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Reconstituting Glioma Perivascular Niches on a Chip for Insights into Chemoresistance of Glioma Caihou Lin, L Lin, Sifeng Mao, Lijuan Yang, Linglu Yi, Xuexia Lin, Junming Wang, Zhixiong Lin, and Jin-Ming Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02133 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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Analytical Chemistry

Reconstituting Glioma Perivascular Niches on a Chip for Insights into Chemoresistance of Glioma Caihou Lin,†,§,#,‡ Ling Lin, ,‡ Sifeng Mao,†,‡ Lijuan Yang, Linglu Yi, † Xuexia Lin,† Junming Wang,† Zhi-Xiong Lin,§,* and Jin-Ming Lin†,* ∥

⊥

†

Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China. § Department of Neurosurgery, the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350005, China. ∥

CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China. ⊥

#

Department of Pharmacology, Fujian Medical University, Fuzhou, Fujian 350005. Department of Neurosurgery, Fujian Medical University Union Hospital, Fuzhou, Fujian 350001, China.

ABSTRACT: In this work, we report the directly diagnosing chemoresistance of glioma stem cells (GSCs) during the chemotherapy on a biomimetric microsystem that reconstitutes glioma perivascular niches on a chip. Glioma stem cells and endothelial cells were specially co-cultured onto the biomimetric system to precisely control stem cell co-culture for the proof-ofprinciple studies. The expression levels of 6-O-methylguanine was confirmed by mass spectrometer, and Bmi-1 gene was also investigated to uncover the chemoresistence of GSCs. The results demonstrated that the formation of perivascular niches effectively maintains the glioma stem cells at a pluripotent status owing to their successful cellular interactions. A stronger chemoresistance of gliomas stem cells was confirmed by the formation of the GSCs neurosphere, the expression levels of 6-O-methylguanine and Bmi1 gene. The vital role of endothelial cells in chemoresistance was demonstrated. The chemoresistence reported in this work will contribute the glioma therapy.

Emerging evidence reveals that GSCs are responsible for tumor propagation even after chemotherapy1. Glioma perivascular niches composed of endothelial cells are wellknown to provide tumor cellular microenvironment for stem cell self-renewal2. Previous studies report that endothelial cells in the perivascular niche may control GSCs maintenance and promote the GSC radio-resistance owing to their ability to activate the Notch and SHH signalling pathways3-9. Traditional cell culture in vitro cannot mimic an efficient cellular microenvironment, whereas animal tests suffer from the disadvantages of high costs, which are also difficult to directly observe the real biological phenomena. Thus, it is highly significant to mimic the organization and complexity of the perivascular niches in vitro. However, the traditional methods are greatly challenged because cell culture on a standard culture dishes cannot effectively mimic the cellular microenvironment in vivo and mostly lose the real tissue functions that occurs in human body, and it is still lack of experimental model systems to reconstitute the glioma perivascular microenvironment with spatial-temporal precision10. Development of microfluidic technologies is increasingly emerging as a powerful strategy to mimic precisely controlled cellular microenvironment, enabling in-situ dynamically monitor of the biological process11-15. Recently, microfluidic

chip creates considerable opportunities for stem cell studies, and provides novel and preferable methods to mimic the cellular microenvironment through constructing well-defined architectures16-19. For example, thousands of microwells in microfluidics were designed to culture single hemopoietic stem cells for studying their gene heterogeneity 20. Significantly, Ingber et al describes a biomimetric microsystem approach to fabricate a multifunctional microdevice that is capable of reproducing fundamental functional units of the living lung, containing key structural, functional, and mechanical properties of human alveolarcapillary interface.21 Niche-on-a-chip was recently reported for glioma studies.22-26 To date, it is of great interest and significance to direct insight into clinical chemoresistance by reconstituting biomimetric system on microfluidic chips. The chemoresistance and the key factor for chemoresistance remain vital issues. In this paper, we report the chemoresistance of GSCs by combination of LC-MS, immunostaining and RT-PCR assay. The bioinspired microdevice was designed to reproduce integrated glioma perivascular niches for studying the chemoresistance on glioma stem cell that is critically important in clinical therapy. The achieved continuous perfusion allows to reduce the flow shear stress and provide sufficient nutrition that are strongly required by glioma stem

