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Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix
Biochemical and Biophysical Research Communications xxx (2018) 1e6
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Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix Xuanzhi Wang a, 1, Xingliang Dai a, 1, Xinzhi Zhang b, c, Xinda Li c, Tao Xu c, d, **, Qing Lan a, * a
Department of Neurosurgery, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, People's Republic of China Medprin Biotech GmbH, Gutleutstraße 163-167, Frankfurt am Main, D-60327, Germany c Department of Mechanical Engineering, Biomanufacturing Center, Tsinghua University, Beijing, 100084, People's Republic of China d Department of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute, Shenzhen, 518055, People's Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2018 Accepted 14 March 2018 Available online xxx
Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies. © 2018 Elsevier Inc. All rights reserved.
Keywords: 3D porous scaffold Cancer stem cell Enrichment Glioma Chitosan Hyaluronic acid
1. Introduction Glioblastoma (GBM) is the most invasive and deadly primary tumor in the brain [1], even after total resection and adjuvant chemoradiotherapy, the prognosis for patients is still poor, and the median survival time was only 14 months after diagnosis [2]. GSCs are thought to be the source of recurrence and chemoradiotherapy resistance in GBM [3]. There are many strategies for GSCs targeted therapy, but the results are still not satisfactory [4]. Further study of
* Corresponding author. ** Corresponding author. Department of Mechanical Engineering, Biomanufacturing Center, Tsinghua University, Beijing, 100084, People's Republic of China. E-mail addresses: [email protected] (T. Xu), [email protected] (Q. Lan). 1 These authors contributed equally to this work.
GSCs is important for the treatment of glioma. However, CSCs account for only less than 1% of cancer cell population [5]. Therefore, how to successfully isolate and enrich CSCs in vitro has been a research focus. Thus far, CSCs have been isolated in vitro by using fluorescence-activated cell sorting or magnetic-activated cell sorting, and serum-free CSC culture medium has been used as the suspension culture of tumorspheres for enriching CSCs [5,6]. However, the application of these techniques often require specialized equipment and costly antibodies, time-consuming cell suspension culture, the yield of CSCs is very low [5]. Most importantly, suspension culture lacks the 3D environment required for cell-extracellular matrix (ECM) interactions to promote cancer heterogeneity [7]. Due to the limitations of traditional CSCs isolation and enrichment methods, researchers have enriched CSCs by seeding cancer cells on 3D porous scaffolds [8,9]. It was found that 3D microenvironment constructed by 3D porous scaffolds could not only promote the formation of tumor cell spheroids, but also the
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invasiveness and chemotherapeutic resistance of tumor cells cultured on 3D scaffolds were significantly improved compared with conventional 2D models [10,11]. At present, a variety of natural and synthetic scaffolds have been used to construct 3D porous scaffolds for the study of CSCs enrichment [10,12,13]. However, the most effective composition that enriches CSCs is still unclear. In this study, glioma cell line U87 cells were grown on CS and CS-HA scaffolds. Cell viability, morphology and the expression of GSC biomarkers and EMT-related genes were analyzed under different culture conditions. Finally, we evaluated the in vivo tumorigenicity of cells derived from both scaffolds and compared with that of cells cultured under 2D conditions. 2. Materials and methods 2.1. Materials Chitosan (practical grade, >90% deacetylated, MW ¼ 250,000) was purchased from Haidebei (Shandong, China) and hyaluronic acid (sodium hyaluronate) was purchased from Freda (Shandong, China). Human glioma cell line U87 was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) at 37 C in humidified air with 5% CO2. 2.2. Scaffolds synthesis CS scaffolds and CS-HA scaffolds were prepared based on previously reported methods [14]. CS scaffolds were obtained by dissolving chitosan in dilute acetic acid and lyophilized. CS-HA scaffolds were prepared by forming a polyelectrolyte complex (PEC) between chitosan and hyaluronic acid molecules, and then froze and lyophilize the PEC solution. Briefly, for CS scaffolds, 2 wt% chitosan and 0.5 wt% acetic acid solution were stirred for 30 min to obtain a homogeneous CS solution. For CS-HA scaffolds, 4 wt% chitosan and 1 wt% acetic acid solution were stirred for 30 min to obtain a homogeneous CS solution, then 1 wt% hyaluronic acid solution dissolved in deionized water was gradually added in at an equal volume and mixed for 3 h to obtain a homogeneous CS-HA solution. Approximately 2 mL of CS solution or CS-HA solution was filled into each well of 24-well cell culture plate, froze overnight at 20 C, 2 h at 80 C, and lyophilized for 48 h using freeze dryer (LYOQUEST-85, TELSTAR). These scaffolds were cut into 2 mm thick discs, and then neutralized with 25v% ammonium hydroxide solution for 1 h. The residual ammonium hydroxide in scaffolds was removed by repeated washing with deionized water. The neutralized CS-HA scaffolds and CS scaffolds were again froze at 20 C overnight and lyophilized for 48 h. Scaffold samples were sterilized by gamma irradiation. 2.3. Live/dead assay Cell viability was evaluated by fluorescent live/dead assay kit (KeyGEN BioTECH, Nanjing, China) according to the protocol. Briefly, medium was removed from the wells and scaffolds were washed with PBS, followed by soaking in PBS solution containing 8 mM propidium iodide and 2 mM Calcein-AM. These scaffolds were cultured for 10 min at room temperature away from light, and washed with PBS. Cells were green (live cells) or red (dead cells). Images were obtained from fluorescence microscope (Olympus IX51,Tokyo, Japan). For each sample (n ¼ 3), live and dead cells were counted at 200 in 5 random spots.
