HEPATOCYTE GROWTH FACTOR IS a MAJOR CYTOKINE FOR NSC HOMING TO GLIOMA

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HEPATOCYTE GROWTH FACTOR IS a MAJOR CYTOKINE FOR NSC HOMING TO GLIOMA

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HEPATOCYTE GROWTH FACTOR IS A MAJOR CHEMOTACTIC FACTOR FOR NEURAL STEM CELLS MIGRATION TO GLIOMA HUANG SIHUA (B.ENG. (Hons), NTU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DPARTMENT OF PHYSIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2015 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information that have been used in thesis. This thesis also not been submitted for any degree in any university previously. ________________________ Huang Sihua 16th Jan 2015 I Acknowledgements This dissertation would not have been possible without the opportunity given by Singapore-MIT Alliance of Research and Technology (SMART), Yong Loo Lin School of Medicine, and the constant support and encouragement from the following people: First and foremost, I would like to thank my supervisors Professor Roger Kamm and Professor Hanry Yu. In spite of his busy schedule, Professor Kamm always made time to talk to me about my problems in research. He promptly and carefully replies all our email requests. He will not blame me when the experiments are not working out. Instead he is always there to help and guide me through my difficulties. I am truly thankful to Professor Kamm for bringing me the courage and inspiration to pursue my studies. Professor Hanry Yu is my local supervisor. He cares for students in a professional and personal level. He would discuss with us about our projects for hours until we have a satisfying answer. He patiently teaches us the skills we needed in research and academia. He has even made time to teach me the specific details on how to write a paper. Professor Yu has given me valuable suggestions and creative ways to solve the problems. He has supported me in so many different ways. I would also like to thank my colleagues in BioSystems and Micromechanics unit (BioSyM) under the Singapore-MIT Alliance of Research and Technology (SMART), Yu Lab (Physiology, NUS), and Kamm Lab (Bioengineering, MIT). My special thanks go to: Dr. Poon Zhiyong, Dr. Lim Sei Hien, Dr. Zhou Yan, Mr. Tu II Ting Yuan, Dr. Andrea Pavasi, Mr. Ng Inn Chuan, Dr. Jacky Lee, Dr. Tan Chin Wen, Mr. Evan Tan, Ms. Michelle Chen, Mr. Kwok Chee Keong, and Ms. Averil Chen, who have been there to assist me strategically, technically, and socially. III Table of Contents Declaration I Acknowledgements II Table of Contents . IV Summary VII List of Tables . IX List of Figures . X CHAPTER INTRODUCTION .1 1.1 Brief background on neural stem cells . 1.1.1 Origin . 1.1.2 Sources of Neural stem cells 1.1.3 Therapeutic use of neural stem cells 1.2 Glioma . 1.2.1 Types of Glioma 1.2.2 Therapies to treat glioma: traditional and new 1.3 Glioma tropism of neural stem cells . 12 CHAPTER . 16 LITERATURE REVIEW 16 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 Cytokines involved in NSC tumor homing . 16 SDF1α/CXCR4 18 VEGF 20 uPA 21 IL-6 21 SCF/c-kit 23 HMGB1 . 24 EGF . 25 HGF/c-Met . 25 MCP1/CCR2 27 Glioma-produced ECM 27 Summary and Outlook 28 CHAPTER . 30 OBJECTIVES 30 3.1 Specific Aim One: Develop in vitro Assays to Study NSC Homing to Glioma31 3.2 Specific Aim Two: Identify the major homing signals using in vitro experiments in terms of gene and protein expression levels in glioma cells versus astrocytes 32 3.3 Specific Aim 3: Validation of the identified major NSC homing signal(s) in vitro and in vivo . 33 CHAPTER . 34 NEURAL STEM CELLS DISPLAY TROPISM TOWARDS GLIOMA IN TRADITIONAL TRANSWELL ASSAYS AND MICROFLUIDIC DEVICES 34 4.1 4.2 Introduction . 34 Materials and Methods . 35 IV 4.2.1 Cell culture 35 4.2.2 Conditioned medium . 37 4.2.3 Transwell Assay 37 4.2.4 Microfluidic devices . 38 4.2.5 Seeding NSCs into microfluidic devices 39 4.2.6 Statistical Analysis . 40 4.3 Results and Discussion . 40 4.3.1 U87 glioma conditioned media induces NSC migration. . 40 4.4 Conclusions 48 CHAPTER . 50 HGF, VEGF, AND IL6 ARE CANDIDATE CYTOKINES THAT REGULATE NSC HOMING TO GLIOMA; . 50 HGF IS THE MOST PROMISING SIGNAL . 50 5.1 Introduction . 50 5.2 Materials & Methods . 52 5.2.1 Cell culture 52 5.2.2 Conditioned media . 52 5.2.3 Transwell Assay 52 5.2.4 Reverse transcription-PCR 53 5.2.5 ELISA (Sandwich Enzyme-Linked Immunosorbent Assay) . 55 5.2.6 Flow Cytometry . 55 5.2.7 Statistical Analysis . 56 5.3 Results & Discussion . 