- Email: [email protected]
Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C Yuan Tian a, Zhixing Wang a, Yuxin Wang a, Bin Yin a, Jiangang Yuan a, Boqin Qiang a, Wei Han a, *, Xiaozhong Peng a, b, ** a
State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Medical Primate Research Center, Neuroscience Center, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005, China b Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2020 Accepted 4 February 2020 Available online xxx
Extracellular vesicles (EV), as the intercellular information transfer molecules which can regulate the tumor microenvironment, promote migration and tumor progression. Previous studies reported that EV from endothelial cells was used to guide the fate and survival of gliomas, but many researches focus on normal human endothelial cells (NhEC) rather than tumor-derived endothelial cells. Our laboratory isolated human endothelial cells from glioma issue (GhEC). We have previously demonstrated that EV from GhEC and NhEC, which both can promote glioma stem cells (GSC) proliferation and tumorsphere formation in vitro and tumourigenicity in vivo by the transfer of CD9. However, NhEC-EV or GhEC-EV could suppress glioma cells (GC) proliferation in vitro. It demonstrates the undifferentiated impact of EV. Here, we first compared GhEC-EV proteins with NhEC-EV (Screening criteria: GhEC-EV/NhEC-EV, FC > 1.5), and obtained 70 differential expression proteins, most of which were associated with invasion and migration. We found that GhEC or GhEC-EV preferred promoting GC migration than treating with NhEC or NhEC-EV. In terms of mechanism, we further revealed that EV-mediated transfer of MYO1C induced glioma cell LN229 migration. Knockdown of MYO1C in GhEC or GhEC-EV suppressed this effect. Overexpression of MYO1C promoted migration on the contrary. MYO1C was also detected in glioma cerebrospinal fluid (CSF), which is more suitable as a liquid biopsy biomarker and contributes to early diagnosis and monitoring in glioma. Our findings provide a new proteindMYO1C in EV to target tumor blood vessels, and bring a new point-cut to the treatment of gliomablastoma (GBM). © 2020 Elsevier Inc. All rights reserved.
Keywords: Glioma Endothelial cells Extracellular vesicles Migration MYO1C
1. Introduction GBM is a highly vascularized tumor in which the tumor vascular system is abnormal in almost every aspect of its structure and function [1]. Affected by tumor microenvironment, tumor blood
Abbreviations: GhEC, Glioma human endothelial cells; NhEC, Normal human brain endothelial cells; GC, Glioma cells; GSC, Glioma stem cells; EV, Extracellular vesicle; CSF, Cerebrospinal fluid; GBM, Gliomablastoma. * Corresponding author. ** Corresponding author. State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005, China. E-mail addresses: [email protected] (W. Han), pengxiaozhong@ pumc.edu.cn, [email protected] (X. Peng).
vessels and their endothelial cells are significantly different from normal blood vessels and normal endothelial cells in morphology, function, protein expression, and gene level [2e4]. The interaction between tumor cells and stromal cells (such as vascular endothelial cells) plays a vital role in the growth, migration, and metastasis of cancer [5e7]. Previous studies indicated that endothelial cells, peripheral cells and astrocytes could form neurovascular units to support the progression of gliomas [8]. Extracellular vesicles (EV), which diameter is between 50 nm and 1000 nm [9]. It is widely distributed in body fluids and mediates many biological and cellular functions, such as cell-to-cell communication. It can carry and transmit important signal molecules to neighboring cells, such as RNA, proteins, nucleic acids, etc [9]. EV is related to the occurrence and progress of various diseases [10]. It has also been reported that EV is involved in the transmission of information between cells [11], and possess
https://doi.org/10.1016/j.bbrc.2020.02.017 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
2
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
diagnosis and therapeutic potential [12]. There are many articles on RNA components of EV [13], few studies pay attention on proteomics of EV. And the mechanism of EV is very complicated. Our laboratory previously illustrated that both NhEC-EV and GhEC-EV could promote GSC self-renewal, proliferation and tumor globule formation in vitro, and promote tumor growth in vivo by passing CD9. Nevertheless, NhEC-EV or GhEC-EV could suppress glioma cells (GC) proliferation in vitro [14]. It demonstrates the undifferentiated impact of EV. The purpose of this article is to compare the functionally different effects of GC treated with NhEC-EV and GhEC-EV in order to find a new method to target the elimination of tumor vascular endothelium. We found that MYO1C was specifically expressed in GhEC-EV and promoted GC migration.
2. Materials and methods 2.1. Human samples and cell culture Cerebrospinal fluid (CSF) samples (Glioma, n ¼ 8; Meningioma, n ¼ 1; Schwannoma, n ¼ 1) were provided by Yanwei Liu from Beijing Tiantan Hospital. Primary Human Brain Microvascular Endothelial Cells (ACBRI 376; NhEC) were purchased from Cell systems. Glioma vascular endothelial cells (GhEC) were separated by our lab [14]. EC were maintained in EBM-2 medium containing 2% fetal bovine serum (FBS)-EV-free, 0.1% hydrocortisone, 0.1% R3IGF, 0.4% hFGF-b, 0.1% VEGF, 0.1% ascorbic acid, 0.1% GA-1000, 0.1% hEGF, 0.1% heparin, 100 U/mL penicillin and 100 mg/mL streptomycin. Human glioma cell lines LN229, T98G and A172 were obtained from American Type Culture Collection (ATCC) and cultured in modified Eagle’s medium (MEM) supplemented with 1 mM sodium pyruvate and 1% (vol/vol) non-essential amino acids (NEAA), 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. HA, HAc and HA-sp were purchased from ScienCell Research Laboratories (Carlsbad, CA) and cultured with astrocyte medium (catalog 1801). Glioma stem cell lines GSC2, GSC5 and U251-SLC were obtained and cultured as previously reported [15]. All kind of cells were cultivated in a 37 C, 5% CO2 incubator.
2.2. Isolation of EV Ultracentrifugation (Beckman, optima L-100XP) was used to extract EV. After 48 h of cell culture, the conditioned media (CM) was centrifuged at 1000 rpm for 5 min to remove cell debris, then supernatant was filtered through a 0.22 mm filter. Next, filtered supernatant was centrifuged as described previously [16]. Resuspend the EV pellet with protein lysate or fresh medium for the next series of experiments.
2.3. Characterization of EV EV protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). EV particle size was detected by Transmission Electron Microscope (TEM). Purified EV were identified as described previously [16]. Images were obtained using Japan Electronics TEM-1400 plus. Particle size distribution of EV was determined by Nanoparticle-tracking analysis (NTA). EV (25 mg) was dissolved in 500 mL PBS, and tested with ZetaView PMX 110 (Particle Metrix, Germany).
