part C: Methods. 2009;15:5312540. 10. Vasita R, Katti DS. Nanofibers and their applications in tissue engineering. Int J Nanomed. 2006;1:15230. 11. Xie, J, Li X, Xia Y. Putting electrospun nanofibers to work for biomedical research. Macromol Rapid Commun. 2008;29:177521792. 12. Chew SY, Mi R, Hoke A, Leong KW. The effect of the alignment of electrospun fibrous scaffolds on Schwann cell maturation. Biomaterials. 2008;29:6532661. 13. Khain E, Katakowski M, Hopkins S, Szalad A, Zheng X, Jiang F, Chopp M. Collective behavior of brain tumor cells: the role of hypoxia. Phys Rev E Stat Nonlin Soft Matter Phys. 2011;E 83:031920. 14. Avila JG, Wang Y, Barbaro B, Gnagemi A, Qi M, Kuechle J, Doubleday N, Doubleday M, Churchill T, Salehi P, Shapiro J,
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Philipson LH, Benedetti E, Lakey JR, Oberholzer J. Improved outcomes in islet isolation and transplantation by the use of a novel hemoglobin-based O2 carrier. Am J Transplant. 2006;6: 286122870. Chen G, Palmer AF. Hemoglobin-based oxygen carrier and convection enhanced oxygen transport in a hollow fiber bioreactor. Biotechnol Bioeng. 2009;102:160321612. Mozzarelli A, Ronda L, Faggiano S, Bettati S, Bruno S. Haemoglobin-based oxygen carriers: research and reality towards an alternative to blood transfusions. Blood Transfus. 2010;8: s592s68. Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport. Br J Pharmacol. 2008;154:5292541. Fronticelli C, Bucci E, Orth C. Solvent regulation of oxygen affinity in hemoglobin. Sensitivity of bovine hemoglobin to chloride ions. J Biol Chem. 1984;259:10841210844. Elmer J, Zorc K, Rameez S, Zhou Y, Cabrales P, Palmer AF. Hypervolemic infusion of Lumbricus terrestris erythrocruorin purified by tangential-flow filtration. Transfusion. 2012;52: 172921740. Terwilliger RC. Structures of invertebrate hemoglobins. Amer Zool. 1980;20:53267. Elmer J, Palmer AF. Biophysical properties of Lumbricus terrestris erythrocruorin and Its potential use as a red blood cell substitute. J Funct Biomater. 2012;3:49260. Erecinska M, Silver, IA. Tissue oxygen tension and brain sensitivity to hypoxia. Respir Physiol. 2001;128:2632276. Palmer AF, Sun G, Harris DR. Tangential flow filtration of hemoglobin. Biotechnol Prog. 2009;25:1892199. Altschul AM, Hogness TR. The hemoglobin-oxygen equilibrium. J Biol Chem. 1939;129:3152331. Winterbourne CC, CRC Handbook of Methods of Oxygen Radical Research (ed. Greenwald, RA). 1985: p. 137–141. Fujiwara, S, Nakagawa K, Harada H, Nagato S, Furukawa K, Teraoka M, Seno T, Oka K, Iwata S, Ohnishi T. Silencing hypoxia-inducible factor-1 alpha inhibits cell migration and invasion under hypoxic environment in malignant gliomas. Int J Oncol. 2007;30:7932802. Agudelo-Garcia PA, De Jesus JK, Willaims SP, Nowicki MO, Chiocca EA, Liyanarachchi S, Li PK, Lanutti JJ, Johnson JK, Lawler SE, Viapiano MS. Glioma cell migration on threedimensional nanofiber scaffolds is regulated by substrate topography and abolished by inhibition of STAT3 signaling. Neoplasia. 2011;13:8312840. Kang SH, Yu MO, Park KJ, Chi SG, Park DH, Chung YG. Activated STAT3 regulates hypoxia-induced angiogenesis and cell migration in human glioblastoma. Neurosurgery. 2010;67: 138621395.