56 5.3.1 HGF, VEGF, and IL6 were upregulated in U87 glioma cells comparing to normal astrocytes. . 56 5.3.2 HGF has higher chemotactic potency than VEGF and IL6 . 59 5.3.3 NSCs express receptors for HGF, VEGF, and IL-6 64 5.3.4 Among three candidate signals, HGF induced maximum migration of NSCs in transwell assay. 66 5.4 Conclusions 73 CHAPTER . 75 BLOCKING HGF RECEPTOR INHIBITS NSC TROPISM TO GLIOMA; NSCs ARE CHEMOTACTIC TO HGF IN LIVE CONDITION 75 6.1 Introduction . 75 6.2 Materials & Methods . 76 6.2.1 Transwell Assay – Blocking 76 6.2.2 Dunn’s chamber 77 6.3 Results & Discussion . 78 6.3.1 Blockades of CMET (HGF receptor) and VEGFR2 (VEGF receptor) were able to reduce NSC migration. Blocking of CMET imposed greater inhibition on NSC migration than blocking of VEGFR2. 78 6.3.2 NSCs are chemotactic to HGF gradient in Dunn Chamber 84 6.4 Conclusions 89 Chapter 91 Additional Results & Future Experiments 91 7.1 Summary . 91 7.2 Methodology 94 7.2.1 Endothelial monolayer . 94 7.2.2 Vascular network & NSC infusion 94 7.2.3 Transfecting U87 cells with RFP . 96 V 7.2.4 In-vivo protocol . 97 7.3 Additional Results, Discussion & Future Plan . 98 7.3.1 Forming endothelial monolayer in microfluidic devices . 98 7.3.2 Forming vascular network within the 3-D gel region of microfluidic devices 101 7.3.3 Observing NSC extravasation from the vascular network and homing to glioma 103 7.3.4 Observing NSC homing to glioma in vivo 106 7.4 Future explorations 108 References 109 VI Summary Neural stem cell (NSC) homing to brain tumor has been exploited for targeted gene therapy, but the underlying mechanism remains unclear. Various signals were proposed by the literature. We hypothesize that among the known signals we chose from the literature, there would be a major homing signal regulating NSC homing to glioma. Our first specific aim is to observe the homing of NSCs towards glioma in vivo and in vitro. Transwell and microfluidic assays are used and homing was confirmed in both assays. Our second specific aim is to determine the major cytokine(s) that regulates NSC glioma tropism among the known cytokines. We screened these factors for up-regulated gene expression in U87 glioma cells relative to non-cancerous astrocytes. Only VEGF, IL6, and HGF were up-regulated in U87 glioma cells, among which HGF showed the highest up-regulation of >400 fold over astrocytes, versus fold for VEGF and 10 fold for IL6. Similar trends were observed in protein expression measured by ELISA. In transwell assays, HGF induced significant NSC migration relative to VEGF and IL6. Through FACS experiment we also found that NSCs have the highest expression of HGF receptor over the receptors for VEGF and IL6. VII Our third specific aim is to further explore the role HGF plays in NSC homing to glioma. We found that blocking HGF receptor inhibited NSC migration towards glioma conditioned medium. In live imaging, NSCs migrated along HGF gradient. We conclude that HGF is a major chemoattractant in NSC homing to glioma. VIII List of Tables Table 1. List of pre-clinical trials that make use of NSCs as cellular carriers to deliver therapeutic transgene into tumor bearing mice . 13 Table 2. List of important signaling pathways involved in NSC homing to glioma . 18 Table 3. Primer sequences of various cytokines used for RT-PCR experiments . 54 Table 4. Amount of HGF, VEGF, SDF-1, and IL-6 secreted by astrocytes and U87 glioma cells . 62 Table 5. List of dissociation constants of HGF, VEGF, and IL6 . 63 Table 6. Molecular Weight of HGF, VEGF, and IL6 68 Table 7. Parameter of migration in control condition and HGF gradient . 87 IX 1) forming a monolayer inside the channel of the device Because endothelial cells have the natural tendency to grow confluent and form junctions among them, we can try to form the monolayer in the channel and introduces NSCs into the monolayer 2) simulate a 3-D vascular network in the gel region of the device When endothelial cells were seeded into the gel, they will self-organize and form vascular network in the 3-D space. The network has openings to the channel, where we can introduce NSCs and observe their extravasation. A C B D channel (monolayer) gel region Figure 20. Forming endothelial monolayer in the channel of microfluidic devices. 