2.4. Protein extraction and western blotting Cells were harvested and lysed by Tris-Nacl-Triton-EDTA (TNTE) buffer containing four protease inhibitors (phenylmethylsulfonyl fluoride [PMSF], leupeptin, pepstatin and aprotinin), and the total protein concentration was quantified using BCA method. The equal amounts of samples were separated by sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and then transferred onto nitrocellulose membranes (Millipore). After being blocked for 1 h with 5% non-fat milk at room temperature, membranes were incubated with primary antibodies at 4 C overnight against Calnexin (ab75801, 1:1000), Flotillin-1 (ab41927, 1:1000), CD9 (ab92726, 1:2000), CD63 (ab134045, 1:1000), TSG101 (ab83, 1:1000), ALIX (ab117600, 1:1000), MYO1C (HPA00176B, 1:1000), MTAP (11475-1-AP, 1:1000), TPM4 (13741-1-AP, 1:500), Cathepsin B (sc-365558, 1:100), Catalase (ab16731, 1:2000), ANKRD50 (ab108219, 1:1000), a-Albumin (AF8065-SP, 1:33333), Flag (AE005, 1:2000) or b-actin (ab8227, 1:5000). Next, the membranes were probed with secondary antibody for 2 h at room temperature. At last, protein signaling was visualized using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific; Amersham) and quantified using ImageJ software. 2.5. Endothelial cell secreted media (conditioned media, CM) EC were seed in complete EBM-2 in 10 cm plates. When cells grow to 90%, then cultured in serum-free EBM-2 media for 36 h. The secreted media was centrifuged for 5 min at 2000 rpm so as to remove cell debris and filtered through a 0.22 mm filter. Then the solution was concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3K membrane (Millipore, Watford, UK). After centrifugation for 100 min at 4000 rpm at 4 C, protein concentration was measured by BCA Protein Assay Kit. 2.6. Transwell assay LN229 (1 105 cells/0.1 ml in FBS-free DMEM) were seed into transwell chambers (Constar, 24-Well Plate, 8.0 mm Polycarbonate Memrance) and put it into 24-well for 30 min in a 37C, 5% CO2 incubator. Next, 600 mL CM or EV suspensions were loaded in the lower chamber. After 24e48 h of conventional culture, 4% paraformaldehyde was used to immobilize cells for 30 min. And wash them three times with PBS. Then, the migrated cells were stained with 1% crystal violet solution for 15 min. Images were obtained by Stereomicroscope (Leica). Migrated cells were counted using ImageJ software. Three replicate wells were set for each experiment and repeated three times. 2.7. Mass spectrometry analysis We used reversed phase nanoliquid chromatographyetandem mass spectrometry (LC-MS/MS) (Ultimate 3000 coupled to QExactive, Thermo Scientific) to analysis proteins of EV as described previously [14,17]. 2.8. Transfection Cells were seed on 12-well plates or 10 cm petri dishes and transfected with siRNA oligonucleotides or plasmid the next day, using INTERFERin® (PolyPlus) or Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The siRNA were as follows: siMYO1C-1 sense:50 -GCUCAAAGAAUCCCAUUAU-3’; 0 siMYO1C-2 sense: 5 -CCUAUCGCCGCAAAUACGAAGCUUU-3’; siNC
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 1. Proteomic analysis of GhEC-EV and NhEC-EV. (A) Mass spectrometry analysis of GhEC-EV and NhEC-EV. 70 proteins showed a >1.5-fold change (FC) in GhEC-EV versus NhEC-EV. (BeD) GO enrichment (B), KEGG pathway enrichment (C) and PPI (D) analysis of EV-associated proteins. GO: Gene oncology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: Protein-Protein interaction.
sense: 50 -UUCUCCGAACGUGUCACGUTT. 2.9. Statistical analysis We conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis by the Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8). PPI was constructed by STRING and Cytoscape (version 3.7.1). Gene expression data and survival curve of GBM samples were obtained from The Cancer Genome Atlas (TCGA) database. Graphing were performed using GraphPad Prism 6.0 Software. Student’s t-test was used for statistical analysis and data are presented as mean ± SD. P-value < 0.05 was defined as the cut-off criteria. 3. Results 3.1. Proteomic analysis of GhEC-EV and NhEC-EV
[14]. Here we named them as glioma human endothelial cells (GhEC). To find differences between GhEC-EV and NhEC-EV. We compared both their appearance and size. Firstly, we detected ALIX, TSG101, CD9, CD63 and Flotillin-1 for EV typical markers, Calnexin for endoplasmic reticulum marker in EC and EC-derived EV (Fig. S1A). Furthermore, EV was identified by Transmission Electron Microscope (TEM) and Nanoparticle-tracking analysis (NTA) (Fig. S1B, S1C). We found there are no differences between GhEC-EV and NhEC-EV in appearance or size. To look for differentially expressed proteins in GhEC-EV and NhEC-EV, we continued to analyze the mass spectrometry of EV. We indicated 70 proteins, which expressed in GhEC-EV higher than in NhEC-EV (GhEC-EV/NhEC-EV, FC > 1.5; Fig. 1A). Then, we analyzed 70 proteins by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Protein-Protein interaction (PPI) analysis. We showed that most of them were related to extracellular exosome and involved in the migration pathway (Fig. 1BeD).
Our lab isolated endothelial cells from glioma tissue previously
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
4
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. GhEC-EV promote migration of glioma cells in vitro. (AeB) Transwell assay was performed by treating LN229 with CM (100 mg/ml) (A) or EV (50 mg/ml) (B) from NhEC or GhEC. Untreated cells were used as a control (n ¼ 3). Percentage of migrated cells was performed using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; data are mean ± SD. Scale bar: 0.1 mm; 0.9 mm. CM: Conditioned medium.
3.2. GhEC-EV promote migration of glioma cells in vitro To determine the effect of EC on glioma cell migration capacity, we treated LN229 cell with 100 mg/ml CM from EC, and we illustrated GhEC-CM could specifically promote LN229 migration compared with the control and NhEC-CM (Fig. 2A). Meanwhile, we analyzed the effect of EV on LN229 migration, we treated LN229 with 50 mg/ml EC-derived EVs. Interestingly, compared with NhECEVs group, GhEC-EV significantly increased cell migration in vitro (Fig. 2B). 3.3. MYO1C is selected as a candidate protein in GhEC-EV Next, we wanted to screen which specific factor regulates this function. We utilised LC-MS/MS to analyze proteins in two biological replicates of EC-derived EV. Our screen criteria is: GhECEV/NhEC-EV, FC > 1.5. We found 9 proteins were enriched in GhEC-EV in comparison with NhEC-EV in both replicates (Fig. 3A). To verify the reliability of mass spectrometry, we detected 6 proteins of them: MYO1C, Catalase, TPM4, MTAP, Cathepsin B and ANKRD50. Western blot analyzed that MYO1C significantly expressed higher in GhEC-EV than NhEC-EV, but MYO1C
expression was not different in NhEC and GhEC (Fig. 3B). We also detected MYO1C expression level in GSC (GSC2, U251-SLC), normal glial cells (HA, HA-c, HA-sp) and GC (T98G, A172, LN229) (Fig. S2A). However, MTAP, Cathepsin B and Catalase did not detect the same result as mass spectrometry (Fig. S2B). TPM4 and ANKRD50 were not detected in EV, the data was not shown. We also detected these proteins expression in EC (Fig. S2C). According to the TCGA databases, MYO1C mRNA expression was highly expressed in GBM (n ¼ 163) than normal brain tissue (n ¼ 207) (Fig. 3C). Moreover, patients with higher MYO1C expression had a shorter survival time than those lower MYO1C expression (p ¼ 0.0042; Fig. 3E). In order to further confirm the role of MYO1C expression in the diagnosis of gliomas, we detected MYO1C expression in cerebrospinal fluid (CSF) of Glioma (n ¼ 8), Meningioma (n ¼ 1) and Schwannoma (n ¼ 1) (Fig. 3D). Although we lack of normal cerebrospinal fluid samples, we hypothesize that MYO1C is likely to be a sign of disease diagnosis. 3.4. Knockdown of MYO1C reduces the migration of glioma cells To ascertain if it was EV-mediated transfer of MYO1C, thereby facilitating the migration of LN229. We knocked down MYO1C by
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 3. MYO1C is selected as a candidate protein in GhEC-EV. (A) Mass spectrometry analysis of GhEC-EV and NhEC-EV in two biological replicates. 9 proteins were selected in this screen criteria (GhEC-EV/NhEC-EV, FC > 1.5). (B) Western blot analysis of MYO1C expression level in EV and EC. (C) TCGA database showed MYO1C gene expression level in GBM (n ¼ 163) compared with normal brain tissue (n ¼ 207). (D) Western blot showing MYO1C protein level in CSF of Glioma (n ¼ 8), Meningioma (n ¼ 1) and Schwannoma (n ¼ 1). (E) TCGA database accessed prognosis for MYO1C in GBM. CSF: Cerebrospinal fluid.