Manuscript received Aug. 13, 2013, and revision received Jun. 17, 2014.
Tyler Nelson Dept. of Biomedical Engineering, The Ohio State University, Columbus, OH 43210
Jed K. Johnson Nanofiber Solutions LLC, 1275 Kinnear Road, Columbus, OH 43212
Ruipeng Xue and John J. Lannutti Dept. of Materials Science and Engineering, The Ohio State University, Columbus, OH 43210
Mariano S. Viapiano Dept. of Neurological Surgery, The Ohio State University Wexner Medical Center, Columbus, OH 43210 DOI 10.1002/btpr.1950 Published online July 24, 2014 in Wiley Online Library (wileyonlinelibrary.com)
Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2)-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(e-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2. Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2, while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2, Hb did not impact aggregate dispersion. Also at 1% O2, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on C 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1214– nanofibers. V 1220, 2014 Keywords: hemoglobin, glioma cells
Introduction Glioblastoma multiforme (GBM) is a form of neurological cancer often associated with a rapid onset of symptoms and a poor prognosis following initial diagnosis.1 The ability of GBM cells to proliferate and invade neural tissue is regulated by multiple growth factors and extracellular matrix (ECM) proteins,2 which are themselves often regulated by oxygen (O2) availability.3 It has been suggested that tumor invasion is triggered by low O2 levels and associated necrosis of tumor tissue.4–6 Therefore, the availability of O2 is critical for the viability of individual cancer cells and overall tumor survival.3 Correspondence concerning this article should be addressed to A. F. Palmer at [email protected]. 1214
Tumor morphology is often characterized by a leaky vasculature containing regions of dense cell mass.7 The centers of these regions possess low O2 tensions, which often leads to cell necrosis in the hypoxic region.7 The cells surrounding the hypoxic regions tend to migrate away from the tumor mass, searching for more nutrients and better O2 availability.6 The use of aligned fibers has been shown to aid in directing cells to migrate along a specific path and presents a suitable substrate for GBM cell migration studies since it mimics the topography of the white matter in the brain.8–10 While natural and synthetic polymers have been used for cell migration studies, poly(e-caprolactone) (PCL) has been shown to be a good scaffold for neural cells and tissues, due to the ability of fibrous PCL to mimic the ECM structure of neural progenitor cells.10,11 Aligned PCL fibers (as opposed to randomly oriented fibers) facilitated the growth and regeneration of nerve C 2014 American Institute of Chemical Engineers V
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gaps.12 Therefore, PCL-aligned fibers present an ideal in vitro system allowing for the effective study of GBM cell migration.9 Previous in vitro experiments showed that hypoxia increased the rate of individual U87 glioma cell migration while aggregate dispersion was relatively constant for cell aggregates.13 However, that study was performed on tissue culture plates with randomly oriented surfaces rather than on aligned fibers. A key limitation to performing extended more realistic GBM cell migration studies is the low solubility of O2 in aqueous cell culture media. Hemoglobin (Hb)-based O2 carriers (HBOCs) have been used to facilitate O2 transport to cells in static tissue culture14–16 due to the ability of HBOCs to store and transport O2. Perfluorocarbons (PFCs) have also been used as O2 carriers due to their ability to dissolve large amounts of O2.17 However, the ability of PFCs to dissolve O2 is a linear function of the dissolved O2 concentration, unlike Hb which exhibits cooperative O2 binding.14 Therefore, HBOCs are better O2 transporters vs. PFCs for the purpose of binding and releasing O2. In this work, it is hypothesized that improved O2 transport via HBOCs to GBM aggregates in vitro will lead to increased cell migration on PCL-aligned fibers. For these experiments, we chose to study bovine Hb (bHb) and human Hb (hHb), for their respective low and high affinities,18 while Lumbricus terrestris (earthworm) Hb (LtHb) was selected for its resistance to oxidation.19–21 Additionally, three O2 levels were used: 19% O2 as an atmospheric control, 5% O2 as a physiological control simulating venous O2 levels in the brain22 and 1% O2 as a condition resembling hypoxia.