2.4. Scanning electron microscopy (SEM) analysis Cell-free scaffolds and cell-laden scaffolds were fixed in 2.5% glutaraldehyde overnight at 4 C. Samples were then dehydrated using a series of ethanol solutions (70%, 80%, 90%, 95%, 100%, and 100%) for 30 min in each solution, then critical point dried using carbon dioxide critical point dryer (LEICA, EM CPD300). Samples were sputter coated with platinum, and images were obtained from the ULTRA 55 Scanning Electron Microscope (ZEISS, Germany). 2.5. Quantitative RT-PCR Cells were detached from porous scaffolds or well plates using versene and completely dissociated with Trizol (Invitrogen, 15,596e026), the total RNA was extracted following the manufacturer's protocol. Reverse transcription was performed using ImProm-IITM Reverse Transcription System (Promega, A3800). DNA transcripts were carried out using SYBR Green qPCR Super Mix. Thermo cycling was performed using ABI PRISM® 7500 Sequence Detection System and 18srRNA was used as the housekeeping gene. Relative gene expression was calculated using 2DDCt method. 2.6. Flow cytometry analysis 2D and 3D cells were digested with trypsin at day 10 and a certain amount of single cell suspension was prepared with PBS. Cells were incubated with 10 mL anti-CD133-PE or mouse IgG-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) per 106 cells in dark at room temperature for 20 min. Subsequently, cells were washed with PBS and resuspended for preparation. The proportion of CD133þ cells was analyzed with a BD FACSArial Flow Cytometer and results were evaluated by FlowJo software program (Treestar, Ashland, OR). 2.7. In vivo tumorigenicity analysis Cells (1 104) were harvested from 2D cultures or 3D scaffolds on day 10 and resuspended in 200 mL serum-free medium with 50% Matrigel (Bedford, USA). Cells were inoculated subcutaneously into the right flank of BALB/c nude mice (4e6weeks old). All animal experiments were carried out in accordance with the approved program of the Animal Care Committee of the Second Affiliated Hospital of Soochow University. Tumorigenesis and growth were monitored three times a week. Tumor size was measured with a caliper and tumor volume (V) was calculated according to the following formula: V ¼ (L W2)/2, where L is length and W is width. All tumors were harvested at 6 weeks after inoculation. 2.8. Statistical analysis Each experiment was carried out in three replicas, and all results are presented as the mean ± standard deviation. The Student's ttest was used to compare the means between two groups. Two-way analysis of variance (ANOVA) and a Bonferroni post-hoc test was used to compare multiple groups. Statistical significance was considered as *p < 0.05, **p < 0.01, and ***p < 0.001. Data analysis was performed using GraphPad Prism 7 software. 3. Results 3.1. Growth characteristics of glioma cell U87 cultured in 2D and 3D Sections and macrostructure morphology of CS scaffolds and CSHA scaffolds are shown in Fig. 1A and B. The average dimension of
Please cite this article in press as: X. Wang, et al., Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.114
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Fig. 1. Morphology of 3D porous scaffolds and live/dead assay of cell-laden scaffolds. (A)e(B) The morphology of CS and CS-HA scaffolds after lyophilization and section; (C)e(E) Cell viability after 5 days of culture; (F)e(H) Cell viability after 15 days of culture. Live cells were stained green and dead cells were stained red. Scale bars: (A)e(B) 1 cm; (C)e(H) 100 mm.
each scaffold was 13 mm in diameter and 2 mm thick. These scaffolds absorb water but do not dissolve easily in water. The wet scaffolds are almost transparent and suitable for microscopic observation. U87 cells were grown directly in 24-well plates (2D environment) and different 3D scaffolds (CS scaffolds or CS-HA scaffolds), each sample was seeded with 2 104 cells and cultured in DMEM. Cells grown on 3D scaffolds had a good survival
rate even at 15 days post-seeding (Fig. 1CeH). SEM imaging showed that the morphology of cells was significantly different between 2D plates and 3D scaffolds culture (Fig. 2AeD). In 3D scaffolds, cells formed multi-cellular clusters and aggregated into tumor cell spheroids. However, cells cultured on 2D plates had an epitheliumlike morphology and grew into sheets.
Fig. 2. SEM images of U87 cells cultured on 2D well plates and 3D scaffolds. (A)e(B) Traditional 2D culture; (C)e(D) 3D scaffolds culture. Scale bars: (A)e(D) 50 mm.
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3.2. Expression of biomarkers of GSCs and EMT-related genes in CS scaffolds and CS-HA scaffolds In order to evaluate the ability of 3D scaffolds to enrich GSCs, qRT-PCR was performed on day 15 of culture. We chose CD133 and Nestin, which are recognized as biomarkers of GSCs. The results were shown in Fig. 3C, compared with 2D controls, the amount of CD133 mRNA in CS-HA scaffolds was increased by 4.25 ± 3.04, 12.56 ± 2.51 and 12.08 ± 3.41 folds on days 5, 10 and 15, respectively. On the other hand, the expression of CD133 mRNA in CS scaffolds was 3.96 ± 2.83, 7.79 ± 1.94 and 8.62 ± 2.29 folds higher than that of 2D monolayer cells at the corresponding culture time. Furthermore, Nestin expression had similar changes. It is noteworthy that the relative expression of CD133 and Nestin in CS-HA scaffolds was higher than that of CS scaffolds at day 10. Recent studies have demonstrated that EMT plays an important role in the transition of non-CSCs to CSCs [15,16]. The expressions of EMT-related genes, including CD44, HIF-1a and Snail were examined by qRT-PCR, as shown in Fig. 3C, it was observed that CD44 mRNA expression of CS-HA scaffolds was increased by 1.74 ± 1.34, 5.72 ± 3.17 and 4.62 ± 1.85 folds on days 5, 10 and 15 compared to 2D cultured cells. Notably, the amount of CD44 mRNA in CS-HA scaffolds was higher than that of CS scaffolds on day 10 and day 15 except for day 5. In addition, the expression of HIF▪1a and Snail in CS-HA scaffolds was similar to that of CD44, which increased to 2.51 ± 1.49, 23.37 ± 3.65, 17.07 ± 4.39 and 3.70 ± 2.36, 18.38 ± 3.02, 16.48 ± 2.74 folds higher than that of 2D control on days 5, 10, and 15, respectively. More importantly, it was found the relative expression of EMT-related genes in CS-HA scaffolds was higher than that of CS scaffolds. In order to further demonstrate the improvement of stemness properties after 3D scaffolds culture,
flow cytometry was used to evaluate the difference in the proportion of GSCs-like cells between 2D monolayer culture and 3D scaffolds culture on day 10. As shown in Fig. 3A, the percentage of CD133þ phenotype cells reached 36.82 ± 3.86%, 50.18 ± 5.34% in CS scaffolds and CS-HA scaffolds, respectively, whereas 2D monolayer culture was 3.34 ± 1.05%. Furthermore, the proportion of GSCs-like cells in CS-HA scaffolds was higher than 2D monolayer culture and CS scaffolds culture (Fig. 3B), which indicated that cell culture in 3D scaffolds, especially in CS-HA scaffolds have increased the proportion of stem cell-like cells.
