Background
In developed countries, cancer is the leading cause of death, whereas in developing countries, it is second only to heart disease [
1]. Breast cancer is the second most common cancer overall (1.7 million cases, 11.9%) but ranks fifth as the cause of death (522,000, 6.4%) [
2]. Substantial research expenditure is applied to develop new anticancer drugs, but most fail in animal models or clinical trials despite showing promise in preclinical testing [
3‐
5].
The tumour microenvironment plays a critical role in tumour initiation, proliferation, and metastasis, and contains potential therapeutic targets [
6‐
9]. Anticancer therapy is undergoing a conversion from a cancer cell-centric to a stroma-centric strategy, mainly because stromal components are more genomically stable [
10]. Angiogenesis, induced by the tumour microenvironment, plays an important role in promoting tumour growth and metastasis as well as providing the tumour with sufficient oxygen and nutrients for growth [
11].
Researchers have long relied on two-dimensional (2D) in vitro cell culture systems to study cancer cells. In 2D culture systems, cells are grown as monolayers on a flat solid surface lacking the cell–cell and cell–matrix interactions that are present in native tumours. Additionally, 2D cultured cells are exposed to much higher oxygen concentrations than in vivo, and are stretched and undergo cytoskeletal rearrangements acquiring artificial polarity, which in turn causes aberrant gene and protein expression [
12]. In contrast, three-dimensional (3D) culture systems offer the unique opportunity to culture cancer cells in a structure that more closely mimics the native environment of tumours [
13‐
15]. 3D culture models also have been used to assess the diffusion, distribution, and efficacy of drugs [
16].
In the last few decades, hydrogel scaffolds, which are cross-linked networks that possess high water contents, have attracted increasing attention in an attempt to mimic in vivo conditions [
17]. However, synthetic polymers such as polylactide and polyglycolide have large fibre diameters and pore sizes that yield poor scaffold structures with mechanical properties that do not accurately mimic the full complexity of the natural environment of cell growth [
18].
Chitosan has been used for the construction of 3D culture models due to its biocompatible, biodegradable, non-immunogenic, and non-inflammatory characteristics [
19,
20]. In addition, it has antitumour properties, haemostatic function, and antibacterial activity [
21,
22]. However, chitosan is only soluble in dilute acidic solutions, which limits its applications. There has been growing interest in the chemical modification of chitosan to improve its solubility and broaden its applications [
23,
24]. However, the selection of appropriate 3D scaffolds and characterization of the physical properties of the scaffolds (such as fibre length, porosity, and stiffness associated with cellular responses) and gene expression profile remain a challenge for the scaffold engineering field and for mimicking the tumour tissue environment [
25]. In our research, we describe, for the first time, a method to prepare a novel chitosan derivative: a hydroxyethyl chitosan–glycidyl methacrylate (HECS–GMA) hydrogel.
Methods
Materials
Glycidyl methacrylate (GMA), fluorescein isothiocyanate labelled phalloidin (FITC-phalloidin), Dulbecco’s phosphate buffered saline (DPBS, pH 7.4) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Shanghai, China). The live/dead® viability/cytotoxicity kit for mammalian cells was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), penicillin–streptomycin, and 0.25% (w/v) trypsin were purchased from Procell (Wuhan, China). Bovine serum albumin (BSA), Triton X-100, crystal violet and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Ding Guo Chang Sheng Biotechnology Co. Ltd. (Beijing, China). Both MCF-7 cells and athymic nude mice (BALB/c-nu) were supplied by Medical Center of Xi’an Jiaotong University (Xi’an, China). All other reagents and solvents were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Xi’an, China). All aqueous solutions were prepared using ultrapure water with a resistance of 18.25 MΩ. Culture plates (24-well plates) and T-25 cm2 tissue culture flasks were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The following antibodies were used: rabbit polyclonal Ab against CD34 (EP373Y; Abcam, Cambridge, UK) at a dilution of 1:500, rabbit polyclonal Ab against vascular endothelial growth factor (VEGF)-A (EP1176Y; Abcam) at a dilution of 1:100, rabbit polyclonal Ab against platelet-derived growth factor (PDGF)-B (EPR6834; Abcam) at a dilution of 1:50, rabbit polyclonal Ab against basic fibroblast growth factor (bFGF) (#36769; Signalway Antibody, College Park, MD, USA) at a dilution of 1:50 and a horseradish peroxidase-conjugated goat anti-rabbit antibody (GB23303; Goodbio Technology, Wuhan, China) at a dilution of 1:200. The diaminobenzidine (DAB) Colour Kit was purchased from DAKO (K5007; Copenhagen, DK). Recombinant human endostatin (Endostar) was purchased from Xian Sheng Mai De Jin Co. Ltd. (Shandong, China), and Bevacizumab (Avastin) was purchased from Shanghai Roche Pharmaceuticals Co. Ltd. (Shanghai, China).