Materials and Methods Purification of Hb hHb and bHb were purified from their respective red blood cells according to Palmer et al.23 LtHb was purified according to Elmer et al.19 All purified Hbs were assayed for Hb concentration, methemoglobin (metHb, an oxidized form of Hb that cannot transport O2) level, O2 affinity (P50, partial pressure of O2 (pO2) where the Hb is half saturated with O2) and cooperativity coefficient (n) according to the Hill equation.24 The Hb and metHb concentration were quantified for bHb and hHb using the Winterbourne equation,25 while the concentration of LtHb and met LtHb were quantified using a heme-reduction assay. The saturation of Hb with O2 was measured using a HEMOX analyzer (TCS Scientific Corporation, New Hope, PA) at 37 C. Purified Hbs had metHb levels less than 1%. Hbs were sterile-filtered using a 0.22 lm filter before addition to the cell culture media. Viability and live cell counts Human U251 glioblastoma cells were obtained from American Type Culture Collection (Manassass, VA) and validated by genomic markers at IDEXX RADIL laboratories (St. Louis, MO). Cells were seeded in 6-well plates at 125,000 live cells/mL with 2 mL/well. Cells were grown for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose, 10% (v/v) fetal bovine serum, 50 U/mL penicillin and 50 lg/mL streptomycin, at 19% O2 and 5% CO2. After 24 h, the medium was replaced with 5 mL of fresh medium containing Hb at concentrations of 0, 0.5, 1, or 5 g/L
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and cultured for three days at 37 C in a humidified cell culture incubator at O2 tensions of 1%, 5%, or 19% O2. Cell viability and live cell counts were measured using a trypan blue exclusion assay (n 5). Migration on polystyrene plates C
Wound healing assay inserts were obtained from IbidiV (Martinsried, Germany) and placed in 24-well plates. Cells were seeded on each side of the inserts at 35,000 cells in a final volume of 350 lL, and allowed to attach for 18–24 h at 37 C in 19% O2. After adherence, Hb from different species was added to the wells and the inserts were removed. Cells were cultured for an additional day in different O2 atmospheres. Images of gap closure were taken 0, 8, and 24 hrs after insert removal using an IX-71 microscope under phase contrast (n 5). The total gap area was calculated using the image analysis software ImageJ. Synthesis of PCL-aligned fiber plates PCL nanofiber-coated culture plates (NanoAligned; Nanofiber Solutions, Columbus, OH) were prepared as described previously.10 Sheets of optically-clear polystyrene film (MultiPlastics, Lewis Center OH), were attached to the side of a rotating drum. Nanofibers were deposited by electrospinning, using a syringe positioned perpendicular to the polystyrene film. Alignment was controlled by drum rotational speed and scaffold thickness by the amount of time used to deposit the nanofibers. Films covered with multilayered nanofiber scaffolds were trimmed, attached to bottomless culture plates and sterilized with UV radiation (350 mJ/cm2). Preparation of cell aggregates Hard-agar plates were prepared by applying agarose dissolved in DMEM (1%, w/v) to the bottom of culture plates (100 lL/well in 24-well plates). Agar plates were rehydrated before seeding with 2 mL of culture medium for 1 h at 37 C and U251 cells were seeded at 50,000 cells/well. Cell tracker dyes (Cell-Tracker Green, 5 nM final concentration, and Calcein, 5 nM final concentration) were added to label the cells. Cells were shaken every 4–8 h following seeding and tumor spheroids were recovered after 24–72 h. Cell migration on aligned nanofibers The 24-well plates containing aligned nanofiber scaffolds (NanoAligned Part#2402; Nanofiber Solutions, Columbus, OH) were sterilized with 70% (v/v) ethanol and allowed to air dry. Culture medium was supplemented at 500 lL/well and warmed to 37 C for 30 min. The 3–12 aggregates per well measuring approximately 30–200 lm in diameter were manually seeded on the scaffolds and verified with an Olympus IX-41 microscope. Hb from different species was added to the wells and the dispersion of fluorescent cells was imaged over 72 h using an Olympus IX-71 microscope. Statistical analysis of samples Means and standard deviations were calculated for all samples. One-way or multiparametric analysis of variance was used to determine significant differences between treatments based on O2 level, Hb type, and Hb concentration. A P < 0.05 was used to identify significant differences between treatments.