siRNA-1 in GhEC, and Western blot detected the knock-down efficiency in GhEC and GhEC-EV (Fig. 4AeB). Then, we observed that LN229 migration was suppressed when we knocked down MYO1C in GhEC (Fig. 4C). Meanwhile, knockdown of MYO1C in GhEC-EV had the same effect (Fig. 4D). In addition, overexpresion of MYO1C in LN229 could promote cell migration (Fig. 4EeF). All statistical histograms showed in Fig. 4G. In summary, our data illustrated that GhEC could promote glioma migration via EVmediated transfer of MYO1C. 4. Discussion There are significant differences between tumor vascular endothelial cells and normal vascular endothelial cells. Previous studies indicated that brain tumor vascular endothelial cells showed a decrease in tight junction proteins expression (such as claudin-1 and occludin), and an increase in pinocytosis and
permeability [18]. Tumor endothelial cells preferred to promote proliferation and motility than normal endothelial cells [19]. These studies suggest that the emergence of vascular heterogeneity is the result of the interaction of endothelial cells with the tissue microenvironment. Although studies have explored the relationship between EC and GC, most studies have focused on the effects of NhEC on GC [20e22]. In our research, we suggested that GhEC specifically promoted GC migration (Fig. 2A). Then, we focus on the intercellular communication between GhEC-EV and GC. We identified the characterization of NhEC-EV and GhEC-EV. Western blot displayed that a few EV typical markers (ALIX and TSG101) were not expressed in EC-derived EV at the same time (Fig. S1A). This is because the origin of EC-derived EV is likely to different. Some of EV come from endosomal system, and some originate from the plasma membrane [9]. MYO1C is an actin-based motor molecular, which is a member of unconventional myosin protein family. Interesting, we found that
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
6
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. Knockdown of MYO1C reduces the migration of glioma cells. (AeB) Western blot analysis the efficiency of knockdown of MYO1C in GhEC (A) and GhEC-EV (B). (CeD) Transwell assay evaluated migration efficiency treating LN229 with siMYO1C-CM (C) or siMYO1C-EV (D) from GhEC. (E) Western blot analysis the efficiency of overexpression of MYO1C in LN229. (F) The effect of MYO1C overexpression in LN229. (G) Statistical histograms of Fig. 4C, D and 4F. Percentage of migrated cells was performed using Student’s t-test.. *P < 0.05, **P < 0.01; data are mean ± SD. Scale bar: 0.1 mm; 0.9 mm.
MYO1C expressed higher in GhEC-EV but not expressed differently in NhEC and GhEC (Fig. 3B). It indicated that MYO1C protein was more wrapped in GhEC-EV rather than NhEC-EV and perform functions. And we had confirmed that knockdown of MYO1C in GhEC would suppress cell migration compared to the control (Fig. 4A). This result was consistent with previous reports [23e25]. MYO1C was also reported to be involved in angiogenic signaling, exocytosis and vesicle shedding [26e28]. Our result demonstrated for the first time that GhEC could promote GC migration via EVmediated transfer of MYO1C (Fig. 4B). This study is to compare the differential functional analysis of GhEC and NhEC, and to find a new mechanism for communicating between EC-derived EV and GC. MYO1C was also detected in glioma-CSF, which can be used as a biopsy indicator and substitute for needle biopsy, and contribute to early diagnosis and monitoring of glioma [29]. The development of tumors requires the formation of new blood vessels, which are related to invasion [30]. That hints that MYO1C may become a key target for tumor vascular therapy.
Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments This research was supported by the National Key Research and Development Program of China (2016YFC0902502), the National Sciences Foundation of China (31671316, 21874156), Beijing Nova Program of Science and Technology (Z191100001119137), the CAMS Innovation Fund for Medical Sciences (CIFMS;2016-I2M-1-001, 2017-I2M-2-004, 2017-I2M-3-010). We also thankful to Yanwei Liu from Beijing Tiantan hospital for providing CSF samples. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.017. References [1] P. Carmeliet, R.K. Jain, Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases, Nat. Rev. Drug Discov. 10 (2011) 417e427, https://doi.org/10.1038/nrd3455. [2] B.S. Croix, Genes expressed in human tumor endothelium, Science 289 (2000) 1197e1202, https://doi.org/10.1126/science.289.5482.1197. [3] B. Bussolati, I. Deambrosis, S. Russo, M.C. Deregibus, G. Camussi, Altered angiogenesis and survival in human tumor-derived endothelial cells, Faseb. J. 17 (2003) 1159e1161, https://doi.org/10.1096/fj.02-0557fje. [4] T. Akino, K. Hida, Y. Hida, K. Tsuchiya, D. Freedman, C. Muraki, N. Ohga, K. Matsuda, K. Akiyama, T. Harabayashi, N. Shinohara, K. Nonomura,
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
