Hyaluronic Acid-Coated Aligned Nanofibers for the Promotion of

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Hyaluronic acid coated aligned nanofibers for promotion of glioblastoma migration Ariane Erickson, Jialu Sun, Sheeny Levengood, and Miqin Zhang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00704 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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ACS Applied Bio Materials

Hyaluronic acid coated aligned nanofibers for promotion of glioblastoma migration

Ariane Erickson, Jialu Sun, Sheeny K. Lan Levengood, Miqin Zhang*

Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA *Corresponding author: Zhang, M. ([email protected])

Keywords: glioblastoma, migration, electrospun nanofibers, chitosan, hyaluronic acid

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Abstract The propensity of glioblastoma multiforme (GBM) cells to migrate along white matter tracts and blood vessels suggest that topographical cues associated with brain parenchyma greatly influence GBM motility and invasion.

In vitro cell culture platforms that mimic the physical and

biochemical characteristics of brain tissue are needed to develop biologically relevant GBM migration models for development of anti-cancer therapies. Here, we fabricated highly-aligned chitosan-polycaprolactone (C-PCL) polyblend nanofibers coated with hyaluronic acid (HA), a glycosaminoglycan commonly found in the brain, to simulate the structure and biochemistry of native brain tissue. The influence of topography on GBM cell behavior was apparent on both HAcoated and uncoated nanofibers where cells aligned axially along nanofibers and displayed an elongated morphology associated with migration. Time lapse imaging revealed that migrating cells on nanofibers were less likely to divide, suggesting a shift to a mesenchymal-like phenotype. Cells cultured on nanofibers coated with 0.5% HA achieved the highest migratory speed relative to uncoated nanofibers and 2D adherent cultures on polystyrene plates. Further, cells on nanofibers were more resistant to cell death, after exposure to the common chemotherapeutic temozolomide, than cells grown on 2D polystyrene plates. These results indicate that HA-coated nanofibers are a promising substrate for characterization of GBM migration and investigation of novel therapies.


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Introduction Glioblastoma (GBM) is a highly invasive and malignant brain cancer with median survival of only 12 to 15 months despite aggressive surgical resection and adjuvant radiation and chemotherapy.1 Poor survival is attributed to infiltration of adjacent brain tissue by a subset of tumor cells with a more migratory phenotype and a propensity for locomotion along white matter tracts and blood vessels.2-4 Despite the similar extracellular matrix (ECM) composition for brain tissue white and gray matter, the migration rate of GBMs in the elongated structures of the brain’s white matter are thought to be greater compared to the randomly organized gray matter ECM.5-6 This suggests that tissue topography and fiber orientation influences tumor cell motility.2, 7 Further, increased concentrations of hyaluronic acid (HA), a key constituent of brain ECM, promote malignant cell characteristics like increased invasiveness and drug resistance.8 Changes in both tissue topography and ECM composition have been implicated in poor patient outcomes, but overall, mechanisms underlying GBM cell migration in vivo are not well understood. Current in vitro models for studying cell migration include 2D micro-patterned surfaces910

and 3D hydrogels11. Although micro-patterned surfaces provide alignment cues and are useful

for quantifying migration, the architecture of micro-grooves does not accurately replicate the curvature of a blood vessel or white matter tract. Hydrogels, such as Matrigel™, are often used to study the dissemination of tumor spheroids and do promote an elongated migratory morphology typical of cells in vivo.11 However, hydrogels cannot directly simulate the directional nature of brain white matter while downstream assays such as immunostaining are difficult to execute. Currently, the most utilized model for characterizing GBM migration is an organotypic slice assay, where malignant cells are cultured on a living brain slice.12-13 Although mechanical, chemical, and



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morphological characteristics of native brain tissue are present, neural cell death within the slice during the culture period may skew results. Electrospinning provides a unique solution for fabricating biomaterials useful for modeling the GBM microenvironment. The resultant nanofibers, with precisely defined alignment, diameter, and morphology, represent a valuable structural model through which GBM cells act to replicate their invasive behavior in vivo.14 Electrospun nanofibers composed of polycaprolactone (PCL)1518

and polyblend chitosan-PCL (C-PCL)19 have been used to study GBM cell migration. The

polyblend nanofibers are particularly advantageous because they combine the biocompatibility of the natural polymer component and the mechanical stability of the synthetic polymer component.20 In previous work , 400 nm diameter C-PCL nanofibers were shown to promote a migratory GBM phenotype with cell migration profiles similar to those in vivo.19 While substrate topography has proven to be a crucial factor for GBM invasion, biochemistry may also play a strong role in promoting more migratory cell behavior. We hypothesize that integrating native biochemical cues with the relevant topography provided by nanofiber substrates may increase migratory potential of cultured cells, providing an enhanced GBM migration model. The influence of topography and biochemistry on GBM cell motility was investigated here using electrospun, polyblend chitosan-polycaprolactone (C-PCL) nanofiber substrates. The C-PCL nanofiber mats were highly-aligned, biocompatible and mechanically stable. C-PCL nanofibers were coated with different concentrations of HA to investigate the influence of biochemical cues on GBM cell attachment and alignment. The model GBM cell line, U-87 MG, was seeded onto nanofiber substrates and imaged after 24, 48, and 96 hours of culture to visualize cell morphology and alignment characteristics. The expression of Ki-67 and the migration versus proliferation potential of GBM cells on each substrate were investigated to 4 ACS Paragon Plus Environment