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Results Characterization of Hb-O2 binding parameters The O2-Hb equilibrium binding curves for the three types of Hb are shown in Figure 1. The O2-Hb equilibrium curves of LtHb and bHb overlap and show lower O2 affinity compared to hHb. Table 1 lists the P50 and n values for the three types of Hb regressed from the Hill equation and the pO2’s for each Hb as it attains 5 and 95% saturation with O2, respectively.
Dispersion of U251 cell aggregates on aligned nanofibers
Cell viability and live cell counts Viability is defined as the percentage of counted cells that did not uptake the trypan blue dye and were thus assumed to be alive. Live cell counts are the total number of living cells obtained from each well after the individualized culture conditions. Cell viability was not significantly affected by O2 concentrations after three days of culture in the absence of Hb, likely reflecting the ability of glioma cells to maintain glycolytic metabolism even when deprived of atmospheric O2. In addition, at 19% O2 cell viability was essentially unaffected in the presence of all Hbs (Figure 2). However, at lower O2 concentrations, cell viability was significantly reduced by hHb due to the precipitation of heme out of the cell culture medium. Total cell growth was not affected in the presence of Hbs at 19% O2 (Figure 3). In contrast with cell viability, cell growth was significantly reduced at 1% compared to 19% O2, suggesting that O2 is required to increase tumor biomass. At 1 and 5% O2, hHb exerted a strongly negative effect. In contrast, bHb and LtHb did not affect cell growth at 5% O2 and improved growth at 1% O2 compared to control cells cultured without Hb. Cell migration Migration was followed for 24 h after removal of the inserts (Figure 4). The results (Figure 5) show no significant
Figure 1.
differences in cell migration under the majority of the O2 and Hb conditions tested. However, addition of 0.5 g/L bHb at 5% O2 improved cell migration compared to control cells cultured under the same O2 concentration. The opposite effect was observed with LtHb: a significant decrease in migration was observed at 5 g/L in 1% O2, compared to the controls. In contrast, hHb did not appear to impact cell migration.
O2-Hb equilibrium curves of various Hbs used in this study.
The maximum distance between cells originating from the same aggregate dispersed along the fibers was expressed as a ratio of the diameter of the original aggregate plotted vs. time (Figure 6). At most time points, aggregates dispersed more slowly on nanofibers at 1% O2 than at 5% or 19% O2. In contrast, on tissue culture polystyrene migration was completely unaffected by O2 level in the absence of Hb. Migration on nanofibers at 5% O2 vs. 19% O2 was not significantly different. Addition of 0.5 g/L bHb at 5% O2 resulted in a significant increase in cell migration to levels above those of the controls, replicating the effect observed in gap-migration assays. Addition of 5 g/L LtHb at 1% O2 also increased cell dispersion compared against control cells at the same O2 level, in stark contrast with the negative effect of this Hb observed in individual cell migration. However, no improvement was seen when these Hbs, or hHb, were added at 19% O2 (data not shown). Overall, addition of Hbs tended to improve cell migration at low O2 levels compared to the absence of Hb.