M. Klagsbrun, M. Shindoh, Cytogenetic abnormalities of tumor-associated endothelial cells in human malignant tumors, Am. J. Pathol. 175 (2009) 2657e2667, https://doi.org/10.2353/ajpath.2009.090202. n-Jua rez, L. Wachsmuth, K. Han, U.C. Müller, B. Strilic, L. Yang, J. Albarra M. Pasparakis, S. Offermanns, Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis, Nature 536 (2016) 215e218, https://doi.org/10.1038/nature19076. B. Tavora, L.E. Reynolds, S. Batista, F. Demircioglu, I. Fernandez, T. Lechertier, D.M. Lees, P.-P. Wong, A. Alexopoulou, G. Elia, A. Clear, A. Ledoux, J. Hunter, N. Perkins, J.G. Gribben, K.M. Hodivala-Dilke, Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy, Nature 514 (2014) 112e116, https://doi.org/10.1038/nature13541. A. Furlan, C. Vercamer, L. Heliot, N. Wernert, X. Desbiens, A. Pourtier, Ets-1 drives breast cancer cell angiogenic potential and interactions between breast cancer and endothelial cells, Int. J. Oncol. 54 (2019) 29e40, https://doi.org/ 10.3892/ijo.2018.4605. N.A. Charles, E.C. Holland, R. Gilbertson, R. Glass, H. Kettenmann, The brain tumor microenvironment, Glia 60 (2012) 502e514, https://doi.org/10.1002/ glia.21264. G. van Niel, G. D’Angelo, G. Raposo, Shedding light on the cell biology of extracellular vesicles, Nat. Rev. Mol. Cell Biol. 19 (2018) 213e228, https:// doi.org/10.1038/nrm.2017.125. X. Huaitong, F. Yuanyong, T. Yueqin, Z. Peng, S. Wei, S. Kai, Microvesicles releasing by oral cancer cells enhance endothelial cell angiogenesis via Shh/ RhoA signaling pathway, Canc. Biol. Ther. 18 (2017) 783e791, https://doi.org/ 10.1080/15384047.2017.1373213. X. Chen, H. Liang, J. Zhang, K. Zen, C.-Y. Zhang, Secreted microRNAs: a new form of intercellular communication, Trends Cell Biol. 22 (2012) 125e132, https://doi.org/10.1016/j.tcb.2011.12.001. W. Zhang, X. Zhou, H. Zhang, Q. Yao, Y. Liu, Z. Dong, Extracellular vesicles in diagnosis and therapy of kidney diseases, Am. J. Physiol. Ren. Physiol. 311 (2016) 844e851, https://doi.org/10.1152/ajprenal.00429.2016. H.L. Lang, G.W. Hu, B. Zhang, W. Kuang, Y. Chen, L. Wu, G.-H. Xu, Glioma cells enhance angiogenesis and inhibit endothelial cell apoptosis through the release of exosomes that contain long non-coding RNA CCAT2, Oncol. Rep. 38 (2017) 785e798, https://doi.org/10.3892/or.2017.5742. D. Li, Y. Tian, Y. Hu, Y. Qi, N. Tian, S. Li, P. Hu, F. Wu, Q. Wei, Z. Wei, S. Wang, B. Yin, T. Jiang, J. Yuan, B. Qiang, W. Han, X. Peng, Glioma-associated human endothelial cell-derived extracellular vesicles specifically promote the tumourigenicity of glioma stem cells via CD9, Oncogene 38 (2019) 6898e6912, https://doi.org/10.1038/s41388-019-0903-6. P.S. Hu, Q.S. Xia, F. Wu, D.k. Li, Y.j. Qi, Y. Hu, Z.Z. Wei, S.S. Li, N.Y. Tian, Q.F. Wei, L.J. Shen, B. Yin, T. Jiang, J.G. Yuan, B.Q. Qiang, W. Han, X.Z. Peng, NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of alltrans retinoic acid via targeting RDH16 in malignant glioma, Oncogene 36 (2017) 4706e4718, https://doi.org/10.1038/onc.2017.34. ry, S. Amigorena, G. Raposo, A. Clayton, Isolation and characterization of C. The exosomes from cell culture supernatants and biological fluids, Curr. Protoc. Cell Biol. (2006), https://doi.org/10.1002/0471143030.cb0322s30. J. Kowal, G. Arras, M. Colombo, M. Jouve, J.P. Morath, B. Primdal-Bengtson, ry, Proteomic comparison defines novel F. Dingli, D. Loew, M. Tkach, C. The
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
7
markers to characterize heterogeneous populations of extracellular vesicle subtypes, Proc. Nati. Acad. Sci. U. S. A. 113 (2016) 968e977, https://doi.org/ 10.1073/pnas.1521230113. D.C. Davies, Bloodebrain barrier breakdown in septic encephalopathy and brain tumours, J. Anat. 200 (2002) 639e646, https://doi.org/10.1046/j.14697580.2002.00065.x. K. Matsuda, N. Ohga, Y. Hida, C. Muraki, K. Tsuchiya, T. Kurosu, T. Akino, S.C. Shih, Y. Totsuka, M. Klagsbrun, M. Shindoh, K. Hida, Isolated tumor endothelial cells maintain specific character during long-term culture, Biochem. Biophys. Res. Commun. 394 (2010) 947e954, https://doi.org/10.1016/ j.bbrc.2010.03.089. D. Hambardzumyan, O.J. Becher, E.C. Holland, Cancer stem cells and survival pathways, Cell Cycle 7 (2008) 1371e1378, https://doi.org/10.4161/ cc.7.10.5954. T.S. Zhu, M.A. Costello, C.E. Talsma, C.G. Flack, J.G. Crowley, L.L. Hamm, X. He, S.L. Hervey-Jumper, J.A. Heth, K.M. Muraszko, F. DiMeco, A.L. Vescovi, X. Fan, Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells, Canc. Res. 71 (2011) 6061e6072, https://doi.org/10.1158/0008-5472.CAN-10-4269. A.B. Hjelmeland, J.D. Lathia, S. Sathornsumetee, J.N. Rich, Twisted tango: brain tumor neurovascular interactions, Nat. Neurosci. 14 (2011) 1375e1381, https://doi.org/10.1038/nn.2955. W.s.E. Edimo, A.R. Ramos, S. Ghosh, J.-M. Vanderwinden, C. Erneux, The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells, Biochem. Biophys. Res. Commun. 476 (2016) 508e514, https://doi.org/ 10.1016/j.bbrc.2016.05.154. Y. Fan, S.M. Eswarappa, M. Hitomi, P.L. Fox, Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells, J. Cell Biol. 198 (2012) 47e55, https://doi.org/10.1083/jcb.201111088. H. Brandstaetter, J. Kendrick-Jones, F. Buss, Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion, J. Cell Sci. 125 (2012) 1991e2003, https://doi.org/10.1242/jcs.097212. A. Tiwari, J.J. Jung, S.M. Inamdar, D. Nihalani, A. Choudhury, The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling, Am. J. Physiol. Heart Circ. Physiol. 304 (2013) 687e696, https:// doi.org/10.1152/ajpheart.00744.2012. H. Brandstaetter, J. Kendrick-Jones, F. Buss, Molecular roles of Myo1c function in lipid raft exocytosis, Commun. Integr. Biol. 5 (2012) 508e510, https:// doi.org/10.4161/cib.21201. M.F. Yip, G. Ramm, M. Larance, K.L. Hoehn, M.C. Wagner, M. Guilhaus, D.E. James, CaMKII-mediated phosphorylation of the myosin motor Myo1c is required for insulin-stimulated GLUT4 translocation in adipocytes, Cell Metabol. 8 (2008) 384e398, https://doi.org/10.1016/j.cmet.2008.09.011. A.M. Miller, R.H. Shah, E.I. Pentsova, M. Pourmaleki, S. Briggs, N. Distefano, Y. Zheng, A. Skakodub, S.A. Mehta, C. Campos, et al., Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid, Nature 565 (2019) 654e658, https://doi.org/10.1038/s41586-019-0882-3. A. Mukwaya, P. Mirabelli, A. Lennikov, M. Thangavelu, L. Jensen, B. Peebo, N. Lagali, Repeat corneal neovascularization is characterized by more aggressive inflammation and vessel invasion than in the initial phase, Invest. Ophthalmol. Vis. Sci. 60 (2019) 2990, https://doi.org/10.1167/iovs.19-27591.