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evaluate the potential of the coated nanofiber substrates to promote enhanced migratory behavior. Resistance to the clinical chemotherapeutic, alkylator temozolomide (TMZ), by nanofibercultured cells relative to cells grown on polystyrene culture plates was evaluated. Finally, longterm migration assays ( 10 hours) were conducted to study cell migration patterns and velocities.

Materials and methods Materials All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Chitosan (from shrimp shells, 75-85% deacetylated, medium molecular weight), polycaprolactone (PCL, average Mn 80,000), and hyaluronic acid sodium salt (from Streptococcus equi, molecular weight 1.5–1.8 x106 Da) were used as received. Nanofiber synthesis and coating Chitosan- polycaprolactone (C-PCL) polyblend nanofibers were synthesized according to previously published procedures.20-21 Briefly, a 7 wt% chitosan stock solution was prepared in trifluoroacetic acid (TFA, Fisher, Reagent grade ≥ 97%) and refluxed at 60°C for 12 hours under constant stirring. A 12 wt% PCL stock solution was prepared in 2,2,2-trifluoroethanol (TFE, ReagentPlus®, 99%) and stirred overnight. The solutions were mixed resulting in a 50 wt% chitosan and 50 wt% PCL polyblend (C-PCL). The polymer solution was electrospun using a 25gauge blunt tip needle on a high throughput centrifugal electrospinning (HTP-CES) system. The collector on the HTP-CES system was composed of 102 parallel electrodes separated by an insulating air gap where the fibers dispersed perpendicularly to the electrodes and aligned 360° around the spinneret.21 The C-PCL solution was centrifugally-fed from a rotating spinneret at 108 RPM and 5 kV applied DC voltage. The chitosan and PCL solutions were combined immediately before electrospinning and refreshed every 2 hours to prevent polymer degradation. Consistent 5 ACS Paragon Plus Environment


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environmental conditions (40–50% humidity and 20–25°C) were maintained throughout the synthesis. Aligned nanofibers were collected on a 9 mm square cover glass, secured to the cover glass using a medical grade silicone adhesive, neutralized for 15 min in 25 vol% NH4OH, and thoroughly washed in DI water to remove all residual base. Coating the C-PCL nanofibers with hyaluronic acid (HA) was performed following the reported procedures.22-23 Briefly, nanofibers were immersed in 0 wt%, 0.1 wt%, 0.5 wt%, or 1 wt% HA solutions at 37°C for 72 hours. Nanofibers were sterilized for 1 hr in 70% ethanol and washed extensively with Dulbecco’s phosphate buffered saline (D-PBS) before cell culture. Nanofiber characterization The HA coating ratio was calculated as the difference in weight between individual nanofiber samples before and after coating, using Equation 1, where Wf represents the weight of the C-PCL nanofibers after the HA coating and Wi is the weight before coating. Coating ratio (%) =

[

]x 100

W𝑓 ― W𝑖 W𝑖

(1)

Characterization of the nanofiber morphology and diameter distribution was conducted using scanning electron microscopy (SEM) images. Nanofibers were collected onto a pedestal coated with double-sided carbon tape, Au/Pd sputter-coated for 30 s at 18 mA and imaged with a SEM (FEI Sirion XL30) at an operating voltage of 3 kV. The nanofiber diameter distribution was determined from ≥ 50 nanofibers in at least 3 representative SEM images and measured using ImageJ. Frequency distributions were generated using Graph Pad Prism 7. Cell seeding and imaging