Discussion The aggressive behavior of malignant gliomas that leads to tissue necrosis and disseminated invasion has been linked to localized hypoxia26 and underscores the dependence of tumor cells on the available O2 needed to grow and migrate. In addition, migration on 2D vs. 3D substrates—in particular nanofiber-based scaffolds that reproduce the aligned topography of brain structures—has revealed differential expression and activity of genes that constitute key regulators of glioma progression.27 This suggests that major properties of glioma cells, such as its O2 dependence for cell migration are influenced by the topography of the substrate that regulates the overall migratory mechanism. Given their high proliferative rate and constitutive migratory properties, glioblastoma cells represent an excellent model to analyze cellular responses to HBOCs under conditions of O2 deprivation. Unlike PFCs, which bind O2 as a linear function of the pO2,17 Hb binds and releases O2 in a cooperative manner depending on the pO2. This means that cells at low O2 levels supplemented with Hb below 5% O2 saturation are limited in growth by O2 availability. However, cells at high pO2s can function well without the presence of an O2-carrier, while cells existing at low O2 levels with Hb at moderate O2 saturation can function similarly to cells grown at high pO2s
Table 1. Hill Parameters for Various Hbs Hb
P50 (mm Hg)
n
pO2, 5% Saturation (mm Hg)
pO2, 95% Saturation (mm Hg)
Bovine Human Earthworm
23.23 6 0.03 11.78 6 0.01 23.76 6 0.04
2.49 6 0.01 2.62 6 0.01 2.60 6 0.01
7.12 3.83 7.65
75.79 36.24 73.74
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Figure 2.
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Cell viability of U251 cells after three days of culture exposed to different O2 and Hb conditions. Cells were cultured at 19% O2 (A), 5% O2 (B), or 1% O2 (C). Asterisks represent statistically significant differences against control cells cultured without Hb.
Figure 3. Total growth of U251 cells after three days of culture exposed to the different concentrations of O2 and Hb shown. Cells were cultured at 19% O2 (A), 5% O2 (B), or 1% O2 (C). Asterisks represent statistically significant differences against control cells cultured without Hb. The color of the asterisk corresponds to the type of Hb.
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Figure 4.
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Wound healing assay for cells cultured in 5 g/L hHb and 1% O2. Images show gap closure at 0 hr (A), 8hr (B), and 24 hr (C) after insert removal. Some Hb precipitate is visible in C.
Figure 5. Percentage of gap area remaining after 8 h (A, C, and E) and 24 h of incubation(B, D, and F). Samples were cultured at 19% O2 (A and B), 5% O2(C and D), or 1% O2 (E and F). Asterisks indicate significant differences between each condition and control cells with no Hb at the corresponding O2 level. The color of the asterisk corresponds to the type of Hb.
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Figure 6.
Dispersion of U251 cells from aggregates seeded on PCL-aligned nanofibers. Cell dispersion was measured at different O2 levels, in presence or absence of bHb and LtHb. Asterisks indicate significant differences between each treatment and control cells with no Hb at the corresponding O2 level.
because of improved O2 transport from Hb that can supply the cells with enough O2. Previous studies with glioma cells have suggested that cell migration and invasion are increased under hypoxia,26,29 although these effects were moderate and only observed following short-term exposures to hypoxia. Under the same O2 levels, we were unable to detect significant increases in cell migration on tissue culture polystyrene while experimental results suggested an effective decrease in cell migration out of spheroids migrating onto nanofiber scaffolds.12 While prolonged O2 depletion in our models did not affect cell viability, which is predominantly dependent on aerobic glycolysis, it is likely that decreased O2 availability affected cell metabolism and O2-dependent genes involved in the regulation of cell motility. This effect was more noticeable on nanofiber scaffolds, where 1% O2 significantly decreased cell migration from aggregates in a manner that was rescued when O2 availability was increased by adding bHb or LtHb. Given the expected saturation of bHb at 5% O2, the improved migration on nanofibers after addition of 0.5 g/L bHb likely indicates an improvement in the availability of dissolved O2, since aggregate dispersion was only improved in the steep region of the Hb-O2 equilibrium curve. The results indicate that precipitation of hHb out of the cell culture media can reduce long term in vivo cell growth and viability. Additionally, while