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C Yuan Tian a, Zhixing Wang a, Yuxin Wang a, Bin Yin a, Jiangang Yuan a, Boqin Qiang a, Wei Han a, *, Xiaozhong Peng a, b, ** a
State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Medical Primate Research Center, Neuroscience Center, Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005, China b Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Kunming, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 January 2020 Accepted 4 February 2020 Available online xxx
Extracellular vesicles (EV), as the intercellular information transfer molecules which can regulate the tumor microenvironment, promote migration and tumor progression. Previous studies reported that EV from endothelial cells was used to guide the fate and survival of gliomas, but many researches focus on normal human endothelial cells (NhEC) rather than tumor-derived endothelial cells. Our laboratory isolated human endothelial cells from glioma issue (GhEC). We have previously demonstrated that EV from GhEC and NhEC, which both can promote glioma stem cells (GSC) proliferation and tumorsphere formation in vitro and tumourigenicity in vivo by the transfer of CD9. However, NhEC-EV or GhEC-EV could suppress glioma cells (GC) proliferation in vitro. It demonstrates the undifferentiated impact of EV. Here, we first compared GhEC-EV proteins with NhEC-EV (Screening criteria: GhEC-EV/NhEC-EV, FC > 1.5), and obtained 70 differential expression proteins, most of which were associated with invasion and migration. We found that GhEC or GhEC-EV preferred promoting GC migration than treating with NhEC or NhEC-EV. In terms of mechanism, we further revealed that EV-mediated transfer of MYO1C induced glioma cell LN229 migration. Knockdown of MYO1C in GhEC or GhEC-EV suppressed this effect. Overexpression of MYO1C promoted migration on the contrary. MYO1C was also detected in glioma cerebrospinal fluid (CSF), which is more suitable as a liquid biopsy biomarker and contributes to early diagnosis and monitoring in glioma. Our findings provide a new proteindMYO1C in EV to target tumor blood vessels, and bring a new point-cut to the treatment of gliomablastoma (GBM). © 2020 Elsevier Inc. All rights reserved.
Keywords: Glioma Endothelial cells Extracellular vesicles Migration MYO1C
1. Introduction GBM is a highly vascularized tumor in which the tumor vascular system is abnormal in almost every aspect of its structure and function [1]. Affected by tumor microenvironment, tumor blood
Abbreviations: GhEC, Glioma human endothelial cells; NhEC, Normal human brain endothelial cells; GC, Glioma cells; GSC, Glioma stem cells; EV, Extracellular vesicle; CSF, Cerebrospinal fluid; GBM, Gliomablastoma. * Corresponding author. ** Corresponding author. State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, 100005, China. E-mail addresses: [email protected] (W. Han), pengxiaozhong@ pumc.edu.cn, [email protected] (X. Peng).
vessels and their endothelial cells are significantly different from normal blood vessels and normal endothelial cells in morphology, function, protein expression, and gene level [2e4]. The interaction between tumor cells and stromal cells (such as vascular endothelial cells) plays a vital role in the growth, migration, and metastasis of cancer [5e7]. Previous studies indicated that endothelial cells, peripheral cells and astrocytes could form neurovascular units to support the progression of gliomas [8]. Extracellular vesicles (EV), which diameter is between 50 nm and 1000 nm [9]. It is widely distributed in body fluids and mediates many biological and cellular functions, such as cell-to-cell communication. It can carry and transmit important signal molecules to neighboring cells, such as RNA, proteins, nucleic acids, etc [9]. EV is related to the occurrence and progress of various diseases [10]. It has also been reported that EV is involved in the transmission of information between cells [11], and possess
https://doi.org/10.1016/j.bbrc.2020.02.017 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
2
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
diagnosis and therapeutic potential [12]. There are many articles on RNA components of EV [13], few studies pay attention on proteomics of EV. And the mechanism of EV is very complicated. Our laboratory previously illustrated that both NhEC-EV and GhEC-EV could promote GSC self-renewal, proliferation and tumor globule formation in vitro, and promote tumor growth in vivo by passing CD9. Nevertheless, NhEC-EV or GhEC-EV could suppress glioma cells (GC) proliferation in vitro [14]. It demonstrates the undifferentiated impact of EV. The purpose of this article is to compare the functionally different effects of GC treated with NhEC-EV and GhEC-EV in order to find a new method to target the elimination of tumor vascular endothelium. We found that MYO1C was specifically expressed in GhEC-EV and promoted GC migration.
2. Materials and methods 2.1. Human samples and cell culture Cerebrospinal fluid (CSF) samples (Glioma, n ¼ 8; Meningioma, n ¼ 1; Schwannoma, n ¼ 1) were provided by Yanwei Liu from Beijing Tiantan Hospital. Primary Human Brain Microvascular Endothelial Cells (ACBRI 376; NhEC) were purchased from Cell systems. Glioma vascular endothelial cells (GhEC) were separated by our lab [14]. EC were maintained in EBM-2 medium containing 2% fetal bovine serum (FBS)-EV-free, 0.1% hydrocortisone, 0.1% R3IGF, 0.4% hFGF-b, 0.1% VEGF, 0.1% ascorbic acid, 0.1% GA-1000, 0.1% hEGF, 0.1% heparin, 100 U/mL penicillin and 100 mg/mL streptomycin. Human glioma cell lines LN229, T98G and A172 were obtained from American Type Culture Collection (ATCC) and cultured in modified Eagle’s medium (MEM) supplemented with 1 mM sodium pyruvate and 1% (vol/vol) non-essential amino acids (NEAA), 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. HA, HAc and HA-sp were purchased from ScienCell Research Laboratories (Carlsbad, CA) and cultured with astrocyte medium (catalog 1801). Glioma stem cell lines GSC2, GSC5 and U251-SLC were obtained and cultured as previously reported [15]. All kind of cells were cultivated in a 37 C, 5% CO2 incubator.
2.2. Isolation of EV Ultracentrifugation (Beckman, optima L-100XP) was used to extract EV. After 48 h of cell culture, the conditioned media (CM) was centrifuged at 1000 rpm for 5 min to remove cell debris, then supernatant was filtered through a 0.22 mm filter. Next, filtered supernatant was centrifuged as described previously [16]. Resuspend the EV pellet with protein lysate or fresh medium for the next series of experiments.
2.3. Characterization of EV EV protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). EV particle size was detected by Transmission Electron Microscope (TEM). Purified EV were identified as described previously [16]. Images were obtained using Japan Electronics TEM-1400 plus. Particle size distribution of EV was determined by Nanoparticle-tracking analysis (NTA). EV (25 mg) was dissolved in 500 mL PBS, and tested with ZetaView PMX 110 (Particle Metrix, Germany).