2.5M/ml of endothelial cells were seeded in Matrigel coated microfludic channel. days after seeding, cells were fixed and stained with DAPI. A) Top view of the monolayer. B) Side view of the monolayer. C) Picture of device. D) Schematic view of high-throughput microfluidic device 99 The result of forming endothelial monolayer in the channel can be found in Figure 20. FigureA shows the top view of the monolayer, which is quite homogeneously distributed. To make sure the endothelial cells have covered the whole channel, a cross-sectional slice view was attached the right end of Figure 20A. We could see that the cells almost formed a rectangle that covers the whole channel including the sidewalls, top, and bottom of the channel. Figure B is the side view of the channel. However there are limitations of this method. It is hard to make sure the monolayer is intact enough to simulate a blood vessel. If there is a slight opening at the side of the layer, NSCs will be allowed to “escape” rather than extravasate. Therefore we used our second method, which is to simulate a 3-D vascular network in the gel region of microfluidic devices. 100 7.3.2 Forming vascular network within the 3-D gel region of microfluidic devices Figure 21. A) Schematic diagram of 3-gel device (top view). B) Schematic diagram of 3-gel device (side view). C) Picture of 3-gel microfluidic device. D) Endothelial cells form vascular network in the gel region of microfluidic devices. E) Endothelial cells form vascular network under the presence of U87 glioma cells. F) 3-D image of glioma vasculature. G) Projected image of glioma vasculature. Green: VE-cadherin; Red: U87-RFP cells; Blue: DAPI; White: Phalloidin. Figure 21 has shown the vascular network formed in a 3-D gel. With endothelial cells alone, we are able to form vascular network that are quite 101 well connected, regular in shape, and forms opening at the end to enable infusion of other cell types (Figure 20A). When U87 cells were injected together with endothelial cells, the vascular network became densier and more complex (Figure 20B), which we refer to as glioma vasculature. When we look at the glioma vasculature closer under 60X enlargement (Figure 20C), we can see clearly the formed lumen shown in green and white. The red U87 RFP cells always lie outside the lumen. This can be confirmed by looking at the projected image (Figure 20D). When the 3-D image were projected at X and Y plane, we can see a clear round shape lumen in green, and the red U87 cells always lie outside the lumen. This is an interesting and important observation. We not know the reason why U87 cells will naturally lie outside the vascular network when they are mixed and injected together with endothelial cells. The same phenomenon was observed when we inject endothelial cells and mesenchymal stem cells together. Having the model of U87 cells spreaded outside the lumen helps to lay the foundation of our future study where NSCs will be infused inside the lumen. They will be observed whether to cross the endothelial barrier and home to the glioma. 102 7.3.3 Observing NSC extravasation from the vascular network and homing to glioma A 0.5hr D C B 1.5hr 2.5hr 3.5hr Figure 22. Process of NSC extravasation from endothelial network and homing towards U87 cells. Image A, B, C, D represent images taken at 0.5 hr, 1.5hr, 2.5hr, and 3.5hr after introducing NSCs into the glioma vasculature. Red: endothelial cells with RFP; Green: NSC GFP Figure 22 has shown the process of NSC crossing the endothelial barrier and homing to glioma. NSCs were infused into the vascular network through the openings of the network to the microfluidic channel. 0.5hr after infusion, NSCs were stabilized and stay inside the lumen (Figure 22A). 1.5hr after, an NSC started to migrate out of the lumen by squeezing part of itself through the vascular wall (Figure 22B). 2.5hr after, almost the whole cell had reached outside the lumen and attached to the outer wall of the lumen (Figure 22C). 3.5 hr after infusion, the NSC was leaving the outer wall and migrating into the gel region where there are plenty of U87 cells (not shown) (Figure 22D). 103 A B Figure 23. A) 3-D image showing NSCs escaping from the vascular network and homing to glioma cells. B) Projection image showing NSCs escaping from vascular network and homing to glioma cells. Red: U87 RFP cells; Green: NSC GFP cells; Blue: DAPI; White: Phalloidin It is also shown in Figure23 that NSCs cross the endothelial barrier and home to glioma. The green NSCs are lying both in and outside the lumen. Some of them are part of the lumen because they are in the process and extravasation. As shown in Figure 23B, the projected lumen and NSCs are shown in the image on the right and at the bottom. When we refer to the image at the bottom, on NSC is attaching to the outer