Human pluripotent stem cell derived cellular vehicles for cancer gene therapy

HUMAN PLURIPOTENT STEM CELL-DERIVED CELLULAR VEHICLES FOR CANCER GENE THERAPY DANG HOANG LAM NATIONAL UNIVERSITY OF SINGAPORE 2012 HUMAN PLURIPOTENT STEM CELL-DERIVED CELLULAR VEHICLES FOR CANCER GENE THERAPY DANG HOANG LAM (B.Sc., University of Natural Science, Vietnam) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE & INSTITUTE OF BIOENGINEERING AND NANOTECHNOLOGY 2012 ACKNOWLEDGMENTS First and foremost, I would like to express my sincere thankfulness to my scientific supervisor, Dr Wang Shu for his constant and invaluable guidance throughout my PhD study This thesis would not have been completed without his support, understanding and motivation I am so grateful to have opportunities to cooperate with Xiao Ying Bak, Zhao Ying, Yang Jing and Yukti Choudhury It was unforgettable experience working with them and together we discovered meaningful findings which will be presented in this dissertation Thank to all lab members Esther Lee, Ghayathri Balasundaram, Jiakai Lin, Seong Loong Lo, Chrishan Julian Alles Ramachandra, Mohammad Shahbazi, Chunxiao Wu, Kai Ye, Jieming Zeng and Detu Zhu for the help and the laugh they brought in my difficult time of research Thank to Yovita Ida Purwanti and Timothy Kwang for accompanying me during lunchtime Special thank to Thomas Ngo for his help during thesis writing process I am also thankful to my friends including Thuc Nha, Quang Vinh, and Thanh Duong, who always encouraged and supported me during my stay in Singapore Last but most important, this thesis is dedicated to my parents and brother who unconditionally loved me and stood by me in any time of troubles I TABLE OF CONTENTS CONTENTS PAGE Acknowledgments I Table of Contents II Summary VIII List of Publications X List of Tables XI List of Figures XII Abbreviations XIV Chapter Introduction 1.1 Gene therapy in cancer treatment 1.1.1 Gene therapy strategies for cancer treatment 1.1.1.1 Suicide gene therapy 2 1.1.1.1.1 Herpes Simplex Virus Thymidine Kinase/Ganciclovir (HSV-tk/GCV) Prodrug System 1.1.1.1.2 Cytosine Deaminase-5- Fluorocytosine (CD-5-FC) Prodrug System 1.1.1.2 Mutant gene correction/ Tumor suppressor gene replacement 1.1.1.3 Cancer immunotherapy 1.1.1.4 Oncolytic viruses/ Virotherapy 1.1.1.5 RNA interference 10 1.1.1.6 Anti-angiogenic cancer gene therapy 11 II 1.1.1.6.1 Gene therapy to inhibit pro-angiogenic pathways 12 1.1.1.6.2 Gene therapy to stimulate anti-angiogenic pathways 12 1.1.2 Gene carriers for cancer therapy 13 1.1.2.1 Viral vectors 13 1.1.2.2 Nonviral vectors 16 1.2 Stem cells- unique candidates for therapeutic platform 1.2.1 What are stem cells 1.2.1.1 Pluripotent stem cells 19 20 20 1.2.1.1.1 Embryonic stem cells 20 1.2.1.1.2 Induced pluripotent stem cells 21 1.2.1.2 Adult stem cells 23 1.2.1.2.1 Mesenchymal stem cells 23 1.2.1.2.2 Neural stem cells 23 1.2.2 Clinical relevance of stem cell vectors for cancer gene therapy 24 1.2.3 New reliable sources for therapeutic stem cells 26 1.3 Current mouse models in cancer research 28 1.3.1 Syngeneic models 28 1.3.2 Xenogeneic models 29 1.3.3 Genetically engineered models 30 1.4 Objectives 31 References 32 Chapter Materials and Methods 42 2.1 Cell culture 43 III 2.2 Mouse strains 44 2.3 Viral vector preparation and cell transduction 44 2.4 Flow cytometry analysis 46 2.5 Western blotting 46 2.6 Quantitative real-time PCR 47 2.7 Microarray and data analysis 49 2.8 In vitro migration assay 49 2.9 Biophotonic imaging of animals and organs 50 2.10 Intracranial xenografting of human cells 50 2.11 In vivo migration assay 50 2.12 Histology 51 2.13 4T1-luc2 tumor establishment 52 2.14 iPS-NSC transplantation 52 2.15 In vivo 4T1 tumor tropism study 52 2.16 Statistical analysis 53 References 53 Chapter Results: Human Embryonic Stem Cell-derived Mesenchymal and Neural Stem Cells as Cellular Vehicles for Suicide Gene Therapy of Glioblastoma 55 3.1 Introduction and specific aim 56 3.2 hESC-MSCs deliver suicide gene in HSV-tk/GCV system to treat intracranial glioma 3.2.1 In vitro and in vivo glioma tropism of hESC-MSCs 59 59 IV 3.2.2 Transgene expression in hESC-MSCs following baculoviral and lentiviral transductions 60 3.2.3 Intratumor transplantation of HSVtk-expressing hESC-MSCs transduced by baculovirus for anti-glioma therapy 61 3.2.4 Migratory HSVtk-expressing hESC-MSCs transduced by lentivirus for anti-glioma therapy 62 3.3 hESC-NSCs deliver suicide gene in HSV-tk/GCV system to treat intracranial glioma 67 3.3.1 Generation and characterization of NSCs derived from hESCs in adherent monoculture 67 3.3.2 Glioma-specific migration property of NSC1 cells 68 3.3.3 Transgene expression in NSC1 cells using baculoviral vector 69 3.3.4 Tumor killing efficacy of HSVtk-expressing NSC1 cells in glioma mouse model 70 3.4 Immunomodulation effects of MSC and NSC on