2.4. Protein extraction and western blotting Cells were harvested and lysed by Tris-Nacl-Triton-EDTA (TNTE) buffer containing four protease inhibitors (phenylmethylsulfonyl fluoride [PMSF], leupeptin, pepstatin and aprotinin), and the total protein concentration was quantified using BCA method. The equal amounts of samples were separated by sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) and then transferred onto nitrocellulose membranes (Millipore). After being blocked for 1 h with 5% non-fat milk at room temperature, membranes were incubated with primary antibodies at 4 C overnight against Calnexin (ab75801, 1:1000), Flotillin-1 (ab41927, 1:1000), CD9 (ab92726, 1:2000), CD63 (ab134045, 1:1000), TSG101 (ab83, 1:1000), ALIX (ab117600, 1:1000), MYO1C (HPA00176B, 1:1000), MTAP (11475-1-AP, 1:1000), TPM4 (13741-1-AP, 1:500), Cathepsin B (sc-365558, 1:100), Catalase (ab16731, 1:2000), ANKRD50 (ab108219, 1:1000), a-Albumin (AF8065-SP, 1:33333), Flag (AE005, 1:2000) or b-actin (ab8227, 1:5000). Next, the membranes were probed with secondary antibody for 2 h at room temperature. At last, protein signaling was visualized using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific; Amersham) and quantified using ImageJ software. 2.5. Endothelial cell secreted media (conditioned media, CM) EC were seed in complete EBM-2 in 10 cm plates. When cells grow to 90%, then cultured in serum-free EBM-2 media for 36 h. The secreted media was centrifuged for 5 min at 2000 rpm so as to remove cell debris and filtered through a 0.22 mm filter. Then the solution was concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-3K membrane (Millipore, Watford, UK). After centrifugation for 100 min at 4000 rpm at 4 C, protein concentration was measured by BCA Protein Assay Kit. 2.6. Transwell assay LN229 (1 105 cells/0.1 ml in FBS-free DMEM) were seed into transwell chambers (Constar, 24-Well Plate, 8.0 mm Polycarbonate Memrance) and put it into 24-well for 30 min in a 37C, 5% CO2 incubator. Next, 600 mL CM or EV suspensions were loaded in the lower chamber. After 24e48 h of conventional culture, 4% paraformaldehyde was used to immobilize cells for 30 min. And wash them three times with PBS. Then, the migrated cells were stained with 1% crystal violet solution for 15 min. Images were obtained by Stereomicroscope (Leica). Migrated cells were counted using ImageJ software. Three replicate wells were set for each experiment and repeated three times. 2.7. Mass spectrometry analysis We used reversed phase nanoliquid chromatographyetandem mass spectrometry (LC-MS/MS) (Ultimate 3000 coupled to QExactive, Thermo Scientific) to analysis proteins of EV as described previously [14,17]. 2.8. Transfection Cells were seed on 12-well plates or 10 cm petri dishes and transfected with siRNA oligonucleotides or plasmid the next day, using INTERFERin® (PolyPlus) or Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The siRNA were as follows: siMYO1C-1 sense:50 -GCUCAAAGAAUCCCAUUAU-3’; 0 siMYO1C-2 sense: 5 -CCUAUCGCCGCAAAUACGAAGCUUU-3’; siNC
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
3
Fig. 1. Proteomic analysis of GhEC-EV and NhEC-EV. (A) Mass spectrometry analysis of GhEC-EV and NhEC-EV. 70 proteins showed a >1.5-fold change (FC) in GhEC-EV versus NhEC-EV. (BeD) GO enrichment (B), KEGG pathway enrichment (C) and PPI (D) analysis of EV-associated proteins. GO: Gene oncology; KEGG: Kyoto Encyclopedia of Genes and Genomes; PPI: Protein-Protein interaction.
sense: 50 -UUCUCCGAACGUGUCACGUTT. 2.9. Statistical analysis We conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis by the Database for Annotation, Visualization and Integrated Discovery (DAVID, version 6.8). PPI was constructed by STRING and Cytoscape (version 3.7.1). Gene expression data and survival curve of GBM samples were obtained from The Cancer Genome Atlas (TCGA) database. Graphing were performed using GraphPad Prism 6.0 Software. Student’s t-test was used for statistical analysis and data are presented as mean ± SD. P-value < 0.05 was defined as the cut-off criteria. 3. Results 3.1. Proteomic analysis of GhEC-EV and NhEC-EV
[14]. Here we named them as glioma human endothelial cells (GhEC). To find differences between GhEC-EV and NhEC-EV. We compared both their appearance and size. Firstly, we detected ALIX, TSG101, CD9, CD63 and Flotillin-1 for EV typical markers, Calnexin for endoplasmic reticulum marker in EC and EC-derived EV (Fig. S1A). Furthermore, EV was identified by Transmission Electron Microscope (TEM) and Nanoparticle-tracking analysis (NTA) (Fig. S1B, S1C). We found there are no differences between GhEC-EV and NhEC-EV in appearance or size. To look for differentially expressed proteins in GhEC-EV and NhEC-EV, we continued to analyze the mass spectrometry of EV. We indicated 70 proteins, which expressed in GhEC-EV higher than in NhEC-EV (GhEC-EV/NhEC-EV, FC > 1.5; Fig. 1A). Then, we analyzed 70 proteins by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Protein-Protein interaction (PPI) analysis. We showed that most of them were related to extracellular exosome and involved in the migration pathway (Fig. 1BeD).
Our lab isolated endothelial cells from glioma tissue previously
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
4
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 2. GhEC-EV promote migration of glioma cells in vitro. (AeB) Transwell assay was performed by treating LN229 with CM (100 mg/ml) (A) or EV (50 mg/ml) (B) from NhEC or GhEC. Untreated cells were used as a control (n ¼ 3). Percentage of migrated cells was performed using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; data are mean ± SD. Scale bar: 0.1 mm; 0.9 mm. CM: Conditioned medium.
3.2. GhEC-EV promote migration of glioma cells in vitro To determine the effect of EC on glioma cell migration capacity, we treated LN229 cell with 100 mg/ml CM from EC, and we illustrated GhEC-CM could specifically promote LN229 migration compared with the control and NhEC-CM (Fig. 2A). Meanwhile, we analyzed the effect of EV on LN229 migration, we treated LN229 with 50 mg/ml EC-derived EVs. Interestingly, compared with NhECEVs group, GhEC-EV significantly increased cell migration in vitro (Fig. 2B). 3.3. MYO1C is selected as a candidate protein in GhEC-EV Next, we wanted to screen which specific factor regulates this function. We utilised LC-MS/MS to analyze proteins in two biological replicates of EC-derived EV. Our screen criteria is: GhECEV/NhEC-EV, FC > 1.5. We found 9 proteins were enriched in GhEC-EV in comparison with NhEC-EV in both replicates (Fig. 3A). To verify the reliability of mass spectrometry, we detected 6 proteins of them: MYO1C, Catalase, TPM4, MTAP, Cathepsin B and ANKRD50. Western blot analyzed that MYO1C significantly expressed higher in GhEC-EV than NhEC-EV, but MYO1C
expression was not different in NhEC and GhEC (Fig. 3B). We also detected MYO1C expression level in GSC (GSC2, U251-SLC), normal glial cells (HA, HA-c, HA-sp) and GC (T98G, A172, LN229) (Fig. S2A). However, MTAP, Cathepsin B and Catalase did not detect the same result as mass spectrometry (Fig. S2B). TPM4 and ANKRD50 were not detected in EV, the data was not shown. We also detected these proteins expression in EC (Fig. S2C). According to the TCGA databases, MYO1C mRNA expression was highly expressed in GBM (n ¼ 163) than normal brain tissue (n ¼ 207) (Fig. 3C). Moreover, patients with higher MYO1C expression had a shorter survival time than those lower MYO1C expression (p ¼ 0.0042; Fig. 3E). In order to further confirm the role of MYO1C expression in the diagnosis of gliomas, we detected MYO1C expression in cerebrospinal fluid (CSF) of Glioma (n ¼ 8), Meningioma (n ¼ 1) and Schwannoma (n ¼ 1) (Fig. 3D). Although we lack of normal cerebrospinal fluid samples, we hypothesize that MYO1C is likely to be a sign of disease diagnosis. 3.4. Knockdown of MYO1C reduces the migration of glioma cells To ascertain if it was EV-mediated transfer of MYO1C, thereby facilitating the migration of LN229. We knocked down MYO1C by
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
5
Fig. 3. MYO1C is selected as a candidate protein in GhEC-EV. (A) Mass spectrometry analysis of GhEC-EV and NhEC-EV in two biological replicates. 9 proteins were selected in this screen criteria (GhEC-EV/NhEC-EV, FC > 1.5). (B) Western blot analysis of MYO1C expression level in EV and EC. (C) TCGA database showed MYO1C gene expression level in GBM (n ¼ 163) compared with normal brain tissue (n ¼ 207). (D) Western blot showing MYO1C protein level in CSF of Glioma (n ¼ 8), Meningioma (n ¼ 1) and Schwannoma (n ¼ 1). (E) TCGA database accessed prognosis for MYO1C in GBM. CSF: Cerebrospinal fluid.