single cell migration was not impacted, aggregate dispersion was impacted by the presence of Hb. Most of the Hbs are almost fully saturated withO2 at 19% O2 based on the Hill equation parameters (Table 1). Furthermore, the O2 solubility in aqueous media is fairly high at 19% O2. Thus, the ability of Hb to carry O2 is impeded by the fact that the cells are already receiving sufficient O2, and the fact that O2 is tightly bound to Hb and is not being released from it. At 5% O2, hHb is mostly fully saturated with O2, while bHb and LtHb are approximately 75–80% saturated with O2. At 1% O2, bHb and LtHb are not effectively binding O2 (under 5% saturation), while hHb exists in the steep region of the Hb-O2 equilibrium curve. 5% O2 was the closest to the physiologically equivalent O2 level that glioma cells experienced in this study,22 while
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at 1% O2, the amount of dissolved O2 in cell culture media is less than what glioma cells are exposed to in vivo. While viability levels and individual cell migration are similar between all O2 levels, dispersion on aligned fibers and cell growth is decreased at 1% O2, which is more apparent after 24 h in cultured media. Without Hb, 5% O2 and 19% O2 did not vary significantly when comparing viability, cell growth, aligned fiber dispersion, and individual cell migration. Significant differences occurred for aligned fiber migration at 5% O2 in the presence of bHb (at all concentrations), indicating that the O2 binding properties and the type of Hb in question does favorably impact U251 migration on aligned fibers. While improvements in migration from LtHb supplementation were not significant at 5% O2, the average displacement and Feret diameter measurements were greater for samples in the presence of LtHb than without Hb. The reduced cell growth also correlates with a decrease in aggregate dispersion, suggesting that the mechanisms for tumor growth and migration are linked to each other and O2 availability. The presence of hHb at 1% O2 corresponds with hHb being in the steep region of the Hb-O2 equilibrium curve, indicating its ability to transport O2. While hHb should function as an effective HBOC, the formation of precipitate in cell culture media supplemented with hHb dramatically decreased cell growth and viability. The reduced viability of cells cultured in the presence of hHb under1% O2 and 5% O2 suggests the production of reactive O2 species, which can kill cells. However, aggregate dispersion on aligned fibers and the wound healing assay results were not affected by the presence of hHb. It is possible that despite significantly reduced viability and decreased cell growth, another mechanism is responsible for cell migration in the presence of hHb. The addition of LtHb at 1% O2 improved cell viability, growth, and aggregate dispersion but decreased individual cell migration. However, the total number of live cells and aggregate dispersion was still less than that observed at 5 or 19% O2 without Hb. The improvements observed in aligned fiber migration and cell growth at 1% O2 in the presence of LtHb could be attributed to a function of LtHb not related to O2 transport, but still dependent on O2. In sharp contrast, the results from wound healing assays suggest that O2 level has little or no effect on individual cell migration. Additionally, these results show that individual cell migration on polystyrene plates does not correlate with cell viability. In this work, cell viability and growth were improved by supplementation of bHb and LtHb in the cell culture medium at 1% O2. hHb reduced cell viability and growth of individual cells, and was ineffective as an HBOC in vitro. Individual cell migration was not dependent on Hb concentration, Hb type, presence of Hb, or O2 levels for 1–19% O2. However, dispersion of individual cells away from cell aggregates on nanofibers was dependent on the O2 level with regards to the presence or absence of Hb and its location on the Hb-O2 equilibrium curve. On an aligned fiber platform, aggregate dispersion increased at higher O2 availability, which is in direct contrast with previous migration studies performed on tissue culture plates.
Acknowledgments This work was partly supported by research grants from the National Science Foundation under Grant Nos. CBET1033991, EEC-0425626, and CMMI-0928315. We would
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like to thank Nanofiber Solutions LLC for the donation of “NanoAligned” fiber plates and Dr. Jessica Winter in the William G. Lowrie Department of Chemical and Biomolecular Engineering at the OSU for use of her Confocal Microscope.
15.
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Manuscript received Aug. 13, 2013, and revision received Jun. 17, 2014.