3.3. In vivo tumorigenicity of 3D cultured cells To compare the tumorigenicity difference between cells in 3D scaffolds and 2D culture, 1 104 U87 cells harvested from 2D and 3D cultures were implanted into the flank of BALB/c nude mice on day 10, respectively and tumor formation was observed after 14 days of inoculation. As shown in Table 1 and Fig. 4A, the tumor incidence of CS-HA and CS scaffolds were 100% and 80%, respectively, which was significantly higher than the 40% of 2D. Moreover, the size of tumor formed by 3D culture cells was significantly larger than 2D cells (Fig. 4B and C).
Table 1 The tumor incidence of U87 cells under different culture conditions. Culture
Incidence
Percentage
2D CS CS-HA
2/5 4/5 5/5
40% 80% 100%
Fig. 3. Analysis of GSCs enrichment induced by 3D scaffolds culture. (A)e(B) The proportion of CD133þcells in CS-HA scaffolds was significantly higher than CS scaffolds and 2D cultures at day 10; (C) Expression of stem cell biomarkers and EMT-related genes was measured by qRT-PCR. mRNA content was calculated using expression of 2D monolayer culture at day 5 as reference. *p < 0.05, **p < 0.01, ***p < 0.001, compared with 2D cells. #p < 0.05, compared with other 3D cultures.
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Fig. 4. In vivo tumorigenicity of U87 cells cultured under different conditions. (A) Tumor formation by U87 cells cultured in CS scaffolds, CS-HA scaffolds or 2D plate; (B) Size change of tumors after inoculation of U87 cells; (C) The solid tumors harvested on day 42 showed different sizes. Scale bars: (C) 1 cm.
4. Discussion CS is obtained by deacetylation of chitin. It is the only hydrophilic cationic nature polymer, with good biocompatibility, antibacterial and biodegradability [17]. CS and its derivatives have been widely used, especially in food [18], and pharmaceutical applications, including drug delivery [19] and tissue engineering [20]. HA is a glycosaminoglycan, widely exists in the ECM of living tissue, especially in the central nervous system [21]. Due to its good biodegradability, biocompatibility and non-immunogenicity, HA has been extensively studied as a biomaterial [22]. HA has low mechanical strength and rapid bioabsorption rate, whereas CS has good mechanical properties and slow bioabsorption. Therefore, HA and CS have similarity and complementarity in structure and properties. When combining these two biopolymers, they exhibit excellent biocompatibility and physical properties [23]. At a given pH, HA is negatively charged and CS is positively charged. In this work, CS scaffolds were obtained by directly lyophilizing CS solution, and CSHA scaffolds were prepared by lyophilizing the PEC formed by these two polysaccharides. PEC is a supramolecular structure formed by ionic interactions between two oppositely charged polyelectrolytes. The SEM images revealed that cells grown on 2D plates as a flat monolayer. However, cells formed tumor spheroids in 3D scaffolds, which is more similar to the in vivo morphological structure of glioma cells and better promote cell-cell and cell-ECM interactions. These results again show that the use of 3D scaffolds can better mimic the in vivo growth environment of tumor. CD44 is a membrane glycoprotein that mediates cells to ECM, and EMT is mediated by the signal cascade induced by the interaction of CD44 with ECM [24]. The main receptor for HA is CD44, and the interaction between CD44 and HA plays a key role in GBM adhesion, proliferation and invasion by activating downstream cell signaling [25]. U87 human GBM cells cultured in 3D scaffolds, especially in CS-HA scaffolds consistently overexpressed CD44 during the 15 days compared to conventional 2D cultures. This might due to the fact that HA promoted the expression of CD44. Nestin and CD133 are widely used as biomarkers for GSCs to
determine the prognosis of GBM [26]. Compared with 2D culture, the mRNA expression of Nestin and CD133 in 3D scaffolds increased significantly. It is noteworthy that the proportion of CD133þ phenotype cells in CS-HA scaffolds was higher than that of CS scaffolds and 2D cultures, which suggested that CS-HA scaffolds culture has better capability increasing the stemness properties of GBM cells. Thus, it is possible that the change in chemical microenvironment of 3D scaffolds could change the stemness properties of cultured GBM cells. Researchers have found that CSCs exposed to hyperoxic environments will reduce their stemness properties. In order to maintain the stemness characteristics, CSCs aggregate to form cell spheroids to preserve the hypoxic environments [27]. Hypoxia tumor microenvironment can directly or indirectly promote the occurrence of EMT, and hypoxia inducible factors can be used to promote the stemness of tumor cells, causing tumor invasion and metastasis [28,29]. Particularly, HIF-1a promotes the development of EMT by activating the transcription factor Snail gene expression [30]. Targeted therapy of HIF-1a in CD133þ GSCs can reduce the survival and tumorigenesis of GBM [31]. Our study found that compared with 2D culture, CS scaffolds and CS-HA scaffolds cultured cells had significantly increased expression of HIF-1a mRNA, which was similar to previously reported results [32,33]. This