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All cell culture reagents were purchased from Life Technologies unless otherwise specified. Minimum essential media (MEM), antibiotic-antimycotic, fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (D-PBS) were purchased from Invitrogen (Carlsbad, CA). The human glioblastoma cell line, U-87 MG, was purchased from American Type Culture Collection (Manassas, VA) and was transfected with pRFP-N2 using Lipofectamine 2000 reagent according to manufacturer’s instructions.24 Cells were washed in D-PBS 48 hours after transfection and cultured in fresh media supplemented with G418 (500 µg/mL) for selection of the stably transfected population. Two weeks after transfection, U-87 MG RFP cells were sorted by fluorescence activated cell sorting (FACS; Vantage SE). Cells were maintained in fully supplemented Minimum Essential Medium (MEM) (Thermo Fisher Scientific, Grand Island, NY) containing 10% FBS and 1% antibiotic-antimycotic at 37C and 5% CO2 in a humidified environment. U87 MG – RFP cells were seeded on sterilized nanofibers in a 24-well plate in 2 mL of fully supplemented MEM at 5,000 cells/mL. Bright field and fluorescence images were obtained 24, 48, and 96 hours after cell seeding to observe alignment and changes in cell morphology. A 4 or 10 objective was used on an upright fluorescence microscope with a Ri1 Color Cooled Camera System and the NIS Elements software package (Nikon Instruments, Melville, NY). Immunostaining was conducted on 4% paraformaldehyde-fixed samples after 96 hours of culture. The cell membrane was permeabilized with 0.25% Triton X-100 in D-PBS for 20 min at room temperature and then blocked in 1% bovine serum albumin (BSA) in D-PBS for 30 min to block non-specific binding. For Ki-67 staining, samples were incubated in rabbit polyclonal antibody to Ki-67 (Abcam, Cambridge, MA) at 1:200 overnight at 4°C. The samples were then incubated with a goat anti-rabbit FITC secondary antibody (1:400) (Abcam, Cambridge, MA) for 7 ACS Paragon Plus Environment


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1 hour at room temperature. Samples were rinsed with D-PBS and stained for 5 min with DAPI before imaging. Quantification of cell number, alignment, and aspect ratio Cell alignment was measured using images obtained with a 4X objective on an upright fluorescence microscope with a Ri1 Color Cooled Camera System with the NIS Elements software package (Nikon Instruments, Melville, NY). Images were processed with ImageJ using the “Li” uniform threshold filter. All cell images were converted to binary 8-bit and the “Analyze Particle” plug-in was used to quantify the cell number and morphology. The cell alignment angle was determined using the “best fit ellipse” measurement and recording the angle of alignment of the major axis from the horizontal. At least 350 cells were measured for each condition and converted to a frequency distribution histogram. Time course migration imaging U87 MG – RFP cells were seeded on the various substrates and cultured for 24 or 96 hours before imaging using an inverted microscope (Zeiss Axiovert 200M) on an automated stage (Marianas Imaging System, Intelligent Imaging Innovations) operated in a heated chamber at 37 °C. Time course images were acquired every 2 min for ≥ 10 hours using a 10 objective. Image sequences were analyzed using the semi-automated TrackMate plug in for ImageJ.25 At least 7 individual cells were tracked for each culture condition (24 hr and 96 hr culture timepoint). Cell displacement for each 2-minute time interval was measured and plotted for each individual cell. Cell migration speed was determined by measuring the cell displacement between imaging timepoints (2 min). Maximum and average cell migration speeds were determined using the trajectory of at least 7 individual cells for each condition. Only cells visible for  10 hours were tracked. Drug response analysis 8 ACS Paragon Plus Environment

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C-PCL nanofibers were immersed in a 0.5 wt% solutions of HA for 72 hours at 37°C. HA-coated and uncoated nanofibers were sterilized for 1 hr in 70% ethanol and washed extensively with DPBS before cell culture. U-87 MG RFP cells were cultured for 96 hours on 2D TCPS, 0% HA or 0.5% HA nanofibers. The samples were then treated with either 0 µM (untreated) or 100 µM temozolomide (TMZ) for 48 hours. After 48 hours, 200 µL of an alamarBlue® working solution (10% resazurin and 90% fully supplemented medium) was added to each well and incubated at 37C for 2 hours. Aliquots of the alamarBlue® solution were transferred to a black-bottom 96well plate and the fluorescence intensity was measured on a microplate reader. Differences in cell viability (percent difference in metabolic activity) was reported as a percent change in fluorescence intensity between TMZ-treated samples relative to untreated controls for each substrate. Statistical analysis All data were analyzed to express the mean ± standard deviation of the mean unless stated otherwise. Statistical significance was analyzed by one-way or two-way analysis of variance (ANOVA) with post hoc Tukey multiple comparisons testing (p ≤ 0.05) in GraphPad Prism 7 (Prism version 7.04, Graph Pad Software, San Diego, CA). All frequency distributions were generated using Graph Pad Prism and statistical significance was determined using the Kolmogorov-Smirnov test (p ≤ 0.05).