wall of the lumen while the other NSC was integrating into the wall of another lumen. Among the pre-clinical studies that use NSCs to deliver tumor-killing drugs, most of them inject the NSCs intracranially [84, 85]. During their intracranial migration towards the tumor, we are not sure whether these cells cross the brain endothelium. However, several studies have injected NSCs intravascularly [37, 40, 86], which requires the NSCs to cross the endothelial wall or even the blood brain barrier. Therefore if we can understand the process of 104 NSC crossing endothelial barrier, we can improve the therapy because currently intravascular injection is much less efficient then intracranial injection. However, intravascular injection is much safer and more convenient which doesn’t need brain surgery. Here using microfluidic devices, we have successfully demonstrated the process of NSC crossing endothelial barrier. We used a 3-D condition simulating in-vivo situation, which is a step further than the study of NSC crossing simulated endothelial barrier in transwell system [87]. For future study, we plan to block the c-MET receptor and see it inhibits the ability for NSCs to cross the endothelial barrier and home to glioma. 105 7.3.4 Observing NSC homing to glioma in vivo A C B Figure 24. A) The set-up of in vivo experiment using stereotaxic instrument. B) Isolated mouse brain with established glioma shown in red. C) Migration of NSCs (Red) from the original cell mass towards the established glioma (unseen) at the right frontal lobe. Figure 24A has shown the setup of the in vivo experiment using stereotaxic instrument. We have successfully implanted and grown glioma in the mouse brain shown in red color in Figure 24B. U87 cells tagged with RFP were injected and 25 days later when glioma was formed. However, the RFP signal was not strong enough to penetrate the skull; therefore we had to take the brain out to image the tumor mass. After the glioma was formed at the right frontal lobe the mouse brain, NSCs stained with xenolight DIR were injected into the left frontal lobe. days after NSC injection, image was taken to track the location of NSCs (Figure 24C). NSCs stained with Xenolight DIR were depicted in red and glioma cells were not shown here but they were present 106 at the right frontal lobe. As shown in the figure, part of NSCs left the main cell mass at the injection site on the left and migrates towards the tumor on the right. Homing of NSCs was observed towards glioma inside the in-vivo model. RFP signal was previously not strong enough to penetrate the skull. Xenolight DIR is in the infrared light range therefore has much better penetration ability than RFP. It was reported in other pre-clinical studies that NSCs home to glioma [29, 85, 88]. We confirmed the results in our in-vivo model. Following up these results, we plan to block the c-MET receptor using si-RNA or functional blocking and observe whether it impair the homing of NSCs. If the migration of NSCs was significantly reduced due to the blocking of c-MET, we could prove in vivo that HGF is a major cytokine for NSC homing to glioma. 107 7.4 Future explorations In addition to following up these experiments we have tried, there are a few directions we can pursue for future experiments. Firstly, we can test other glioma cell lines to see whether they also secrete high level of HGF. If HGF is the only major cytokine for NSC homing, types of tumors that not secrete HGF may not be the best option to receive NSC-mediated therapy. Secondly, we can over-express c-MET receptor on NSC to see whether homing will be increased in vitro and in vivo. Thirdly, mesenchymal stem cells (MSCs) can be used in the study as a competitor of NSCs because MSCs were also reported to home to tumor [89]. MSCs are more abundant in number but they are not of neuron origin, which may cause problems. Lastly, we may also incorporate the cytotoxic gene in NSCs to see how effectively they kill glioma cells. Then we modify the sensitivity of HGF by overexpressing or blocking c-MET receptors on NSCs, to see how it affects the tumor killing efficiency. 108 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Andressen, C., Neural stem cells: from neurobiology to clinical applications. 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Gene Therapy (Nature Publishing Group), 2008. 15: p. 739-752. 114 [...]