suggested that the porous structure of CS and CS-HA scaffolds may form a local hypoxic environment during in vitro culture. Furthermore, the tumor cell spheroids were surrounded by a number of layers of cells to avoid the internal hypoxic environment suffered from external hyperoxia damage, which might enhance the stem cell-like characteristics of GBM [34,35]. Interestingly, HIF-1a expression in CS-HA scaffolds was also higher than that in CS scaffolds, which may be due to the fact that CS-HA scaffolds have relatively small and regular pores and have the potential to create hypoxic environments. Recently, Snail family was found to be the main regulator of EMT, it is a key factor in the EMT process [36]. In our study, compared with 2D culture, the expression of Snail mRNA in CS scaffolds and CS-HA scaffolds was upregulated at three time points. Increased expression of EMT-related genes associated with GSCs-
Please cite this article in press as: X. Wang, et al., Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.114
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like cells in 3D scaffolds indicated that 3D scaffolds might promote the conversion of non-CSCs to CSCs by EMT [16,37]. Additionally, the chemical structure of the scaffold matrix has a key effect on the promotion of GSCs-like cells. Hyaluronic acid is a natural polymer that makes up the ECM. More importantly, it is thought to activate the downstream signaling pathway by binding to the cell surface receptor to play a central role in the development of EMT [38]. This should be one of the key reasons that the expression of GBM related stem cell biomarkers in CS-HA scaffolds was higher than that of CS scaffolds. It was also shown that the 3D structure and HAmimicking ECM were necessary for the occurrence of EMT and enrichment of GSCs-like cells. Easier tumorigenesis in vitro was considered to be an important feature of CSCs [39]. Our results showed that cells derived from CSHA and CS scaffolds had a significantly higher tumorigenicity in nude mice than 2D cells. Remarkably, cells obtained from CS-HA scaffolds had greater tumorigenicity and larger tumor size compared to 2D cells or CS scaffold cells, which suggests that GSCslike cells in CS-HA scaffolds were more strongly enhanced. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This study is financially supported by the following programs: 1.Clinical Cutting-edge Technology, Social Development Projects in Jiangsu Province (BE2016668); 2.The National High Technology Research and Development Program of China (863 Program, No. 2015AA020303); 3.Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_1993); 4. The National Natural Science Foundation of China (grant nos. 81702457); 5.Suzhou Science and Technology Project (SYS201723). References [1] A. Omuro, L.M. Deangelis, Glioblastoma and other malignant gliomas: a clinical review, J. Am. Med. Assoc. 310 (2013) 1842e1850. [2] H.G. Wirsching, M. Weller, The role of molecular diagnostics in the management of patients with gliomas, Curr. Treat. Options Oncol. 17 (2016) 1e16. [3] A. Bradshaw, A. Wickremsekera, S.T. Tan, L. Peng, P.F. Davis, T. Itinteang, Cancer stem cell hierarchy in glioblastoma multiforme, Front. Sci. 3 (2016) 21. [4] T. Seymour, A. Nowak, F. Kakulas, Targeting aggressive cancer stem cells in glioblastoma, Front. Oncol. 5 (2015) 159. [5] S.E. Kelly, A.D. Benedetto, A. Greco, C.M. Howard, V.E. Sollars, D.A. Primerano, J.V. Valluri, P.P. Claudio, Rapid selection and proliferation of CD133þ cells from cancer cell lines: chemotherapeutic implications, PLoS One 5 (2010) e10035. [6] B.B. Zhou, H. Zhang, M. Damelin, K.G. Geles, J.C. Grindley, P.B. Dirks, Tumourinitiating cells: challenges and opportunities for anticancer drug discovery, Nat. Rev. Drug Discov. 8 (2009) 806e823. [7] W. Rao, S. Zhao, J. Yu, X. Lu, D.L. Zynger, X. He, Enhanced enrichment of prostate cancer stem-like cells with miniaturized 3D culture in liquid corehydrogel shell microcapsules, Biomaterials 35 (2014) 7762e7773. [8] S.J. Florczyk, F.M. Kievit, K. Wang, A.E. Erickson, R.G. Ellenbogen, M. Zhang, 3D porous chitosan-alginate scaffolds promote proliferation and enrichment of cancer stem-like cells, J. Mater. Chem. B Mater. Bio Med. 4 (2016) 6326e6334. [9] F.M. Kievit, S.J. Florczyk, M.C. Leung, K. Wang, J.D. Wu, J.R. Silber, R.G. Ellenbogen, J.S. Lee, M. Zhang, Proliferation and enrichment of CD133(þ) glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds, Biomaterials 35 (2014) 9137e9143. [10] L. Chen, Z. Xiao, Y. Meng, Y. Zhao, J. Han, G. Su, B. Chen, J. Dai, The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs, Biomaterials 33 (2012) 1437e1444. [11] B.H. Smith, L.S. Gazda, B.L. Conn, K. Jain, S. Asina, D.M. Levine, T.S. Parker, M.A. Laramore, P.C. Martis, H.V. Vinerean, E.M. David, S. Qiu, C. Cordon-Cardo, R.D. Hall, B.R. Gordon, C.H. Diehl, K.H. Stenzel, A.L. Rubin, Three-dimensional culture of mouse renal carcinoma cells in agarose macrobeadsselects for a subpopulation of cells with cancer stem cell or cancer progenitorproperties, Canc. Res. 71 (2011) 716e724. [12] C. Martínez-Ramos, M. Lebourg, Three-dimensional constructs using hyaluronan cell Carrier as a tool for the study of cancer stem cells, J. Biomed. Mater.