siRNA-1 in GhEC, and Western blot detected the knock-down efficiency in GhEC and GhEC-EV (Fig. 4AeB). Then, we observed that LN229 migration was suppressed when we knocked down MYO1C in GhEC (Fig. 4C). Meanwhile, knockdown of MYO1C in GhEC-EV had the same effect (Fig. 4D). In addition, overexpresion of MYO1C in LN229 could promote cell migration (Fig. 4EeF). All statistical histograms showed in Fig. 4G. In summary, our data illustrated that GhEC could promote glioma migration via EVmediated transfer of MYO1C. 4. Discussion There are significant differences between tumor vascular endothelial cells and normal vascular endothelial cells. Previous studies indicated that brain tumor vascular endothelial cells showed a decrease in tight junction proteins expression (such as claudin-1 and occludin), and an increase in pinocytosis and
permeability [18]. Tumor endothelial cells preferred to promote proliferation and motility than normal endothelial cells [19]. These studies suggest that the emergence of vascular heterogeneity is the result of the interaction of endothelial cells with the tissue microenvironment. Although studies have explored the relationship between EC and GC, most studies have focused on the effects of NhEC on GC [20e22]. In our research, we suggested that GhEC specifically promoted GC migration (Fig. 2A). Then, we focus on the intercellular communication between GhEC-EV and GC. We identified the characterization of NhEC-EV and GhEC-EV. Western blot displayed that a few EV typical markers (ALIX and TSG101) were not expressed in EC-derived EV at the same time (Fig. S1A). This is because the origin of EC-derived EV is likely to different. Some of EV come from endosomal system, and some originate from the plasma membrane [9]. MYO1C is an actin-based motor molecular, which is a member of unconventional myosin protein family. Interesting, we found that
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
6
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 4. Knockdown of MYO1C reduces the migration of glioma cells. (AeB) Western blot analysis the efficiency of knockdown of MYO1C in GhEC (A) and GhEC-EV (B). (CeD) Transwell assay evaluated migration efficiency treating LN229 with siMYO1C-CM (C) or siMYO1C-EV (D) from GhEC. (E) Western blot analysis the efficiency of overexpression of MYO1C in LN229. (F) The effect of MYO1C overexpression in LN229. (G) Statistical histograms of Fig. 4C, D and 4F. Percentage of migrated cells was performed using Student’s t-test.. *P < 0.05, **P < 0.01; data are mean ± SD. Scale bar: 0.1 mm; 0.9 mm.
MYO1C expressed higher in GhEC-EV but not expressed differently in NhEC and GhEC (Fig. 3B). It indicated that MYO1C protein was more wrapped in GhEC-EV rather than NhEC-EV and perform functions. And we had confirmed that knockdown of MYO1C in GhEC would suppress cell migration compared to the control (Fig. 4A). This result was consistent with previous reports [23e25]. MYO1C was also reported to be involved in angiogenic signaling, exocytosis and vesicle shedding [26e28]. Our result demonstrated for the first time that GhEC could promote GC migration via EVmediated transfer of MYO1C (Fig. 4B). This study is to compare the differential functional analysis of GhEC and NhEC, and to find a new mechanism for communicating between EC-derived EV and GC. MYO1C was also detected in glioma-CSF, which can be used as a biopsy indicator and substitute for needle biopsy, and contribute to early diagnosis and monitoring of glioma [29]. The development of tumors requires the formation of new blood vessels, which are related to invasion [30]. That hints that MYO1C may become a key target for tumor vascular therapy.
Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments This research was supported by the National Key Research and Development Program of China (2016YFC0902502), the National Sciences Foundation of China (31671316, 21874156), Beijing Nova Program of Science and Technology (Z191100001119137), the CAMS Innovation Fund for Medical Sciences (CIFMS;2016-I2M-1-001, 2017-I2M-2-004, 2017-I2M-3-010). We also thankful to Yanwei Liu from Beijing Tiantan hospital for providing CSF samples. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.017. References [1] P. Carmeliet, R.K. Jain, Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases, Nat. Rev. Drug Discov. 10 (2011) 417e427, https://doi.org/10.1038/nrd3455. [2] B.S. Croix, Genes expressed in human tumor endothelium, Science 289 (2000) 1197e1202, https://doi.org/10.1126/science.289.5482.1197. [3] B. Bussolati, I. Deambrosis, S. Russo, M.C. Deregibus, G. Camussi, Altered angiogenesis and survival in human tumor-derived endothelial cells, Faseb. J. 17 (2003) 1159e1161, https://doi.org/10.1096/fj.02-0557fje. [4] T. Akino, K. Hida, Y. Hida, K. Tsuchiya, D. Freedman, C. Muraki, N. Ohga, K. Matsuda, K. Akiyama, T. Harabayashi, N. Shinohara, K. Nonomura,
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017
Y. Tian et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
M. Klagsbrun, M. Shindoh, Cytogenetic abnormalities of tumor-associated endothelial cells in human malignant tumors, Am. J. Pathol. 175 (2009) 2657e2667, https://doi.org/10.2353/ajpath.2009.090202. n-Jua rez, L. Wachsmuth, K. Han, U.C. Müller, B. Strilic, L. Yang, J. Albarra M. Pasparakis, S. Offermanns, Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis, Nature 536 (2016) 215e218, https://doi.org/10.1038/nature19076. B. Tavora, L.E. Reynolds, S. Batista, F. Demircioglu, I. Fernandez, T. Lechertier, D.M. Lees, P.-P. Wong, A. Alexopoulou, G. Elia, A. Clear, A. Ledoux, J. Hunter, N. Perkins, J.G. Gribben, K.M. Hodivala-Dilke, Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy, Nature 514 (2014) 112e116, https://doi.org/10.1038/nature13541. A. Furlan, C. Vercamer, L. Heliot, N. Wernert, X. Desbiens, A. Pourtier, Ets-1 drives breast cancer cell angiogenic potential and interactions between breast cancer and endothelial cells, Int. J. Oncol. 