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temperature before the cells were seeded. The HBMECs and Eahy926 cells were trypsinized and resuspended at nearly density of 1.26 × 106 cells/ml before seeding. A 3 μL cell suspension was added into the microchannels inlet with a pipe filling in the outlet. Then, co-culture medium was gently injected into the channels, and the inlet and outlet were covered with pipe and additional media. Finally, the device was placed in a 37 ℃ humidified incubator with 5 % CO2. The medium was changed every 12 h. Since the cells with diameters in the range of 16-22 μm is larger than the 3 μm polycarbonate membrane, different types of cells could not hybridize in the cell seeding step. Following, experiments were carried out within 3 days after cell seeding.

cells and beneficial to mimic real stem cell niches in the normal tissue. Our experimental results demonstrated that the GSCs and endothelial cells were successfully co-cultured on a porous membrane-integrated microdevice, which was further established the feasibility of dynamically monitoring cell-cell commutation. Then, this biomimetric system was achieved to explore the chemoresistance of glioma stem cell in the artificial glioma perivascular niches. Temozolomide (TMZ), extensively used as a standard chemotherapy for patients with malignant gliomas 1,7, was introduced into the microdevices to mimic and establish models of chemotherapy. Consequently, this technology facilitated dynamically observing the chemoresistance of TMZ on glioma stem cells protected by endothelial cells and investigating the molecular metabolism by using mass spectrometry27,28. Besides, several key biomarkers indicated chemoresistance were successfully monitored in the biomimetric system to explain the biological mechanism. The role of endothelial cells in chemoresistance was successfully demonstrated.

Real-time PCR analysis for MGMT, Bim, Sox2 mRNA levels for GSCs and tube formation assay for HBMECs in vitro. After cultured for 72 h, the GSCs were trypsinized to get RNA. Total RNA was isolated and purified by E. Z. N. A. Total RNA Ki t II (Omega). RNA yield was determined by spectroscopy. Complementary DNA (cDNA) was made from 1-5 μg of total RNA using TIANScript RT Kit (Tiangen Biotech (Beijing) Co., LTD.). Real-time PCR was done on StepOnePlusTM system (Life Technologies) by SuperRealPreMix Plus with SYBR Green (Tiangen Biotech (Beijing) Co., LTD.). Briefly, reaction volume was 20 μl and contained 2× Super Real PreMix Plus, 50× ROX Reference Dye, 10-100 pg of cDNA template and 0.2-1.0 μM of primer. The following primers were designed for MGMT, forward 5’GTTTTCCAGCAAGAGTCGTTCA-3’ and reverse 5’CAGGATTGCCTCTCATTGCTC-3’; for Bim1, forward 5’GACAAATGCTGGAGAACTGGA-3’ and reverse 5’GGCAAACAAGAAGAGGTGGA-3’; for Sox2, forward 5’GCCGAGTGGAAACTTTTGTC-3’ and reverse 5’GGCAGCGTGTACTTATCCTTCT-3’. All the primers were synthesized by Sangon Biotech. LTD. (China). The thermocycling conditions were as follows: 95 ℃ for 10 min, followed by 40 cycles at 95 ℃ for 10 s, 55 ℃ for 30 s, 72℃ for 30 s. Tube formation assay for HBMEC was performed in matrigel29.

EXPERIMENTAL SECTION

Fabrication of microfluidic devices. The microfluidic device was designed and fabricated with alignment and permanent bonding of a 3 μm (pores) polycarbonate (PC) membrane between two PDMS layers with microchannels (Supplementary Fig. 1). The design and fabrication details are elaborated in the Supporting Information. Cell culture. GSCs derived from GBM patients, U251 cell strain and SU3-RFP (The Second Affiliated Hospital of Soochow University) were cultured and maintained in DMEM/F12 (Corning) supplemented with 20 ng/ml epidermal growth factor (EGF; Pepro Tech), 20 ng/ml FGFb (Pepro Tech), 2% B27 (Invitrogen). Human brain microvascular endothelial cells (HBMECs) and fusion cell line Eahy926 were purchased from Sciencell Corporation and ATCC respectively. HBMECs were maintained in ECM complete medium (ScienCell Corporation) which containing ECM medium supplemented with 5% FBS, 1% penicillinstreptomycin (PS, Invitrogen) and 1% endothelial cell growth supplement (ECCS), and Eahy926 were cultured in Medium RPMI 1640 (RPMI 1640, Invitrogen) with 10% FBS and 1% penicillin-streptomycin (PS, Invitrogen). All cells were cultured at 37 ℃ in a humidified incubator containing 5% CO2. For co-culture system, the attached endothelial cells were seeded on the top channel with GSCs on the bottom channel in microfluidics chip and maintained in the co-culture medium which contains serum-free ECM complete medium supplemented with 20 ng/ml of EGF, 20 ng/ml of FGFb, and 1% of PS. For co-culture in the microfluidics chip, all cells were then cultured on it at the density of 1.26 × 106/ml. The GSCs were utilized respectively after co-culture for 72 h.