Results and discussion The selective migration of GBM cells down white matter tracts and blood vessels suggests that the physical architecture of the brain may be a key factor in migratory tumor cell behavior.26 Recapitulating the native brain ECM in an in vitro platform can result in a useful tool for studying GBM cell migration. Studies using these platforms can provide insight into the cues that initiate 9 ACS Paragon Plus Environment


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phenotypic changes resulting in invasive, malignant cells. Further, materials optimized to mimic brain ECM represent a reliable substrate for screening anti-invasion therapeutics. Characterization and coating of C-PCL nanofibers Thirty identical, highly-aligned C-PCL nanofiber samples were fabricated by electrospinning with a custom-made fiber spinning system and collected on glass coverslips after 2 hours. Previously, the presence of both chitosan and PCL in the resultant nanofibers was confirmed using Fourier Transform Infrared spectroscopy (FTIR).19, 21 Figure 1a shows the typical fiber density of a sample with a high degree of fiber alignment. At higher magnifications (Figure 1b), nanofiber diameter uniformity and a bead-less structure are visible. Conventional electrospinning can result in defects in the aligned nanofiber morphology and such defects interfere with cell locomotion and hinder the characterization of migratory cell behavior.17 Nanofiber fabrication using the HTP-CES allows for superior nanofiber alignment and diameter uniformity relative to conventional electrospinning. This ensures that cell locomotion is uninterrupted by morphological changes allowing for characterization cell-cell and cell-ECM interactions.


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Figure 1. Morphology of uncoated nanofibers comprised of 50 wt% chitosan and 50 wt% PCL (C-PCL). SEM images depicting (a) nanofiber mat and (b) individual nanofiber morphology. Scale bars represent (a) 500 µm and (b) 750 nm.

Both biochemical and topographical cues in the ECM play key roles in influencing cell behavior. Importantly, the ECM, through which GBM cells invade, is rich in hyaluronic acid (HA) and proteoglycans.26 To mimic this HA-rich, nanoscale-diameter fibrous structure, C-PCL nanofibers were coated with HA. The optimal amount of HA necessary to adequately mimic the biochemical cues of brain ECM in vitro was determined by comparing U87-MG cell behavior when cultured on C-PCL nanofiber mats exposed to different HA coating concentrations with culture on uncoated C-PCL nanofibers. Specifically, the influence of HA surface coating on cell migration and proliferation was characterized. C-PCL nanofibers were soaked in solutions with HA concentrations ranging from 0–1 wt% and the resultant nanofiber morphologies were characterized by SEM (Figure 2). The 0–1 wt% HA solution range was selected based on reported experiments,22-23 where the same range resulted in a water insoluble HA-surface coating due to the polysaccharide ionic complex between chitosan and HA. The change in weight of nanofiber mats after coating and lyophilization was examined to evaluate the coating ratio, which reflects the difference in nanofiber substrate weight before and after coating (Figure 2a). The uncoated (0% HA) C-PCL nanofibers weighed significantly less than the coated substrates. Although not all conditions were statistically different, the weight of the nanofiber mat increased with increasing HA concentration signifying successful coating. HA coating is used to mimick the native microenvironment where neurons, axons, and glia cells are embedded in HA27. The nanofiber diameter distribution associated with dehydrated, uncoated C11 ACS Paragon Plus Environment


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PCL or dehydrated, HA-coated C-PCL nanofiber mats was visualized and measured using SEM images (Figure 2b). The mean diameter of uncoated and coated nanofibers ranged from 326 nm to 382 nm with no significant difference among the various conditions. The diameter of white matter tracts in the human brain varies, but generally ranges from 0.3 μm to 10 μm28-29 with the majority of white matter tract diameters less than 1 μm.29-30 Based on a previously study aimed at optimizing nanofiber diameter for GBM migration, our nanofiber diameter range encompasses the diameter range reported to promote migratory GBM behavior.19 Light microscopy was used to visually evaluate physical changes in the hydrated coated nanofibers (Figure 2c). A highly-aligned, nanofibrous morphology was observed for all conditions over a large field of view with no visible nanofiber agglomeration or macro-scale changes identified due to the HA coating. At higher magnification using SEM, a nanofibrous morphology was observed for all HA coating conditions (Figure 2d) although slight agglomeration of nanofibers into multi-nanofiber bundles occurred as HA content increased. This may be an adverse effect of the lyophilization procedure post-hydration to prepare SEM imaging samples, as no multi-nanofiber bundles were identified under the light microscope. Taken together, these results indicate that the overall morphology was preserved, and the physical properties of the nanofibers were not drastically altered by the HA coating.


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Figure 2. Uncoated and HA-coated C-PCL nanofiber characteristics. (a) HA coating ratio (wt%) (n  4) and (b) average nanofiber diameter (n  50) as a function of HA solution concentration. *Statistically significant from all or specified. (p ≤ 0.05) Morphological analysis of lyophilized nanofibers using (c) brightfield optical imaging for large field-of-view and (d) SEM images for morphological changes. The scale bars represent (c) 200 µm and (d) 750 nm.