Synthesis of HECS–GMA hydrogels
Synthesis of HECS–GMA
Three types of HECS–GMA were synthesized according to a method reported previously with some changes [
24]. Chitosan was prepared by adding chitosan (10 g, 62 mmol) slowly to a 50% w/v NaOH (16.7 g) solution and mechanically stirring at room temperature to obtain alkaline chitosan. The alkaline chitosan was stored at 4 °C overnight. Then 133 mL of isopropanol was added and the mixture was stirred mechanically for 90 min at 85 °C under reflux using a Dean–Stark trap. After this step, 18.3 mL of ethylene chlorohydrin, dissolved in isopropanol (33.3 mL), was added to the aforementioned mixture and stirring continued for 5 h under reflux at 65 °C. The desired product of HECS was obtained after filtration, washing with ethyl alcohol, dialysis against water and lyophilisation. The chemical structure of HECS was confirmed from the
13C NMR (Bruker, Karlsruhe, Germany) spectrum. Solutions of GMA (0.98, 1.46, and 1.95 mmol) in dimethyl sulphoxide (10 mL) were added to HECS solution (1 g in 90 mL water). The three reaction mixtures were incubated at 70 °C for 6 h with stirring. The product from each mixture was purified by dialysis (molecular weight cut-off: 3500) for 3 days against distilled water. The aqueous solutions were filtered, evaporated, and lyophilized. The three products were confirmed by
1H NMR spectra.
Synthesis of HECS–GMA hydrogels
Each of the three HECS–GMAs (35 mg), prepared as described above, was dissolved in 1 mL of distilled water. Irgacure 2959 [0.1% (w/w)] was added to the solutions. Polymerization was initiated by ultraviolet irradiation for 120 s at approximately 400 mW/cm2. According to the mole percentages of grafted GMA in HECS (20, 30 and 40%, relative to the number of repeating HECS units), the corresponding hydrogels were designated as HECS–GMA20, HECS–GMA30, and HECS–GMA40, respectively.
Characterization of the HECS–GMA hydrogels
Rheological analysis
Both rheological measurements (time sweep test and oscillation frequency sweep test) were performed on a Malvern Kinexus Pro + rotational rheometer (Malvern, UK) by using parallel plate geometry with a diameter of 20 mm and a sample gap of 1 mm. For the time sweep test to monitor the gelation process, 320 µL of fresh solution (ensuring a gap size of 1 mm) was irradiated by an Omnicure S2000 lamp, which was auxiliary equipment for the rheometer, for 120 s at a frequency of 1 Hz and a strain of 1%. For the oscillation frequency sweep test, HECS–GMA hydrogels were exposed to a shear frequency increasing from 0.1 to 10 Hz while maintaining the shear amplitude at 1% and the temperature at 37 °C. As the shear frequency changed, variation of the elastic (storage, G′) and viscous (loss, G′′) moduli was observed.
Swelling behaviour
To investigate the swelling ratios, lyophilized HECS–GMA hydrogels were weighed in the dry state (Wd) and then immersed in Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4) at 37 °C. At regular time intervals, samples were removed to obtain the wet weight (Ws) after removal of the water on the surface. This step was repeated until the weight did not change. The swelling ratios were inferred from the following formula: swelling ratio = (Ws − Wd)/Wd.
Morphology
Scanning electron microscopy was performed on the HECS–GMA hydrogels (lyophilized to maintain the porous structure without any collapse) to obtain information on the pore structure. The lyophilized hydrogels were sputter-coated with gold and investigated by using a hitachi (Tokyo, Japan) S-3400N scanning electron microscope at an accelerating voltage of 20 kV.