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Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix Xuanzhi Wang a, 1, Xingliang Dai a, 1, Xinzhi Zhang b, c, Xinda Li c, Tao Xu c, d, **, Qing Lan a, * a
Department of Neurosurgery, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, People's Republic of China Medprin Biotech GmbH, Gutleutstraße 163-167, Frankfurt am Main, D-60327, Germany c Department of Mechanical Engineering, Biomanufacturing Center, Tsinghua University, Beijing, 100084, People's Republic of China d Department of Precision Medicine and Healthcare, Tsinghua-Berkeley Shenzhen Institute, Shenzhen, 518055, People's Republic of China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2018 Accepted 14 March 2018 Available online xxx
Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies. © 2018 Elsevier Inc. All rights reserved.
Keywords: 3D porous scaffold Cancer stem cell Enrichment Glioma Chitosan Hyaluronic acid
1. Introduction Glioblastoma (GBM) is the most invasive and deadly primary tumor in the brain [1], even after total resection and adjuvant chemoradiotherapy, the prognosis for patients is still poor, and the median survival time was only 14 months after diagnosis [2]. GSCs are thought to be the source of recurrence and chemoradiotherapy resistance in GBM [3]. There are many strategies for GSCs targeted therapy, but the results are still not satisfactory [4]. Further study of
* Corresponding author. ** Corresponding author. Department of Mechanical Engineering, Biomanufacturing Center, Tsinghua University, Beijing, 100084, People's Republic of China. E-mail addresses: [email protected] (T. Xu), [email protected] (Q. Lan). 1 These authors contributed equally to this work.
GSCs is important for the treatment of glioma. However, CSCs account for only less than 1% of cancer cell population [5]. Therefore, how to successfully isolate and enrich CSCs in vitro has been a research focus. Thus far, CSCs have been isolated in vitro by using fluorescence-activated cell sorting or magnetic-activated cell sorting, and serum-free CSC culture medium has been used as the suspension culture of tumorspheres for enriching CSCs [5,6]. However, the application of these techniques often require specialized equipment and costly antibodies, time-consuming cell suspension culture, the yield of CSCs is very low [5]. Most importantly, suspension culture lacks the 3D environment required for cell-extracellular matrix (ECM) interactions to promote cancer heterogeneity [7]. Due to the limitations of traditional CSCs isolation and enrichment methods, researchers have enriched CSCs by seeding cancer cells on 3D porous scaffolds [8,9]. It was found that 3D microenvironment constructed by 3D porous scaffolds could not only promote the formation of tumor cell spheroids, but also the
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invasiveness and chemotherapeutic resistance of tumor cells cultured on 3D scaffolds were significantly improved compared with conventional 2D models [10,11]. At present, a variety of natural and synthetic scaffolds have been used to construct 3D porous scaffolds for the study of CSCs enrichment [10,12,13]. However, the most effective composition that enriches CSCs is still unclear. In this study, glioma cell line U87 cells were grown on CS and CS-HA scaffolds. Cell viability, morphology and the expression of GSC biomarkers and EMT-related genes were analyzed under different culture conditions. Finally, we evaluated the in vivo tumorigenicity of cells derived from both scaffolds and compared with that of cells cultured under 2D conditions. 2. Materials and methods 2.1. Materials Chitosan (practical grade, >90% deacetylated, MW ¼ 250,000) was purchased from Haidebei (Shandong, China) and hyaluronic acid (sodium hyaluronate) was purchased from Freda (Shandong, China). Human glioma cell line U87 was purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco) at 37 C in humidified air with 5% CO2. 2.2. Scaffolds synthesis CS scaffolds and CS-HA scaffolds were prepared based on previously reported methods [14]. CS scaffolds were obtained by dissolving chitosan in dilute acetic acid and lyophilized. CS-HA scaffolds were prepared by forming a polyelectrolyte complex (PEC) between chitosan and hyaluronic acid molecules, and then froze and lyophilize the PEC solution. Briefly, for CS scaffolds, 2 wt% chitosan and 0.5 wt% acetic acid solution were stirred for 30 min to obtain a homogeneous CS solution. For CS-HA scaffolds, 4 wt% chitosan and 1 wt% acetic acid solution were stirred for 30 min to obtain a homogeneous CS solution, then 1 wt% hyaluronic acid solution dissolved in deionized water was gradually added in at an equal volume and mixed for 3 h to obtain a homogeneous CS-HA solution. Approximately 2 mL of CS solution or CS-HA solution was filled into each well of 24-well cell culture plate, froze overnight at 20 C, 2 h at 80 C, and lyophilized for 48 h using freeze dryer (LYOQUEST-85, TELSTAR). These scaffolds were cut into 2 mm thick discs, and then neutralized with 25v% ammonium hydroxide solution for 1 h. The residual ammonium hydroxide in scaffolds was removed by repeated washing with deionized water. The neutralized CS-HA scaffolds and CS scaffolds were again froze at 20 C overnight and lyophilized for 48 h. Scaffold samples were sterilized by gamma irradiation. 2.3. Live/dead assay Cell viability was evaluated by fluorescent live/dead assay kit (KeyGEN BioTECH, Nanjing, China) according to the protocol. Briefly, medium was removed from the wells and scaffolds were washed with PBS, followed by soaking in PBS solution containing 8 mM propidium iodide and 2 mM Calcein-AM. These scaffolds were cultured for 10 min at room temperature away from light, and washed with PBS. Cells were green (live cells) or red (dead cells). Images were obtained from fluorescence microscope (Olympus IX51,Tokyo, Japan). For each sample (n ¼ 3), live and dead cells were counted at 200 in 5 random spots.