Drug exposure and acid hydrolysis of alkylated DNA. For TMZ treatment, cells were exposed to 400 μM, 800 μM and 1,200 μM. After cultured for 72 h, the activity of GSCs were observed after treating with Calcein AM/EthD-1 by confocal laser scanning microscope. The medium was changed every 12 h to make the drug level constantly. The GSCs were obtained from the microfluidics chip 72 h after exposed to TMZ, and DNA was extracted from GSCs for follow-up study. Total DNA was isolated and purified by TIANamp Genomic DNA kit (DP 304-02). DNA yield was determined by spectroscopy. DNA solution 30 μl was mixed 30 μl HPLC grade water and 6 μl formic acid (90% in water) was added to the mixture. After mixing, the mixture was heating at 85 ℃ for 60 min by digital dry baths and then cool down to room temperature. The hydrolytes were used for quantitation of 6O-MeG by LC/MS immediately.

Co-culture of different endothelial cells and GSCs on the microfluidic device. Immunofluorescent staining of GSCs and GFP transduction were stated in the supporting information (Supplementary Fig. 2). After the HBMECs or Eahy926 cells were seeded on the top microchannels, the GSCs were cultured on the bottom microchannels one day latter. For the endothelial cells growing better, the fibronectin was placed on the microchannels for 12 h, rinsed with PBS buffer and coculture medium for three times each, and then dried at room

LC/MS Conditions. Analysis was performed on single valve two column - reverse flushing mode, including a pretreatment column (PC hilic S5 2.0 mm× 150 mm) and an analytical column (PC hilic S 52.0 mm× 50 mm) from Shiseido Co Ltd.

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Analytical Chemistry composed of three parallel channels on a single chip (Fig. 1b, c and Supplementary Fig. 2); each top-bottom channel was separated by a polycarbonate porous membrane between two PDMS microchannels that was capable of precisely controlling stem cell co-cultures. The endothelial cells and glioma stem cells can be respectively introduced into the upbottom channels and were cultured on the membrane and bottom channel. Specially, the porous property and the 10 µm thickness of porous membrane will allow free exchange of signal molecules from the neighboring channels, thus mimicking the microenvironment for cellular interactions. After the cells were grown to confluence, the compartmentalized microchannels in the microdevice makes it possible to more precisely mimic the perivascular niche of glioma tumor microenvironment, allowing dynamically observing the changes of cells, as well as delivery of drugs and nutrients to both of cells.

Mobile phases consisted of pump A (0.1% formic acid, 90% acetonitrile) and pump B (0.1% formic acid, 70% acetonitrile). The flow rate was 0.2 ml/min. MS detection was done in the positive ESI mode on AB SCIENCE QTRAP 5,500. 6.0 (Media Cybernetics, USA). Methylation detection on microchip electrophoresis (MCE) and High-resolution melting (HRM) analysis. For MGMT published primer30 sequences were used. PCR was performed on Gradient Cycler (Eppendorf, Hamburg, Germany). All amplified DNAs were analyzed by themicrochip electrophoresis system according to our prior report31. HRM analysis was performed on LightCycler 480 (Roche).The details are elaborated in Supporting Information. RESULTS AND DISCUSSION

Design of Biomimetric Microdevices for Mimicking Perivascular Niches. In normal perivascular niches, glioma (stem) cells reside in the microenvironment of tumor vasculature that composed of endothelial cells (Supplementary Fig. 1). Glioma (stem) cells are tightly grown to the neighboring endothelial cells, enabling to normal cell-cell communications. The tumor vasculature supplies sufficient cytokines and essential drugs for glioma stem cells (Fig. 1a). Base on this principle, we specially design a biomimetric microdevice for mimicking the glioma perivascular niches after chemotherapy in clinical. This biomimetric system