3D and 2D cell cultures
MCF-7 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in 5% CO2. The disc-like sterile HECS–GMA40 hydrogels with a certain thickness of 2–3 mm and a diameter of 15 mm close to the 24-well culture plate were infiltrated with 0.5 mL of cell suspension (106 cells/mL) and incubated at 37 °C for 4 h. 1 mL of culture medium was added to each disc. The culture medium was replaced every other day. Traditional 2D cell cultures with the same number of cells were used as a control.
Cell morphology
To evaluate the attachment of MCF-7 cells onto HECS–GMA hydrogels, cells were seeded onto the hydrogels and incubated for 1, 4 and 7 days in 24-well culture plates. The hydrogels (HECS–GMA40) were then washed gently with DPBS to remove non-adherent cells. The hydrogels were viewed with an inverted microscope (Leica, Wetzlar, Germany). The experiments were repeated three times.
Cell morphology was observed with a confocal laser scanning microscope (CLSM, Leica) using fluorescent staining. After 1, 4 and 7 days of cultivation, MCF-7 cells in the HECS–GMA hydrogels were rinsed with DPBS twice, fixed in 4% paraformaldehyde for 20 min and permeabilised with 0.2% Triton X-100 for 10 min. To prevent non-specific labelling, the cells were treated with blocking buffer (1% BSA) for 20 min and then washed with DPBS three times. The actin cytoskeleton was stained with 5 μg/mL FITC-phalloidin for 1 h. Nuclei were stained with DAPI for 10 min and visualized by CLSM. The experiments were repeated three times.
Cell viability
The viability of cells in 3D and 2D cultures was determined on days 1, 4 and 7 using a live/dead® viability/cytotoxicity kit according to the manufacturer’s recommendations. Calcein AM (0.5 μM) and ethidium homodimer-1 (1 μM) were used to stain viable and dying cells, respectively. The hydrogel/cell constructs were observed using an inverted fluorescence microscope (Leica, Wetzlar, Germany). The experiments were repeated three times.
Cell proliferation
Cell proliferation was evaluated using the MTT assay. After 1, 4 and 7 days of incubation, cell culture supernatants were removed and the hydrogel/cell constructs were washed completely with DPBS culture medium (0.9 mL). Then, 0.1 mL of MTT (5 mg/mL) was added into each sample well and incubated for 4 h at 37 °C. After removal of the culture medium, the blue formazan crystals were dissolved in 1.0 mL of dimethyl sulphoxide with shaking for 30 min. The absorbance of the solutions was measured at 570 nm using an enspire microplate reader (PerkinElmer, Shelton, CT, USA). The blank control groups lacked cells and were treated identically to the experimental groups. Each experiment was repeated three times.
Cell migration
The migration abilities of MCF-7 cells were investigated using transwell (8 μm, EMD Millipore, Billerica, MA, USA) assays. After incubation for 1, 4 and 7 days, MCF-7 cells were digested and rinsed with DPBS, and suspended at 5 × 106 cells/mL in DMEM containing 0.2% BSA. Cell suspensions (100 μL) were placed in each upper transwell chamber and 500 μL of medium containing 20% FBS was used as the chemoattractant in the lower chamber. After 24 h, cells failing to invade through the pores were removed using a cotton swab. Invasive cells on the lower surface of the membrane were fixed with methanol, stained with 0.1% crystal violet and counted under an inverted microscope. The experiment was repeated three times.
Induction of in vivo xenograft tumours
All animal experiments were performed according to the Chinese Ministry of Public Health Guide and US National Institutes of Health guidelines. The tumour-forming capabilities of MCF-7 cells cultured in HECS–GMA40 hydrogels and 2D monolayers were examined following subcutaneous injection of the cells into five BALB/c nude mice. MCF-7 cells were cultured for 7 days in T-25 cm2 tissue culture flasks or without HECS–GMA40 hydrogels and harvested. Cells (1 × 107) were resuspended in 100 μL of DMEM and injected into the right (3D) and left (2D) flank of each 4–5-week-old mouse. The mice were kept in a specific pathogen free facility with a 12-h light/dark cycle and had free access to food and water. Tumour volumes were measured with a vernier calliper in two dimensions every 5 days. Tumour volumes were calculated using the formula ab2/2, where a and b are the largest and smallest diameters, respectively. After 6 weeks, the animals were sacrificed and the tumours were harvested.