2.4. Scanning electron microscopy (SEM) analysis Cell-free scaffolds and cell-laden scaffolds were fixed in 2.5% glutaraldehyde overnight at 4 C. Samples were then dehydrated using a series of ethanol solutions (70%, 80%, 90%, 95%, 100%, and 100%) for 30 min in each solution, then critical point dried using carbon dioxide critical point dryer (LEICA, EM CPD300). Samples were sputter coated with platinum, and images were obtained from the ULTRA 55 Scanning Electron Microscope (ZEISS, Germany). 2.5. Quantitative RT-PCR Cells were detached from porous scaffolds or well plates using versene and completely dissociated with Trizol (Invitrogen, 15,596e026), the total RNA was extracted following the manufacturer's protocol. Reverse transcription was performed using ImProm-IITM Reverse Transcription System (Promega, A3800). DNA transcripts were carried out using SYBR Green qPCR Super Mix. Thermo cycling was performed using ABI PRISM® 7500 Sequence Detection System and 18srRNA was used as the housekeeping gene. Relative gene expression was calculated using 2DDCt method. 2.6. Flow cytometry analysis 2D and 3D cells were digested with trypsin at day 10 and a certain amount of single cell suspension was prepared with PBS. Cells were incubated with 10 mL anti-CD133-PE or mouse IgG-PE (Miltenyi Biotec, Bergisch Gladbach, Germany) per 106 cells in dark at room temperature for 20 min. Subsequently, cells were washed with PBS and resuspended for preparation. The proportion of CD133þ cells was analyzed with a BD FACSArial Flow Cytometer and results were evaluated by FlowJo software program (Treestar, Ashland, OR). 2.7. In vivo tumorigenicity analysis Cells (1 104) were harvested from 2D cultures or 3D scaffolds on day 10 and resuspended in 200 mL serum-free medium with 50% Matrigel (Bedford, USA). Cells were inoculated subcutaneously into the right flank of BALB/c nude mice (4e6weeks old). All animal experiments were carried out in accordance with the approved program of the Animal Care Committee of the Second Affiliated Hospital of Soochow University. Tumorigenesis and growth were monitored three times a week. Tumor size was measured with a caliper and tumor volume (V) was calculated according to the following formula: V ¼ (L W2)/2, where L is length and W is width. All tumors were harvested at 6 weeks after inoculation. 2.8. Statistical analysis Each experiment was carried out in three replicas, and all results are presented as the mean ± standard deviation. The Student's ttest was used to compare the means between two groups. Two-way analysis of variance (ANOVA) and a Bonferroni post-hoc test was used to compare multiple groups. Statistical significance was considered as *p < 0.05, **p < 0.01, and ***p < 0.001. Data analysis was performed using GraphPad Prism 7 software. 3. Results 3.1. Growth characteristics of glioma cell U87 cultured in 2D and 3D Sections and macrostructure morphology of CS scaffolds and CSHA scaffolds are shown in Fig. 1A and B. The average dimension of
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Fig. 1. Morphology of 3D porous scaffolds and live/dead assay of cell-laden scaffolds. (A)e(B) The morphology of CS and CS-HA scaffolds after lyophilization and section; (C)e(E) Cell viability after 5 days of culture; (F)e(H) Cell viability after 15 days of culture. Live cells were stained green and dead cells were stained red. Scale bars: (A)e(B) 1 cm; (C)e(H) 100 mm.
each scaffold was 13 mm in diameter and 2 mm thick. These scaffolds absorb water but do not dissolve easily in water. The wet scaffolds are almost transparent and suitable for microscopic observation. U87 cells were grown directly in 24-well plates (2D environment) and different 3D scaffolds (CS scaffolds or CS-HA scaffolds), each sample was seeded with 2 104 cells and cultured in DMEM. Cells grown on 3D scaffolds had a good survival
rate even at 15 days post-seeding (Fig. 1CeH). SEM imaging showed that the morphology of cells was significantly different between 2D plates and 3D scaffolds culture (Fig. 2AeD). In 3D scaffolds, cells formed multi-cellular clusters and aggregated into tumor cell spheroids. However, cells cultured on 2D plates had an epitheliumlike morphology and grew into sheets.
Fig. 2. SEM images of U87 cells cultured on 2D well plates and 3D scaffolds. (A)e(B) Traditional 2D culture; (C)e(D) 3D scaffolds culture. Scale bars: (A)e(D) 50 mm.
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3.2. Expression of biomarkers of GSCs and EMT-related genes in CS scaffolds and CS-HA scaffolds In order to evaluate the ability of 3D scaffolds to enrich GSCs, qRT-PCR was performed on day 15 of culture. We chose CD133 and Nestin, which are recognized as biomarkers of GSCs. The results were shown in Fig. 3C, compared with 2D controls, the amount of CD133 mRNA in CS-HA scaffolds was increased by 4.25 ± 3.04, 12.56 ± 2.51 and 12.08 ± 3.41 folds on days 5, 10 and 15, respectively. On the other hand, the expression of CD133 mRNA in CS scaffolds was 3.96 ± 2.83, 7.79 ± 1.94 and 8.62 ± 2.29 folds higher than that of 2D monolayer cells at the corresponding culture time. Furthermore, Nestin expression had similar changes. It is noteworthy that the relative expression of CD133 and Nestin in CS-HA scaffolds was higher than that of CS scaffolds at day 10. Recent studies have demonstrated that EMT plays an important role in the transition of non-CSCs to CSCs [15,16]. The expressions of EMT-related genes, including CD44, HIF-1a and Snail were examined by qRT-PCR, as shown in Fig. 3C, it was observed that CD44 mRNA expression of CS-HA scaffolds was increased by 1.74 ± 1.34, 5.72 ± 3.17 and 4.62 ± 1.85 folds on days 5, 10 and 15 compared to 2D cultured cells. Notably, the amount of CD44 mRNA in CS-HA scaffolds was higher than that of CS scaffolds on day 10 and day 15 except for day 5. In addition, the expression of HIF▪1a and Snail in CS-HA scaffolds was similar to that of CD44, which increased to 2.51 ± 1.49, 23.37 ± 3.65, 17.07 ± 4.39 and 3.70 ± 2.36, 18.38 ± 3.02, 16.48 ± 2.74 folds higher than that of 2D control on days 5, 10, and 15, respectively. More importantly, it was found the relative expression of EMT-related genes in CS-HA scaffolds was higher than that of CS scaffolds. In order to further demonstrate the improvement of stemness properties after 3D scaffolds culture,
flow cytometry was used to evaluate the difference in the proportion of GSCs-like cells between 2D monolayer culture and 3D scaffolds culture on day 10. As shown in Fig. 3A, the percentage of CD133þ phenotype cells reached 36.82 ± 3.86%, 50.18 ± 5.34% in CS scaffolds and CS-HA scaffolds, respectively, whereas 2D monolayer culture was 3.34 ± 1.05%. Furthermore, the proportion of GSCs-like cells in CS-HA scaffolds was higher than 2D monolayer culture and CS scaffolds culture (Fig. 3B), which indicated that cell culture in 3D scaffolds, especially in CS-HA scaffolds have increased the proportion of stem cell-like cells.