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Cell behavior on HA-coated nanofibers Cell behavior on HA-coated nanofibers was characterized by observing cultures of an RFPtransfected human glioblastoma cell line (U-87 MG RFP) using bright field and fluorescence microscopy. U-87 MG was selected as the representative cell line due to well-documented migration and invasion characteristics.19, 31 U-87 MG RFP cells were seeded on 2D tissue culture polystyrene (TCPS), uncoated (0% HA) nanofibers, and HA-coated nanofibers. Cells were cultured for 24, 48, or 96 hours and then imaged to evaluate cell morphology and alignment. Merged bright field and fluorescence images reveal cell responses to substrates (Figure 3a and b). After 24 hours (Figure 3a), cells on all substrates were sparse, but adherent. After 96 hours (Figure 3b), cell density increased on all substrates, signifying proliferation. On 2D TCPS, no cell directionality was observed with cell protrusions visible in all directions under high magnification (Figure 3c). The directional influence of the aligned nanofibers, independent of coating, was apparent with cells displaying a highly elongated, spindle morphology with visible protrusions consistently stretching longitudinally along the nanofiber. At high magnification (Figure 3c), for all nanofiber conditions, aligned and elongated cell morphology was maintained even if cell-cell interactions were visible, suggesting that cells strongly respond to the physical directionality of the nanofibers. Cell elongation and alignment is common for aligned nanotopographies,32-33 and in GBM, the elongated morphology and cell polarization observed on the uncoated and HA-coated nanofibers is similar to native migrative cell morphology.34 To quantify the cell alignment on each substrate, the “Li” automated threshold filter was applied to the fluorescence images in Figure 3b using ImageJ. After 96 hours of culture, cells cultured on the 2D TCPS did not preferentially align (Figure 3d). However, in all nanofiber conditions, a strong correlation between cell alignment angle and nanofiber alignment was 14 ACS Paragon Plus Environment

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displayed with approximately one-third or greater of the cell population aligning within ± 15° of the horizontal. The highest degree of alignment was exhibited in cells on the 0.5% HA substrates (45.1% of cells aligned within ± 15° of the horizontal).

Figure 3. U-87 MG-RFP cells (red) cultured on 2D TCPS, uncoated C-PCL nanofibers, or HAcoated C-PCL nanofibers. Representative fluorescent and bright field images were merged to show cell alignment and orientation relative to the nanofiber directionality after (a) 24 hours and (b) 96 hours of culture. On 2D TCPS, cells were randomly oriented. On nanofiber substrates, cells were 15 ACS Paragon Plus Environment


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aligned and elongated parallel to the aligned nanofibers. Scale bar in (b) represents 250 µm. (c) A high magnification panel shows further directionality and cell elongation at 96 hours. Scale bar represents 50 µm. (d) Angle of U87 MG RFP cell alignment after 96 hours of culture on various substrates. Nanofibers were aligned along the horizontal (0°) (n ≥ 1067, n represents the number of cells measured). All conditions statistically differ from each other (p ≤ 0.05).

Analysis of cell alignment and morphology indicate that both uncoated and HA-coated nanofibers support attachment and proliferation of U-87 MG cells and the nanofiber topography appears to influence cell morphology and alignment relative to flat adherent culture. Cell morphology, quantified by aspect ratio measurements, displayed a shift to higher aspect ratios and increased cell elongation in the 0% HA and 0.5 HA nanofiber conditions (Figure S1, Supplemental Information). Compared against all nanofiber substrates, the 0.5% HA exhibited the largest population of cells with an aspect ratio ≥ 3.0 (24.3% of the population), meaning the cell length elongated along the nanofibers was three times greater than cell width. The differences in extent of cell alignment and elongation among the HA-coated substrates may indicate differences in the effect of HA coating concentration on cell behavior. Longer cell exposure to the nanofiber topography resulted in an increase in directionality, alignment, and cell elongation for all nanofiber cultures with the greatest alignment and elongation displayed in the 0.5% HA nanofiber substrate. Cell elongation is an indicator of mesenchymal-like change often indicating a more invasive cell phenotype. Further, the shift to a more spindle-like morphology suggests that the U-87 MG cells may be undergoing a mesenchymal change to a more malignant and invasive phenotype. Work by others shows that elongation of cells cultured on C-PCL nanofibers was coupled with an increase in invasion-related genes.19 In our current study, at 96 hours, 0.5% HA 16 ACS Paragon Plus Environment

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coated nanofibers appear most conducive to promotion of cell elongation and alignment and were therefore used for further experiments.