54 (2019) 29e40, https://doi.org/ 10.3892/ijo.2018.4605. N.A. Charles, E.C. Holland, R. Gilbertson, R. Glass, H. Kettenmann, The brain tumor microenvironment, Glia 60 (2012) 502e514, https://doi.org/10.1002/ glia.21264. G. van Niel, G. D’Angelo, G. Raposo, Shedding light on the cell biology of extracellular vesicles, Nat. Rev. Mol. Cell Biol. 19 (2018) 213e228, https:// doi.org/10.1038/nrm.2017.125. X. Huaitong, F. Yuanyong, T. Yueqin, Z. Peng, S. Wei, S. Kai, Microvesicles releasing by oral cancer cells enhance endothelial cell angiogenesis via Shh/ RhoA signaling pathway, Canc. Biol. Ther. 18 (2017) 783e791, https://doi.org/ 10.1080/15384047.2017.1373213. X. Chen, H. Liang, J. Zhang, K. Zen, C.-Y. Zhang, Secreted microRNAs: a new form of intercellular communication, Trends Cell Biol. 22 (2012) 125e132, https://doi.org/10.1016/j.tcb.2011.12.001. W. Zhang, X. Zhou, H. Zhang, Q. Yao, Y. Liu, Z. Dong, Extracellular vesicles in diagnosis and therapy of kidney diseases, Am. J. Physiol. Ren. Physiol. 311 (2016) 844e851, https://doi.org/10.1152/ajprenal.00429.2016. H.L. Lang, G.W. Hu, B. Zhang, W. Kuang, Y. Chen, L. Wu, G.-H. Xu, Glioma cells enhance angiogenesis and inhibit endothelial cell apoptosis through the release of exosomes that contain long non-coding RNA CCAT2, Oncol. Rep. 38 (2017) 785e798, https://doi.org/10.3892/or.2017.5742. D. Li, Y. Tian, Y. Hu, Y. Qi, N. Tian, S. Li, P. Hu, F. Wu, Q. Wei, Z. Wei, S. Wang, B. Yin, T. Jiang, J. Yuan, B. Qiang, W. Han, X. Peng, Glioma-associated human endothelial cell-derived extracellular vesicles specifically promote the tumourigenicity of glioma stem cells via CD9, Oncogene 38 (2019) 6898e6912, https://doi.org/10.1038/s41388-019-0903-6. P.S. Hu, Q.S. Xia, F. Wu, D.k. Li, Y.j. Qi, Y. Hu, Z.Z. Wei, S.S. Li, N.Y. Tian, Q.F. Wei, L.J. Shen, B. Yin, T. Jiang, J.G. Yuan, B.Q. Qiang, W. Han, X.Z. Peng, NSPc1 promotes cancer stem cell self-renewal by repressing the synthesis of alltrans retinoic acid via targeting RDH16 in malignant glioma, Oncogene 36 (2017) 4706e4718, https://doi.org/10.1038/onc.2017.34. ry, S. Amigorena, G. Raposo, A. Clayton, Isolation and characterization of C. The exosomes from cell culture supernatants and biological fluids, Curr. Protoc. Cell Biol. (2006), https://doi.org/10.1002/0471143030.cb0322s30. J. Kowal, G. Arras, M. Colombo, M. Jouve, J.P. Morath, B. Primdal-Bengtson, ry, Proteomic comparison defines novel F. Dingli, D. Loew, M. Tkach, C. The
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
7
markers to characterize heterogeneous populations of extracellular vesicle subtypes, Proc. Nati. Acad. Sci. U. S. A. 113 (2016) 968e977, https://doi.org/ 10.1073/pnas.1521230113. D.C. Davies, Bloodebrain barrier breakdown in septic encephalopathy and brain tumours, J. Anat. 200 (2002) 639e646, https://doi.org/10.1046/j.14697580.2002.00065.x. K. Matsuda, N. Ohga, Y. Hida, C. Muraki, K. Tsuchiya, T. Kurosu, T. Akino, S.C. Shih, Y. Totsuka, M. Klagsbrun, M. Shindoh, K. Hida, Isolated tumor endothelial cells maintain specific character during long-term culture, Biochem. Biophys. Res. Commun. 394 (2010) 947e954, https://doi.org/10.1016/ j.bbrc.2010.03.089. D. Hambardzumyan, O.J. Becher, E.C. Holland, Cancer stem cells and survival pathways, Cell Cycle 7 (2008) 1371e1378, https://doi.org/10.4161/ cc.7.10.5954. T.S. Zhu, M.A. Costello, C.E. Talsma, C.G. Flack, J.G. Crowley, L.L. Hamm, X. He, S.L. Hervey-Jumper, J.A. Heth, K.M. Muraszko, F. DiMeco, A.L. Vescovi, X. Fan, Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells, Canc. Res. 71 (2011) 6061e6072, https://doi.org/10.1158/0008-5472.CAN-10-4269. A.B. Hjelmeland, J.D. Lathia, S. Sathornsumetee, J.N. Rich, Twisted tango: brain tumor neurovascular interactions, Nat. Neurosci. 14 (2011) 1375e1381, https://doi.org/10.1038/nn.2955. W.s.E. Edimo, A.R. Ramos, S. Ghosh, J.-M. Vanderwinden, C. Erneux, The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells, Biochem. Biophys. Res. Commun. 476 (2016) 508e514, https://doi.org/ 10.1016/j.bbrc.2016.05.154. Y. Fan, S.M. Eswarappa, M. Hitomi, P.L. Fox, Myo1c facilitates G-actin transport to the leading edge of migrating endothelial cells, J. Cell Biol. 198 (2012) 47e55, https://doi.org/10.1083/jcb.201111088. H. Brandstaetter, J. Kendrick-Jones, F. Buss, Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion, J. Cell Sci. 125 (2012) 1991e2003, https://doi.org/10.1242/jcs.097212. A. Tiwari, J.J. Jung, S.M. Inamdar, D. Nihalani, A. Choudhury, The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling, Am. J. Physiol. Heart Circ. Physiol. 304 (2013) 687e696, https:// doi.org/10.1152/ajpheart.00744.2012. H. Brandstaetter, J. Kendrick-Jones, F. Buss, Molecular roles of Myo1c function in lipid raft exocytosis, Commun. Integr. Biol. 5 (2012) 508e510, https:// doi.org/10.4161/cib.21201. M.F. Yip, G. Ramm, M. Larance, K.L. Hoehn, M.C. Wagner, M. Guilhaus, D.E. James, CaMKII-mediated phosphorylation of the myosin motor Myo1c is required for insulin-stimulated GLUT4 translocation in adipocytes, Cell Metabol. 8 (2008) 384e398, https://doi.org/10.1016/j.cmet.2008.09.011. A.M. Miller, R.H. Shah, E.I. Pentsova, M. Pourmaleki, S. Briggs, N. Distefano, Y. Zheng, A. Skakodub, S.A. Mehta, C. Campos, et al., Tracking tumour evolution in glioma through liquid biopsies of cerebrospinal fluid, Nature 565 (2019) 654e658, https://doi.org/10.1038/s41586-019-0882-3. A. Mukwaya, P. Mirabelli, A. Lennikov, M. Thangavelu, L. Jensen, B. Peebo, N. Lagali, Repeat corneal neovascularization is characterized by more aggressive inflammation and vessel invasion than in the initial phase, Invest. Ophthalmol. Vis. Sci. 60 (2019) 2990, https://doi.org/10.1167/iovs.19-27591.
Please cite this article as: Y. Tian et al., Glioma-derived endothelial cells promote glioma cells migration via extracellular vesicles-mediated transfer of MYO1C, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.017