IHC staining
IHC staining was performed by the standard streptavidin-peroxidase method. The nude mouse xenograft tumours were fixed with 4% paraformaldehyde for 24 h followed by decalcification, dehydration and embedding in paraffin. After hematoxylin and eosin staining for tumour confirmation, IHC staining was performed on 4-µm sections. The slides were incubated with the primary antibodies for CD34 (EP373Y; dilution of 1:500), VEGF-A (EP1176Y; dilution of 1:100), PDGF-B (EPR6834; dilution of 1:50), bFGF (#36769; dilution of 1:50). A horseradish peroxidase-conjugated goat anti-rabbit antibody (GB23303; dilution of 1:200) was used as the secondary antibody overnight at 4 °C. Negative control slices were incubated in the antibody solution but without antibodies. After staining with 3,3-diaminobenzidine, the slices were counterstained with hematoxylin, dehydrated, cleared and mounted. The stained slides were observed and photographs were obtained using an invenio 1D microscope camera (Deltapix, Smorum, Denmark).
To quantify angiogenesis, an anti-CD34 antibody was used to stain microvessel epithelial cells. Positive reactions were indicated by a reddish-brown precipitate in the cytoplasm. The intensity of positive staining was measured through Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA). All images were taken using the same microscope and camera sets. The intensity of positive staining in blood vessels or cells was valued by the mean integrated optical density (mean IOD) according to the following formula: mean IOD = IOD/area of the tumour section.
Effect of drugs on in vivo 3D culture xenograft tumours
All animal experiments were performed according to the Chinese Ministry of Public Health Guide and US National Institutes of Health guidelines. MCF-7 cells were cultured for 7 days in T-25 cm2 tissue culture flasks with HECS–GMA40 hydrogels and harvested. Cells (1 × 107) were resuspended in 100 μL of DMEM and injected into the right flank of each 4–5-week-old BALB/c nude mice. The 20 mice were kept in a specific pathogen free facility with a 12-h light/dark cycle and had free access to food and water. When tumours became palpable (100–200 mm3), 15 mice were randomized into three groups of five: saline control [1 mL/day, intraperitoneally (i.p.)], Endostar (10 mg/kg/day, i.p.) and Bevacizumab (5 mg/kg/twice weekly, i.p.). Tumour volumes and body weights were measured twice weekly. After 2 weeks, the animals were sacrificed and the tumours were harvested. Tumour volumes were calculated as described above. The rate of tumour inhibition in each treatment was evaluated according to the following formula: tumour inhibition rate (%) = (average tumour volume of control group − average tumour volume of experimental group)/average tumour volume of control group × 100. IHC staining was performed as described above.
Statistical analysis
Data are presented as the mean ± standard deviation. Student’s t test was used to determine the statistical significance between two groups. SPSS 18.0 for Windows (IBM, Chicago, IL, USA) was used for the statistical analysis. Data were considered as statistically significant when P < 0.05.
Discussion
Breast cancer is a heterogeneous disease with complex tissue environments that affect cancer initiation, metastasis, angiogenesis, and resistance to therapy [
30,
31]. The discovery of tumour angiogenesis opened a new path in fighting cancer that increased the effectiveness of standard chemotherapy, or even replaced it, by offering better patient outcomes [
32]. Over the past decade, extensive studies of 3D cultures have demonstrated differences compared to the behaviour of cells on 2D surfaces [
33]. A good tumour microenvironment model that closely simulates the real tumour construct would not only enhance the study of physiological mechanisms in vitro, but also dramatically improve the translation of novel chemotherapeutics from in vitro to in vivo testing [
13].