Proliferative state of GBM on HA-coated nanofibers Changes in the proliferative potential of GBM cells have been linked to chemoresistance and invasive behavior. To evaluate proliferative potential, cells cultured on 2D TCPS, uncoated nanofibers, and 0.5% HA-coated nanofibers were stained with DAPI (blue) to visualize cell nuclei and for Ki-67 (green), a proliferative cell marker (Figure 4). The Ki-67 protein is found in nuclei during the active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in the G0 phase of resting cells.35-36 Ki-67 expression has previously been utilized to predict breast cancer patient outcomes in vivo.37 In gliomas, high Ki-67 expression in some studies has been associated with poor survival outcomes,38 yet further investigation of its prognostic potential is required as the correlation of Ki-67 with tumor grade has largely been inconclusive.36, 39 To identify possible effects of nanofiber topography on U-87 MG proliferative state, nanofiber culture conditions were compared relative to 2D TCPS where expression of Ki-67 is indicative of a proliferative cell (Figure 4a). Fewer proliferative cells were present on uncoated (0% HA) nanofiber substrates (45 ± 14% of cell population expressing Ki-67) relative to 2D TCPS (74 ± 12%) which suggests that the nanofiber topography may suppress cell proliferation. Further, when comparing 0.5% HAcoated to uncoated nanofibers, similar Ki-67 expression was observed for cells in both conditions. Only 40–45% of the cells expressed Ki-67 indicating that less than half of the cell population on the nanofiber substrates were proliferative. When cultured on nanofibers, cells either migrated as single cells or formed small clusters (Figure 4b and c). Differences in cell behavior and morphology between the clustered cells and 17 ACS Paragon Plus Environment


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single migratory cells appeared predictive of the cell proliferative state. Single cells migrating along coated or uncoated nanofibers did not generally express Ki-67 (blue nuclei absent of green stain shown in Figure 4b). In cell clusters on the nanofibers, expression of Ki-67 was predominant (Figure 4c). Importantly, these types of morphological differences are prevalent in the native tumor microenvironment, where cells are generally either found in proliferative multi-cellular clusters or as single migrating cells.40 Cells in 2D TCPS culture did not cluster or form highly migratory single cells so therefore these differences in Ki-67 expression and cell behavior were not mirrored in 2D TCPS culture.

Figure 4. Evaluation of cell proliferative states on 2D TCPS, uncoated (0% HA) nanofibers, and 0.5% HA-coated nanofibers after 96 hours of culture. (a) Quantification of Ki-67 expression in


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cells as a fraction of total cell population on each substrate (n ≥ 150, n represents the number of individual cells counted). (b) Single cells migrating along the nanofibers generally did not express Ki-67 (green) and appeared blue from nuclear DAPI stain. (c) Conversely, most cells in cell clusters expressed Ki-67 (green). Scale bars represent 100 µm. (b,c) Images were taken on 0.5% HA coated nanofibers, but are representative of cell behavior on both nanofiber substrates. (d, e) Proliferative cell behavior was characterized over time (at 24 h and 96 h of culture). Fraction of observed cell population migrating (represented by blue on graph) or dividing (represented by green on graph) over a ≥10-hour time lapse period following (d) 24 and (e) 96-hour culture period (n ≥ 20, n represents the number of individual cells counted for each substrate).

To further investigate the influence of HA-coated nanofibers on GBM cells, the migratory and proliferative behavior was tracked for at least 20 individual cells on each substrate for  13 hours using time-course imaging (Figure 4d and e). Cell behavior was monitored to determine if cells were dividing or migrating. After 24 hours of culture on each substrate, more than 50% of observed cells cultured on 2D TCPS and 0% HA nanofibers exhibited at least one division event (Figure 4d). These division events were less frequent in cells cultured on 0.5% HA nanofibers. Notably, at 96 hours, the division events decreased for both nanofiber conditions and a more migratory phenotype was observed (Figure 4e). Two

challenges

in

clinical

treatment

of

GBM

include

aggressive

tumor

proliferation/growth and diffuse local invasion.41 However, proliferative and invasive cell phenotypes are rarely expressed in the same cells, giving rise to the “go versus grow” hypothesis.4243

Previous work by others demonstrated that cells at the leading edge of a tumor have a lower



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proliferative capacity than cells at the tumor core.44-45 Further, proliferation is reduced in leading edge tumor cells when the cells migrate from the tumor site on aligned nanofiber substrates relative to 2D flat films.16 Although the mechanisms inducing a proliferative versus invasive phenotypes are not well understood, the “go versus grow” hypothesis has been experimentally validated where glioma cell proliferation abates and migration is promoted and vice versa.41, 44 In this nanofiber system, a decline in proliferation of cells cultured on aligned nanofibers demonstrates that the aligned nanotopography mimics the native tumor microenvironment with cells behaving according to the “go versus grow” hypothesis, and potentially enhancing migration at the partial exclusion of proliferation. More insight is necessary to determine the exact mechanism governing malignant phenotype induction. However, the nanofiber platform could be a useful tool for parsing these mechanisms and determining phenotype-specific drug interactions.