tumor growth 76 References 78 Chapter Results: Human Induced Pluripotent Stem Cell-derived Neural Stem Cells as Cellular Vehicles for Suicide Gene Therapy of Invasive Glioma and Metastatic Breast Cancer 80 4.1 Introduction and specific aim 81 4.2 Human Induced Pluripotent Stem Cell-derived Neural Stem Cells as Cellular Vehicles for Suicide Gene Therapy of Invasive Glioma 85 4.2.1 Establishment of highly metastatic and invasive glioblastoma model V in mouse using experimental lung metastasis (ELM) assay 85 4.2.2 In vitro and in vivo migration of iPS-NSCs towards invasive 2M1 88 4.2.3 2M1 glioma growth inhibition by engineered iPS-NSCs 89 4.3 Gene therapy application of intravenously injected human iPS-NSCs for metastatic breast cancer 96 4.3.1 In vivo distribution of human iPS-NSCs in normal mice without tumors 96 4.3.2 In vivo distribution of human iPS-NSCs in 4T1 breast tumor-bearing mice 98 4.3.3 Systemic injection of HSVtk-expressing iPSNSCs inhibits 4T1 breast cancer growth and metastasis 99 References 110 Chapter Discussion 112 5.1 hESCs as source for MSCs and NSCs in suicide gene therapy 113 5.1.1 Baculoviruses as gene therapy vectors for human cancer 113 5.1.2 HESC- derived MSCs and NSCs as gene delivery vehicles for glioma therapy 116 5.1.3 NSCs may be a better option for glioma therapy 118 5.2 iPS cells as source for NSCs in treating invasive glioblastoma and metastatic breast cancer 121 5.2.1 2M1 glioma cell line expresses similarities with human GBM 121 5.2.2 iPS-NSCs – promising gene carriers for highly metastatic cancers 122 5.3 General Discussion and Future directions 125 References 127 VI Chapter Conclusion 131 Appendix 136 VII SUMMARY Cancer, also known as malignant neoplasm, is one of the leading causes of morbidity and mortality throughout the world Hitherto cancer therapies that have been widely accepted and practiced are skillful surgery or microsurgery, radiotherapy and chemotherapy or combination Those treatments, however, are too invasive for normal and healthy tissues and lead to further patient weakening and shortened survival By conventional methods, cancer is still a deadly disease with poor prognosis and the outcome is variable and depends on many factors such as age, gender, tumor type and stage Hence, more efficacious system for cancer treatment is desirable Of all candidates, gene therapy to achieve therapeutic effects through transgene expression is a promising treatment Unfortunately, although gene therapy using viral vectors produced encouraging results in various animal models, the same accomplishments have not been observed in clinical trials owing to low gene expression and more importantly poor distribution of vectors in patients Stem cells have recently become an emerging solution to serve as fascinating vehicles for gene delivery in cancer therapy because they show favorable migration towards local and metastatic tumors This allows more specific targeting of transgenes to tumor sites, thus increasing efficacy However, one major issue with stem cell based gene therapy is the use of adult stem cells which causes limited cell sources and variable quality of cells from donors To overcome these obstacles, alternative large scale production of therapeutic stem cells must be identified Pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells may hold the answer to this question VIII thymidine kinase and oncolytic adenoviruses showed an enhanced therapeutic effect (Rogulski et al., 2000) Second, the use of pluripotent stem cells has certain risks Pluripotent stem cells are known for the ability to form teratomas in transplanted animals (Jung et al., 2012) Hence, methods that ensure culture purity need to be developed as the contamination of undifferentiated pluripotent stem cells in the culture could potentially cause tumorigenesis in downstream applications Besides that, the generation of iPS cells with retroviral and lentiviral vectors provide high efficiency, but carry the risk of genome integration possibly causing gene disruption and oncogene activation (Jung et al., 2012) Recent papers have proposed many approaches that avoid transgene integration or pesistence such as adenoviral vector transductions, DNA plasmid vector transfections, Cre-LoxP excision of reprogramming vector cassettes transferred by lentiviral vectors, transposons, episomal Epstein-Barr virus, mRNA transfections, and protein transfections (Jung et al., 2012; González et al., 2011) Third, to some extent, suicide gene therapy may introduce cytotoxicity in healthy tissue In glioblastoma treatment, stem cells, which are not able to reach or en route to the tumors, still release therapeutic drugs Moreover, the majority of stem cells through systemic delivery in breast cancer therapy will be trapped in the lung and liver because of the narrow diameters of lung capillaries and liver