Reconstitution of Glioma Perivascular Niches on Cell Cocultured Microdevices. In the glioma microenvironment, perivascular niches regulate transport of soluble factors, cytokines and secreted proteins from neighbouring cells for the spatial and temporal control of glioma cell growth32-34. To reconstitute this experimental model system, after the stem cell pluripotency of GSCs was confirmed by CD133 and Nestin identification (Supplementary Fig. 3), the GSCs and endothelial cells (HBMECs or Eahy926) were respectively introduced into the up-bottom channels. After they attached onto the ECM-coated microchannels, cell viabilities and proliferation were evaluated by Calcein AM/EthD-1 (cell live/dead kit) staining. Supplementary Fig. 2 g-j show excellent cell viabilities when GSCs and HBMECs (or Eahy926) were co-cultured in the microfluidic systems. To observe the cellular morphology in the biomimetric system, green fluorescence protein expressed HBMECs (GFP-

Figure 2. Cellular viability and 3D morphology of glioma stem cells and endothelial cells on the microdevice. (a-b) The cell viabilities of HBMECs and GSCs were conducted by GFP or RFP at day 3; scale bar, 100 μm. (c-e) 3D morphology of the cell co-cultures was conducted by 3D scan confocal microscopy; scale bar, 100 μm. (f-g) The GFP-HBMECs cells migrated to the bottom-layer of GSCs due to the inspired cellular interactions; scale bar, 100 μm. (h) The different distance between HBMECs and GSCs by 3D scan confocal microscopy in different times after co-cultured.

Figure 1. Biomimetric design of miniaturized artificial perivascular niche on a chip. (a) During chemotherapy process in gliomas, chemoresistance in perivascular niche composed of glioma cells and endothelial cells. (b) GSCs and endothelial cells were respectively co-cultured in the topbottom channels of a porous membrane-integrated biomimetric microdevice. (c) After the GSCs and endothelial cells were co-cultured on microfluidic chip, relative metabolites were analyzed by LC-MS.

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Figure 3. Characterization of cellular morphology between GSCs and endothelial cells on the biomimetric microsystems. (ac) endothelial cells promote the tumor spheres of GSCs in microfluidics; (d-g) Formation of the vascular structure of HBMECs and HBMECs/GSC cultured on Matrix gel by transwell or microfluidic. (h-l) The Nestin protein expression in HBMECs with or without GSCs in microfluidic and transwell. Scale bars, 100 μm (a-e). HBMECs) and red fluorescence protein expressed GSCs (RFP-GSCs) were used for dynamically tracking of cellular growth and co-culture (Fig. 2). Figure. 2a and 2b shows obvious cellular proliferation when cultured in the microdevice for 3 days. These results indicate that the designed microsystem can provide an excellent microenvironment for cell growth and co-culture. Further, the 3D morphology of cell co-culture between GFP-HBMECs and RFP-GSCs were characterized after 3 days by laser confocal fluorescence microscopy. We obviously observe the distance between HBMECs and GSCs is become shorter as the time longer due to the inspired cellular interactions when the pore size in the porous membrane is 0.4 μm (Fig. 2c-e and 2h). Furthermore, when the pore size in the porous membrane is 12 μm, the GFP-HBMECs migrated into the neighboring layer of the GSCs (Fig. 2f and 2g). This result suggests the successful reconstitution of glioma perivascular niches on cell cocultured microdevices, which will be suitable for further insight into the chemoresistance of glioma stem cells.

GSCs were aggregated as tumor neurospheres which was recognized as the behavior of stem cell pluripotency (Fig. 3b and 3c). Besides, we also find that the GSCs are capable of promoting the formation of vascularstructure of the endothelial cells, especially of HBMECs (Fig. 3d-g). Compared to the HBMECs cultured itself, the co-cultured HBMECs can express the stem cell pluripotency-relative biomarkers such as nestin proteins especially in microfluidic system while the HBMECs (Fig. 3h-l). These results demonstrated the successful achievement of cellular interactions on the cell co-culture system. Furthermore, the mRNA expressions of Bmi-1, SOX2 and MGMT in GSCs were examined to evaluate the stem cell pluripotency. Compared to the traditional system such as transwell, the mRNA expressions of Bmi-1, CD133, Nestin, SOX2 and MGMT in GSCs and the protein expression of CD133 and Nestion in GSCs are significantly increased (Fig 4). As a result, the biomimetric cellular interactions were successfully confirmed by the co-culture of GSCs and the endothelial cells. This evidence also suggests that the formation of perivascular niches can more effectively maintain the glioma stem cells at a pluripotent status compared to the traditional method35. Thus, the reconstitution of the perivascular niches on the biomimetric microsystems will be used to study the chemoresistance of glioma stem cells.