Cell migration, speed, and displacement on various HA-coated nanofibers To determine the influence of the HA coating on U-87 MG-RFP cell motility, cell migration on each substrate was tracked using time-course imaging. Tracking commenced after 96 hours of culture. Images were acquired every 2 min for  10 hours and examined with the semiautomated TrackMate plugin for ImageJ.25 After 96 hours of culture, cells on aligned nanofibers were attached, elongated, and aligned correlating with the work of others who showed upregulation of invasion-related gene markers.19 Representative, individual cell migration pathways on 2D TCPS (Figure 5a) were compared to 0% HA (Figure 5b) and 0.5% HA (Figure 5c) coated nanofiber substrates after 96 hours of culture. On 2D TCPS, no directional preference in cell elongation or migration is observed for one representative cell during a ~4-hour period (Figure 5a), while cells cultured on both 20 ACS Paragon Plus Environment

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nanofiber substrates display an elongated spindle morphology and a consistent linear, directional motion (Figure 5b,c). In Figure 5c, cell elongation and long cell protrusions result in a large cell displacement over a short time period (as seen between 56 min and 58 min). Increased cell migration and an elongated spindle morphology have been linked to a highly invasive and drug resistant phenotype. Figure 5d-f summarize the migration pathways of several cells on each substrate, where different colors represent unique paths of individual cells. Overall, cells cultured on 2D TCPS migrated randomly with no directionality (Figure 5d) whereas cells on nanofiber substrates migrated along a linear path corresponding to fiber alignment (aligned along the horizontal) (Figure 5e,f). GBM cells expressed elongated and directional behavior on both nanofiber substrates regardless of coating, indicating that topographical cues play a key role in cell migration and the nanofiber platforms were effective at directing cell locomotion. Migratory behavior was also quantified to further characterize the influence of HA coatings on cell migration. Average cell speed associated with each culture condition was determined over the course of 10 hours by measuring the distance individual cells traveled during consecutive twominute intervals (images captured every two minutes). On all substrates, spikes in cell speed occurred intermittently (Figure 5g). Bursts of increased cell speed, also observed during live imaging of gliomas in mice,46 correspond to daughter cell migration after cell division. Bursts of speed observed here also corresponded with cell-cell interactions, or in highly migratory cells, after extension of lamellipodia. Importantly, bursts in cell speed were more frequent and of higher magnitude for cells cultured on nanofibers relative to 2D TCPS.



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Figure 5. Representative cell migration characteristics associated with 2D TCPS and the nanofiber topography. Representative migration pathways of an individual cells tracked on (a) 2D TCPS, (b) 0% HA, or (c) 0.5% HA nanofibers. Nanofibers were aligned parallel to the horizontal. Images are time stamped to show the displacement over time. (d) Representation of the migration pathways of several cells on 2D TCPS for  10 hours starting after 96 hours of culture where cell 22 ACS Paragon Plus Environment

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locomotion and elongation does not display directionality. Conversely, (e,f) the migration pathway of cells on 0% HA and 0.5% HA nanofiber substrates showing cell locomotion with clear directionality (nanofibers were aligned parallel to the horizontal). (g) Representative migration speed versus time measured for an individual cell on 2D TCPS, 0% HA, or 0.5% HA nanofiber substrates. Speed was determined by quantifying cell displacement every 2 min for ≥ 10 hours after 96 hours of cell culture.

The influence of substrate on GBM motility was analyzed by averaging the cell speed from  7 cells per substrate (Figure 6a). A general increase in average cell speed is demonstrated between cells on 2D TCPS and the nanofiber substrates, with cells on the 0.5% HA substrate exhibiting the highest average speed of approximately 35 µm/hr. Maximum cell speed for each individual cell tracked was quantified (Figure 6b) and averaged for each condition. Cells cultured on the 0.5% HA substrate also exhibited the highest maximum speeds. This maximum velocity is slightly greater than that seen in previous studies on C-PCL nanofibers17, 19 and organotypic brain slice cultures40.



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Figure 6. Quantification of (a) average cell speed ± standard error of mean and (b) maximum cell speed ± standard error of mean for cells cultured on 2D TCPS, 0% HA (uncoated), and 0.5% HA nanofibers. *Statistically significant from all conditions (p ≤ 0.05).

Tracking and quantifying the migratory behavior of cells cultured on 2D TCPS, 0% HA nanofibers and 0.5% HA-coated nanofibers shows a difference due to topography (TCPS and 0% HA nanofibers substrates) and differences due to the presence of HA (0% HA and 0.5% HA). It is well known that HA is a targeting molecule that binds with CD44 receptors, and CD44 receptors are often overexpressed in various cancer cells.47 Yet, the influence of HA on cancer cell behavior is currently understudied with published findings reporting both enhancement48 and impedance18 of cell adhesion and migration due to the presence of HA. Further, studies by Rao et al. using coreshell nanofibers revealed that GBM cells were highly sensitive to variations in nanofiber mechanical properties and surface chemistry.18 Similarly, we found the GBM cells in our system were sensitive to both topography and surface chemistry as cells on 0.5% HA-coated C-PCL nanofibers migrated consistently faster and had large bursts of cell speed when compared to cells on uncoated nanofibers and 2D cultures. This type of data can be used to determine the optimal HA-coating for in vitro brain ECM mimics. After 96 hours of cell culture, the most consistent migration, in terms of speed and distance traveled, was observed of cells cultured on 0.5% HAcoated nanofibers. This culture condition resulted in the most consistent cell displacement and migration at relevant velocities. Thus, the nanofiber substrates appear to promote migration behavior similar to GBM tumor cells in vivo and provide an unhindered migration path allowing for greater speeds in vitro.