... of atypical and mitotic cells Grade 4 tumors are the most malignant and happen most frequently They include glioblastoma and 7 gliosarcoma They have properties of micro-vascular proliferation or pseudopalisading necrosis [17] 1.2.2 Therapies to treat glioma: traditional and new The current standard of care for patients of glioma includes maximal safe resection, followed by radiation therapy (RT) to. .. (SDF1), hepatocyte growth factor (HGF), monocyte chemoattractant protein-1 (MCP-1), urokinase plasminogen activator (uPA), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), stem cell factor (SCF), epidermal growth factor (EGF), and high mobility group box 1 protein (HMGB1) SDF1 is an inflammatory chemoattractant that can be sensed by NSCs through CX chemokine receptor 4 (CXCR4) NSCs have... Therefore the study concludes that uPA activation of uPAR in cancer cells is a critical mechanism in regulating stem cell tropism to malignant solid tumors [48] 2.5 IL-6 Zhao et al [47] has identified IL-6 as a major cytokine responsible for NSC homing to breast cancer cells Firstly, cytokine levels in breast cancer cellconditioned media were detected by cytokine antibody arrays It was found that Il-6 and... barrier provides an additional advantage in treating CNS related cancer [33] Researchers have been using NSCs as a cellular agent to carry transgenic products and target the tumor at the site Since brain tumor is highly migratory, selective targeting of the residue by NSCs has the great advantage of getting to the cancer cells without damaging the healthy brain tissue 12 Figure 2 Cell based anti-cancer... with a slow proliferation rate It includes the most common glioma of children: pilocytic astrocytoma Grade 2 glioma includes astrocytoma, oligodendroglioma, and oligoastrocytoma They also have relatively slow growth rates but these tumors start to diffuse to normal brain and have a higher potential of malignant progression Grade 3 tumor is characterized by a higher cellular density and the existence... external beam radiation therapy to the surgical resection cavity and to a 2cm margin of surrounding 8 brain tissue [21] Ionizing radiation induces single strand and double strand DNA breaks in proliferating cells TMZ is an oral alkylating chemotherapeutic agent It derives its therapeutic effect from adding a methyl group to purine bases of DNA, causes DNA damage, and triggers a cascade of events, leading... therapeutics Transplanted neural stem cells (NSCs) homing to tumor cells in rodent models of brain neoplasia [34] A number of pre-clinical studies have been conducted to evaluate NSCs as tumor selective therapy Various rodent and human NSC cell lines are administered into a mouse model bearing glioma either intracranially or through the circulation NSCs carry cytotoxic genes or an enzyme that transforms... and extracellular peptides [22, 24] These molecular targeted therapies can inhibit growth factor pathways such as EGFR pathway Amplification of EGFR signaling is one the most common genetic alteration seen in GBM [25] Some of them inhibit angiogenic 9 pathways Glioma is a highly vascularized tumor characterized by extensive angiogenesis VEGF is critical for angiogenesis and is highly expressed in glioma. .. common primary intracranial tumor 7 out of 100,000 individuals are diagnosed with glioma every year Despite tremendous efforts to understand the development of the disease, this is still no cure And the median survival rate for patients with glioma is 12-15 months [16, 17] 1.2.1 Types of Glioma According to the World Health Organization (WHO), gliomas are graded from 1 to 4 Grade 1 glioma is benign and circumscribed... tumorlocalized chemotherapy against brain tumor, NSCs are genetically engineered to carry enzymes, which can transform systematically or orally administered 6 prodrug into an active form at the tumor foci and kill the tumor cells In this study, we focus on tumor-localized chemotherapy and we aim to improve the therapy through studying of the mechanisms of homing to brain cancers 1.2 Glioma Glioma is the . HEPATOCYTE GROWTH FACTOR IS A MAJOR CHEMOTACTIC FACTOR FOR NEURAL STEM CELLS MIGRATION TO GLIOMA HUANG SIHUA (B.ENG. (Hons), NTU) A THESIS SUBMITTED FOR THE DEGREE. migrated along HGF gradient. We conclude that HGF is a major chemoattractant in NSC homing to glioma. IX List of Tables Table 1. List of pre-clinical trials that make use of NSCs as. microfluidic assays are used and homing was confirmed in both assays. Our second specific aim is to determine the major cytokine( s) that regulates NSC glioma tropism among the 9 known cytokines.

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