Biomimetric Cellular Interactions on the Cell Co-culture System. The cellular interactions between GSCs and neighboring endothelial cells play key roles in regulating the tumor microenvironment. Thus, in this study, the co-culture of GSCs and endothelial cells were conducted to achieve a biomimetric model system and precisely mimic perivascular niches. Our experiments clearly show that, compared with GSCs culture only (Fig. 3a), the endothelial cells co-cultured

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Figure 4. Comparative analysis of GSCs associated gene in different co-culture methods. (a) How to characterize the mRNA of stem associate gene of GSCs. (b)The mRNA expression of CD133, Nestin, Bmi-1, MGMT and Sox-2 mRNA in GSCs in microfluidic compare to it in transwell. The same condition of transwell was define as 1. Immuno-fluorescence Investigation of Glioma Stem Cell Chemoresistance on Biomimetric Microsystems. TMZ is commonly used to perform chemotherapy for patients with malignant gliomas. However, the chemoresistance of TMZ on glioma cells is still an important factor in the therapy failure. For this reason, we contributed our efforts to study this issue by developing biomimetric systems on microdevices for insights into the biological mechanism of TMZ’s chemoresistance. Firstly, we investigated the effect of TMZ on GSCs in the biomimetric systems and we found the TMZ that 800 μM of TMZ led to 50% death rate of GSCs (Supplementary Fig. 5). After the co-culture of GSCs and the endothelial cells for 72 h, we find that the cell survival rate of co-cultured GSCs are stronger than that of GSCs culture only (Supplementary Fig. 5). It is worth mentioning that CD133 protein and Nestin protein are two well-known biomarkers for correlating with chemoresistance of GSCs1. Thus, immunefluorescence staining was conducted to quantify the expression of CD133 and Nestin on the GSCs (Fig. 5). After the co-culture of GSCs and the endothelial cells for 72 h, we find that the expresses of CD133 and Nestin on co-cultured GSCs are stronger than that of GSCs culture only (Fig. 5). The results might be reasonable because the reconstituted perivascular niches provide the microenvironment chemoresistance of GSCs. Additionally, the chemoresistance of GSCs were identified by the formation of GSCs neurosphere after the treatment of 0 μM and 800 μM TMZ, respectively. Our results show that the formation of the GSCs neurosphere was destroyed when the treatment of TMZ on the GSCs (Supplementary Fig. 6). Our experiments confirmed that the co-culture of GSCs with the endothelial cells led to stronger chemoresistance than the GSCs without co-cultured with the endothelial cells (Fig. 5, Supplementary Fig. 6). Thus, the co-culture of the GSCs and the endothelial cells lead to the chemoresistance of the GSCs against the TMZ.

Figure 5. Comparative analysis of GSCs associated proteins between the GSCs and GSCs/HBMECs with treatment of TMZ. (a-f) Fluorescent imaging of GSCs and GSCs/HBMECs with 400 μM of TMZ after 3 days. (e) Statistic analysis of ration of CD133 positive cells and Nestin positive cells in GSCs. Scale bars, 100 μm (a-d). * means P＜ 0.05, # means P＜0.01, & means P≧0.05. Molecular Mechanism Analysis of GSCs Chemoresistance. Previous studies reported that 6-O-methylguanine (6-O-MeG) and 7-methylguanine (7-MeG) are the two important biomarkers of chemoresistance diagnosis when the GSCs were treated by TMZ (Fig. 6). In our study, microfluidic device were coupled with LC/MS system to achieve the quantitative detection of 6-O-MeG and 7-MeG. After the co-culture of GSCs and the endothelial cells was exposed into TMZ for 72 h, DNA was extracted from GSCs and then acid hydrolysis of DNA was conducted. The amounts of 6-O-MeG and 7-MeG of GSCs were successfully analyzed by qualitative and quantitative analysis using LC/MS at the mode of precursor product ion multiple-reaction monitoring (MRM) transition (m/z 166.2>149.2) (Fig. 6). As shown in Fig. 6, the expression levels of 6-O-MeG and 7-MeG from co-cultured GSCs are obviously lower than that from GSCs culture only. The amount of 6-O-MeG and 7-MeG at different concentrations of