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Drug resistance on various HA-coated nanofibers To evaluate the response of U-87 MG cells cultured on nanofiber substrates to a therapeutic alkylator agent, the relative resistance to temozolomide (TMZ)-induced cell death was evaluated (Figure 7). Experimental groups included 2D TCPS, uncoated nanofibers, and 0.5% HA-coated nanofibers. After 96 hours of culture, media was changed for all culture conditions and the replacement media contained either 0 µM (untreated) or 100 µM TMZ. After an additional 48 hours of culture, cell metabolic activity was evaluated by measuring alamarBlue® fluorescence intensity of treated and untreated cultures per condition and calculating the percent reduction in fluorescence intensity for each condition. Increased fluorescence intensity is indicative of relatively more viable, metabolically active cells. The 2D TCPS condition exhibited the most significant degree of cell death following TMZ exposure represented by a 42% ± 9% reduction in fluorescence intensity. Comparatively, cells cultured on both nanofiber conditions and treated with TMZ exhibited a slight decrease in fluorescence intensity relative to the corresponding untreated control, ranging from a 1% ± 6% reduction (0.5% HA coated nanofibers) to an 11% ± 3% reduction (uncoated nanofibers). Cells cultured on nanofibers and exposed to 100 M TMZ were more resistant to cell death compared to control cells cultured on the 2D TCPS. An in vitro model capable of encouraging the rapid development of TMZ-resistance in GBM cells may be a valuable tool in elucidating the molecular mechanisms behind acquired TMZ resistance.49



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Figure 7. Drug resistance of U-87 MG RFP to TMZ-induced cell death evaluated using alamarBlue®. All conditions were either untreated or treated with 100 µM TMZ for 48 hours prior to analysis. Percent difference in cell metabolic activity was evaluated by quantifying the fluorescence intensity of the treated sample relative to the untreated controls (n ≥ 3). *Statistically significant from all conditions (p ≤ 0.05).

Conclusion Polyblend chitosan-PCL nanofiber substrates, fabricated using an HTP-CES system, are characterized by highly-aligned nanofibers with superior diameter uniformity relative to conventional electrospinning. C-PCL nanofiber substrates with biologically-relevant topography were coated with hyaluronic acid to mimic an important biochemical characteristic of brain ECM. The nanofiber substrates were evaluated as biomimetic in vitro substrates for characterizing U-87 MG cell behavior. GBM cells cultured on aligned C-PCL nanofibers and nanofibers coated with 26 ACS Paragon Plus Environment

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0.1%–1.0% HA displayed a shift at 96 hours to a highly aligned and elongated cell with a spindle morphology. Relative to 2D TCPS, cells cultured on nanofibers were characterized by lower proliferative potential coupled with increased resistance to TMZ induced cell death. Additionally, coating nanofibers with 0.5% HA increased cell displacement and cell speed suggesting a shift of the GBM cell population toward a migratory phenotype. The ability to enrich migratory cells in vitro could simplify phenotypic investigation of these highly invasive cells for development of therapeutics targeted at the migrative subpopulation. Due to the unique morphology and surface chemistry of 0.5% HA-coated C-PCL nanofibers reported here, these nanofibers may represent a better in vitro platform for studying cell migration and anti-migration therapeutics.

Supporting Information Cell aspect ratio after 96 hours of culture.

Conflicts of Interest There are no conflicts to declare.

Acknowledgements The authors acknowledge support for this work by the Kyocera Professor Endowment and NIH grant (NIH/NCI R01CA172455) to Miqin Zhang. Ariane E. Erickson acknowledges support from the National Science Foundation Graduate Research Fellowship Program (DGE – 1256082). Time lapse microscopy was conducted at the Digital Microscopy Center at the University of Washington. Part of this work was conducted at the Molecular Analysis Facility, a National



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Nanotechnology Coordinated Infrastructure site at the University of Washington which is supported in part by the National Science Foundation (ECC-1542101), the University of Washington, the Molecular Engineering & Sciences Institute, and the Clean Energy Institute.

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Graphical Abstract


Hyaluronic Acid-Coated Aligned Nanofibers for the Promotion of

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