3.3. In vivo tumorigenicity of 3D cultured cells To compare the tumorigenicity difference between cells in 3D scaffolds and 2D culture, 1 104 U87 cells harvested from 2D and 3D cultures were implanted into the flank of BALB/c nude mice on day 10, respectively and tumor formation was observed after 14 days of inoculation. As shown in Table 1 and Fig. 4A, the tumor incidence of CS-HA and CS scaffolds were 100% and 80%, respectively, which was significantly higher than the 40% of 2D. Moreover, the size of tumor formed by 3D culture cells was significantly larger than 2D cells (Fig. 4B and C).
Table 1 The tumor incidence of U87 cells under different culture conditions. Culture
Incidence
Percentage
2D CS CS-HA
2/5 4/5 5/5
40% 80% 100%
Fig. 3. Analysis of GSCs enrichment induced by 3D scaffolds culture. (A)e(B) The proportion of CD133þcells in CS-HA scaffolds was significantly higher than CS scaffolds and 2D cultures at day 10; (C) Expression of stem cell biomarkers and EMT-related genes was measured by qRT-PCR. mRNA content was calculated using expression of 2D monolayer culture at day 5 as reference. *p < 0.05, **p < 0.01, ***p < 0.001, compared with 2D cells. #p < 0.05, compared with other 3D cultures.
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Fig. 4. In vivo tumorigenicity of U87 cells cultured under different conditions. (A) Tumor formation by U87 cells cultured in CS scaffolds, CS-HA scaffolds or 2D plate; (B) Size change of tumors after inoculation of U87 cells; (C) The solid tumors harvested on day 42 showed different sizes. Scale bars: (C) 1 cm.
4. Discussion CS is obtained by deacetylation of chitin. It is the only hydrophilic cationic nature polymer, with good biocompatibility, antibacterial and biodegradability [17]. CS and its derivatives have been widely used, especially in food [18], and pharmaceutical applications, including drug delivery [19] and tissue engineering [20]. HA is a glycosaminoglycan, widely exists in the ECM of living tissue, especially in the central nervous system [21]. Due to its good biodegradability, biocompatibility and non-immunogenicity, HA has been extensively studied as a biomaterial [22]. HA has low mechanical strength and rapid bioabsorption rate, whereas CS has good mechanical properties and slow bioabsorption. Therefore, HA and CS have similarity and complementarity in structure and properties. When combining these two biopolymers, they exhibit excellent biocompatibility and physical properties [23]. At a given pH, HA is negatively charged and CS is positively charged. In this work, CS scaffolds were obtained by directly lyophilizing CS solution, and CSHA scaffolds were prepared by lyophilizing the PEC formed by these two polysaccharides. PEC is a supramolecular structure formed by ionic interactions between two oppositely charged polyelectrolytes. The SEM images revealed that cells grown on 2D plates as a flat monolayer. However, cells formed tumor spheroids in 3D scaffolds, which is more similar to the in vivo morphological structure of glioma cells and better promote cell-cell and cell-ECM interactions. These results again show that the use of 3D scaffolds can better mimic the in vivo growth environment of tumor. CD44 is a membrane glycoprotein that mediates cells to ECM, and EMT is mediated by the signal cascade induced by the interaction of CD44 with ECM [24]. The main receptor for HA is CD44, and the interaction between CD44 and HA plays a key role in GBM adhesion, proliferation and invasion by activating downstream cell signaling [25]. U87 human GBM cells cultured in 3D scaffolds, especially in CS-HA scaffolds consistently overexpressed CD44 during the 15 days compared to conventional 2D cultures. This might due to the fact that HA promoted the expression of CD44. Nestin and CD133 are widely used as biomarkers for GSCs to
determine the prognosis of GBM [26]. Compared with 2D culture, the mRNA expression of Nestin and CD133 in 3D scaffolds increased significantly. It is noteworthy that the proportion of CD133þ phenotype cells in CS-HA scaffolds was higher than that of CS scaffolds and 2D cultures, which suggested that CS-HA scaffolds culture has better capability increasing the stemness properties of GBM cells. Thus, it is possible that the change in chemical microenvironment of 3D scaffolds could change the stemness properties of cultured GBM cells. Researchers have found that CSCs exposed to hyperoxic environments will reduce their stemness properties. In order to maintain the stemness characteristics, CSCs aggregate to form cell spheroids to preserve the hypoxic environments [27]. Hypoxia tumor microenvironment can directly or indirectly promote the occurrence of EMT, and hypoxia inducible factors can be used to promote the stemness of tumor cells, causing tumor invasion and metastasis [28,29]. Particularly, HIF-1a promotes the development of EMT by activating the transcription factor Snail gene expression [30]. Targeted therapy of HIF-1a in CD133þ GSCs can reduce the survival and tumorigenesis of GBM [31]. Our study found that compared with 2D culture, CS scaffolds and CS-HA scaffolds cultured cells had significantly increased expression of HIF-1a mRNA, which was similar to previously reported results [32,33]. This suggested that the porous