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Figure 6. Analysis of 6-O-methylguanine and 7-methylguanineat different concentrations of TMZ using LC/MS. (a, b) Product ion spectra of 6-O-MeG and 7-MeG. (c) Chromatogram of 6-O-MeG and 7-MeG. (c) The retention times of 6-O-MeG and 7-MeG at 4.56 min and 4.72 min, respectively. (d) The linear analysis of 6-O-MeG and 7-MeG with a limit of detection of 30 pg/mL and 62.5 pg/mL, respectively. (d) Metabolic analysis of 6-O-MeG and 7-MeG from the GSCs at 200 μM TMZ. TMZ (400 µM, 800 µM and 1200 µM) were detected when the GSCs were cultured in the different perivascular niche. Additionally, we also find the methylation of MGMT is consistent with the amount of 6-O-MeG and 7-MeG by high resolution melting (HRM) and microchip electrophoresis system (Supplementary Fig. 7). Combined with the genetic engineering of Bmi-1 in GSCs, we also found that the Bmi-1 may play a key role in the process of endothelial promoting the chemoresistance of GSCs (Supplementary Fig. 8-11). As a result, we inferred that the endothelial cells may contribute to enhance the chemoresistance of GSCs in perivascular niche. In this artificial perivascular niche, the successful 3D morphology of the endothelial cells and GSCs co-cultures was reconstituted to mimic the cellular communications between GSCs and neighboring endothelial cells just like the glioma

model in vitro. We found that the endothelial cells are capable of promoting co-cultured GSCs the aggregation of GSCs neurospheres. Simultaneously, the vascularstructure also interestingly occurred when the endothelial cells were cocultured with GSCs. Furthermore, the gene expresses of stem cell pluripotency-relative biomarkers especially Bmi-1 was up-regulated when the GSCs were co-cultured with the endothelial cells. The simple and effective approach of artificially controlled is directly applicable to explore the chemoresistance in clinical chemotherapy of gliomas. TMZ is commonly used DNA alkylating agent of guanine for gliomas chemotherapy by inducing mis-pairing and DNA damage of GSCs. Chemoresistance of TMZ was demonstrated for investigation of biological mechanism in therapy failure. Our experimental results have clearly confirmed the effective

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Analytical Chemistry Neurosurgery Department of The Second Affiliated Hospital of Soochow University) and Peisen Yao (The First Affiliated Hospital of Fujian Medical University) for assistance with cell culture. This work was financially supported by National Natural Science Foundation of China (Nos. 21435002, 21727814, 21621003) and National Key R&D Program of China (2017YFC0906800).

promotion role of perivascular niche during the chemoresistance process by quantify the expression of CD133 and Nestin on the GSCs. The amount of 6-O-methyl-guanine, 7-guanine and 6-O-methyl-guanine-DNA methyl-transferase also inferred that the endothelial cells may contribute to enhance the chemoresistance of GSCs in perivascular niche. CONCLUSIONS

REFERENCES

In conclusion, we have successfully uncovered the chemoresistance of GSCs on a biomimetric system using the technologies of RT-PCR, immunostaining, and LC-MS. The bioinspired microdevice was designed for co-culture of GSCs and the endothelial cells, mimicking the microenvironement of gliomas. Our results demonstrated that the formation of perivascular niches can effectively maintain the glioma stem cells at a pluripotent status owing to their cellular interactions. Significantly, after the treatment of the TMZ, deep insights into chemoresistance of GSCs were achieved to explore the biological mechanisms during chemotherapy. We concluded that the co-culture of GSCs with the endothelial cells led to stronger chemoresistance than the GSCs without co-cultured with the endothelial cells, which were confirmed by the formation of the GSCs neurosphere, the expression levels of 6O-MeG and 7-MeG. The contribution of endothelial cells to chemoresistance was demonstrated.

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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Nestin-positive tumor cells are colocalized with bmi-1-expressing cells in primary GBM and have been in the perivascular; fabrication of co-cultured microfluidic chip; identification of CD133 /Nestin and screening of RFP-GSCs and GFP-ECs; effect of TMZ in different concentration for GSCs/HBMECs; high resolution melting (HRM) analysis and Methylation identification by microchip electrophoresis (MCE); the genetic engineering of Bmi-1 in the glioma stem cells; endothelial cells promote the Bmi-1 expression in glioma stem cells; Bmi-1 expression is associated with the methylation of MGMT of GSCs (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Fax/Tel: +86 10 62792343. *E-mail: [email protected].

Author Contributions ‡

C.L., L.L., S.M.: These authors contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We thanks Yanli Guo, Qian Yang (The Forntier Science Department of Shiseido China Co, ltd) and Ying Zhang (Department of Chemistry, Tsinghua University) for assistance with MS analysis. We also thank Jun Dong, Xinliang Dai (The

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We have successfully uncovered the chemoresistance of GSCs on a biomimetric system using the technologies of RT-PCR, immunostaining, and LC-MS.

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Reconstituting Glioma Perivascular Niches on a ... - ACS Publications

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