Hydrogels are cross-linked networks of the same or different types of polymers with a high capacity for water absorption [
34]. Natural gels derived from extracellular matrix components and other biological sources are biocompatible and inherit the bioactivity of their starting material, which is something that synthetic polymers usually lack [
35‐
37]. To model the breast cancer microenvironment, and study the growth of breast cancer MCF-7 cells and the targeting of anti-angiogenic agents in 3D cultures, we constructed 3D HECS–GMA hydrogels using chitosan as a platform. The modification of chitosan to HECS–GMA hydrogels increased the water solubility and viscoelasticity, while enabling control of the pore size and enhancing biocompatibility, which was different from other methods [
38]. Additional research is needed to explore the ability of cross-linking to prevent dissolution of the hydrogel polymer chains by rheological processes [
39]. The G′ and G′′ values of our hydrogels had a low frequency dependency, indicating that the hydrogel networks were highly stable. These results indicate that the proportion of energy stored elastically in the hydrogels was much higher than that of dissipated energy. Furthermore, the swelling ratio of the hydrogels was small, indicating that the network had a significant influence on the swelling ratio.
The pore sizes of the hydrogels were suitable for material exchange by cells by providing the necessary space and nutrition channels. The HECS–GMA hydrogels achieved unique structural (fibro-porous, non-toxic) and mechanical (high tensile strength with flexibility) properties, providing oxygen and water permeability to support cell adhesion and growth.
In 3D cultures, breast cancer cells formed spheres embedded in the hydrogels. These spheres could be observed with an inverted microscope and by CLSM. The encapsulation of living cells, and chemical cross-linking, provide hydrogels with excellent mechanical strength, which is different from other scaffolds [
25,
38]. In 2D cultures, MCF7 cells can initially obtain sufficient nutrients and oxygen to grow quickly. However, there are limitations on proliferation in 2D cultures as the number of cells increases, and space and nutrients are constrained. With increasing time in 3D cultures, cell spheroids grew, and hypoxic cells in the core grew more slowly than peripheral cells. This phenomenon, consistent with tumour growth in vivo, showed that the HECS–GMA hydrogels had good cytocompatibility [
40,
41].
To further study the characteristics of 3D cultured cells, their growth in vivo as xenograft tumours and their expression of CD34, VEGF-A, PDGF-B and bFGF were examined. CD34 and VEGF-A expression but not PDGF-B and bFGF expression were significantly different between 3D-cultured cells and 2D cultures. Possible reasons for the lack of difference in the expression of PDGF-B and bFGF include high expression masking the differences, the use of a high antibody concentration, or the short time in 3D culture.
Differential intracellular signalling and transcription of genes has been reported between 2D and 3D cultures [
25]. Although 3D cultures are widely used to study the effect of drugs in breast cancer, their use with anti-angiogenic agents is not common [
42]. Endostar inhibited breast cancer tumour growth in our study, which was consistent with previous studies [
43]. The risks associated with Bevacizumab combination therapy, the lack of a survival benefit, and inconsistencies in the magnitude of the progression free survival time across studies led the US Food and Drug Administration to remove the metastatic breast cancer indication for this drug in November 2011 [
44]. Despite this setback, some studies continue to explore a role for Bevacizumab in treating breast cancer [
45]. The expression differences that we observed among the various angiogenesis-related growth factors indicate the presence of specific drug targets. However, consistent with the results in clinical practice, our results showed that Bevacizumab was ineffective against breast cancer cells.
Our HECS–GMA hydrogels have several advantages over other 3D scaffolds. They are biocompatible, biodegradable, non-immunogenic and non-inflammatory. These characteristics make it possible to provide an in vitro biomimetic microenvironment for breast cancer culture and drug studies. However, there are some limitations to the hydrogel modelling system. Specifically, the hydrogels will deform and degrade after 7 days, and intrinsic opaqueness and autofluorescence may impair morphologic observation. If the stability of HECS–GMA hydrogels can be elevated, it may become possible to replace xenograft models.
Three-dimensional cultures may promote the expression of CD34, VEGF-A, PDGF-B, and bFGF better than 2D cultures in xenograft tumours in vivo. However, 3D cultures provide an integrated environment, and their effects on cells should be further studied. The gradually increasing use of anti-angiogenic agents makes it important to perform additional research on their efficacy [
46]. The 3D culture platform may provide a system suitable for such research, although there may be others factors affecting the efficacy of Endostar and Bevacizumab. Thus, further studies are needed.
Authors’ contributions
AS, JQ, HW, and JX contributed to project design and manuscript drafting. WX and YZ took part in designing and producing hydrogels. The experiments of cells and animals were accomplished by HW, AS. HW, and YZ have contributed significantly in drafting the manuscript, literature review and revising it critically. All authors read and approved the final manuscript.