structure of CS and CS-HA scaffolds may form a local hypoxic environment during in vitro culture. Furthermore, the tumor cell spheroids were surrounded by a number of layers of cells to avoid the internal hypoxic environment suffered from external hyperoxia damage, which might enhance the stem cell-like characteristics of GBM [34,35]. Interestingly, HIF-1a expression in CS-HA scaffolds was also higher than that in CS scaffolds, which may be due to the fact that CS-HA scaffolds have relatively small and regular pores and have the potential to create hypoxic environments. Recently, Snail family was found to be the main regulator of EMT, it is a key factor in the EMT process [36]. In our study, compared with 2D culture, the expression of Snail mRNA in CS scaffolds and CS-HA scaffolds was upregulated at three time points. Increased expression of EMT-related genes associated with GSCs-
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like cells in 3D scaffolds indicated that 3D scaffolds might promote the conversion of non-CSCs to CSCs by EMT [16,37]. Additionally, the chemical structure of the scaffold matrix has a key effect on the promotion of GSCs-like cells. Hyaluronic acid is a natural polymer that makes up the ECM. More importantly, it is thought to activate the downstream signaling pathway by binding to the cell surface receptor to play a central role in the development of EMT [38]. This should be one of the key reasons that the expression of GBM related stem cell biomarkers in CS-HA scaffolds was higher than that of CS scaffolds. It was also shown that the 3D structure and HAmimicking ECM were necessary for the occurrence of EMT and enrichment of GSCs-like cells. Easier tumorigenesis in vitro was considered to be an important feature of CSCs [39]. Our results showed that cells derived from CSHA and CS scaffolds had a significantly higher tumorigenicity in nude mice than 2D cells. Remarkably, cells obtained from CS-HA scaffolds had greater tumorigenicity and larger tumor size compared to 2D cells or CS scaffold cells, which suggests that GSCslike cells in CS-HA scaffolds were more strongly enhanced. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This study is financially supported by the following programs: 1.Clinical Cutting-edge Technology, Social Development Projects in Jiangsu Province (BE2016668); 2.The National High Technology Research and Development Program of China (863 Program, No. 2015AA020303); 3.Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_1993); 4. The National Natural Science Foundation of China (grant nos. 81702457); 5.Suzhou Science and Technology Project (SYS201723). References [1] A. Omuro, L.M. Deangelis, Glioblastoma and other malignant gliomas: a clinical review, J. Am. Med. Assoc. 310 (2013) 1842e1850. [2] H.G. Wirsching, M. Weller, The role of molecular diagnostics in the management of patients with gliomas, Curr. Treat. Options Oncol. 17 (2016) 1e16. [3] A. Bradshaw, A. Wickremsekera, S.T. Tan, L. Peng, P.F. Davis, T. Itinteang, Cancer stem cell hierarchy in glioblastoma multiforme, Front. Sci. 3 (2016) 21. [4] T. Seymour, A. Nowak, F. Kakulas, Targeting aggressive cancer stem cells in glioblastoma, Front. Oncol. 5 (2015) 159. [5] S.E. Kelly, A.D. Benedetto, A. Greco, C.M. Howard, V.E. Sollars, D.A. Primerano, J.V. Valluri, P.P. Claudio, Rapid selection and proliferation of CD133þ cells from cancer cell lines: chemotherapeutic implications, PLoS One 5 (2010) e10035. [6] B.B. Zhou, H. Zhang, M. Damelin, K.G. Geles, J.C. Grindley, P.B. Dirks, Tumourinitiating cells: challenges and opportunities for anticancer drug discovery, Nat. Rev. Drug Discov. 8 (2009) 806e823. [7] W. Rao, S. Zhao, J. Yu, X. Lu, D.L. Zynger, X. He, Enhanced enrichment of prostate cancer stem-like cells with miniaturized 3D culture in liquid corehydrogel shell microcapsules, Biomaterials 35 (2014) 7762e7773. [8] S.J. Florczyk, F.M. Kievit, K. Wang, A.E. Erickson, R.G. Ellenbogen, M. Zhang, 3D porous chitosan-alginate scaffolds promote proliferation and enrichment of cancer stem-like cells, J. Mater. Chem. B Mater. Bio Med. 4 (2016) 6326e6334. [9] F.M. Kievit, S.J. Florczyk, M.C. Leung, K. Wang, J.D. Wu, J.R. Silber, R.G. Ellenbogen, J.S. Lee, M. Zhang, Proliferation and enrichment of CD133(þ) glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds, Biomaterials 35 (2014) 9137e9143. [10] L. Chen, Z. Xiao, Y. Meng, Y. Zhao, J. Han, G. Su, B. Chen, J. Dai, The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs, Biomaterials 33 (2012) 1437e1444. [11] B.H. Smith, L.S. Gazda, B.L. Conn, K. Jain, S. Asina, D.M. Levine, T.S. Parker, M.A. Laramore, P.C. Martis, H.V. Vinerean, E.M. David, S. Qiu, C. Cordon-Cardo, R.D. Hall, B.R. Gordon, C.H. Diehl, K.H. Stenzel, A.L. Rubin, Three-dimensional culture of mouse renal carcinoma cells in agarose macrobeadsselects for a subpopulation of cells with cancer stem cell or cancer progenitorproperties, Canc. Res. 71 (2011) 716e724. [12] C. Martínez-Ramos, M. Lebourg, Three-dimensional constructs using hyaluronan cell Carrier as a tool for the study of cancer stem cells, J. Biomed. Mater.
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Please cite this article in press as: X. Wang, et al., Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.03.114