sinusoids (Meyerrose et al., 2007; Kidd et al., 2009; Fischer et al., 2009) While this could be useful in eliminating metastatic cancer cells in the organs, it may also lead to damage for healthy cells To ameliorate this undesirable problem that may exasperate the patient’s health, it would 126    be important to employ an inducible regulatory system that triggers transgene expression in the injected stem cells in a tumor-specific manner A number of cancerspecific promoters, that may be useful for this purpose, have been reported, such as those of the probasin, human TERT, surviving, ceruloplasmin, HER-2, osteocalcin, and carcinoembryonic antigen (Hui-Wen Lo et al., 2005) Another option is based on the fact that hypoxia is a key factor in determining directed migration of NSCs to tumors (Zhao et al., 2008) We are currently developing a baculoviral transgene expression cassette containing hypoxia-responsive elements to control the expression of therapeutic genes References Aboody, K S., Brown, A., Rainov, N G., Bower, K A., Liu, S., Yang, W., Small, J E., Herrlinger, U., Ourednik, V., and Black, P M., et al (2000) Neural stem cells display extensive tropism for pathology in adult brain: 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Polewski, M D., Gilchrist, M., Glackin, C A., and Kim, S U., et al (2012) Human neural stem cell tropism to metastatic breast cancer Stem Cells 30, 314-325 Zhao, D., Najbauer, J., Garcia, E., Metz, M Z., Gutova, M., Glackin, C A., Kim, S U., and Aboody, K S (2008) Neural stem cell tropism to glioma: critical role of tumor hypoxia Mol Cancer Res 6, 1819-1829 Zhao, Y., Lam, D H., Yang, J., Lin, J., Tham, C K., Ng, W H., and Wang, S (2012) Targeted suicide gene therapy for glioma using human embryonic stem cell-derived neural stem cells genetically modified by baculoviral vectors Gene Ther 19, 189-200 130    CHAPTER CONCLUSION 131    This study aimed to develop alternative, large scale cell sources of mesenchymal stem cells (MSCs) and neural stem cells (NSCs) for cancer gene therapy with pluripotent stem cells It was proved that cells derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS cells) were capable of targeting tumor foci and effectively inhibiting the tumor growth when transduced with baculoviral vectors carrying HSVtk gene As described in Chapter 3, we assessed the efficacy of hESC-MSCs and -NSCs in glioma therapy Both cell lines were modified to express HSVtk gene under CMV promoter and introduced into the brain of nude mice intra-tumorally and contra-laterally after the tumor formation by U87-luciferase cell inoculation All data including tumor volume monitored under IVIS device and histological analysis with H&E staining showed that tumors remained remarkably smaller in the groups with modified hESCNSCs and -MSCs compared with control groups Furthermore, survival analysis confirmed that the shrinkage of tumor size in treatment groups resulted in significantly prolonging animal lifespan It is possible that achieved therapeutic effects were due to preferential migration of NSCs and MSCs toward glioma tumors as histological analysis from harvested brain tissue displayed streams of MSCs and NSCs migrating towards tumor beds These results suggest that cells derived from embryonic stem cells are competent as cellular vehicles to deliver suicide gene for glioblastoma treatment In comparison with previous studies using adult sources, the obtained survival data in tumor bearing animals indicate a similar efficacy by hESC- derived cells Strong immunosuppressive properties toward lymphocytes of either adaptive or innate immune systems by mesenchymal stem cells have been well documented in various 132    studies Furthermore, other studies have demonstrated that MSCs facilitate tumor growth by modulating immune system Recently, there was evidence that NSCs may also influence anti-inflammatory activity through T-cell suppression This can be somewhat of a double - edged sword when those stem cells are introduced into patients for cancer therapy With this in mind, our experiments were conducted to verify whether NSCs favor tumor progression in the same manner with MSCs We have shown for the first time that the tumor support leads to animal death in a short period by both hESCMSCs and NSCs in 4T1 tumor model In the case of MSCs, their powerful effects on tumor growth observed in this study are consistent with the latest reports NSCs, however, appeared to have less impact on tumor development Further research needs to be done to verify this issue.These understandings will serve as useful guidelines to pick the right cell lines for cancer therapy From supporting data to prove hESC derived cells as efficient gene delivery vectors, we moved forward to confirm the feasibility of using iPS-NSCs for invasive glioma and metastatic breast cancer as shown in Chapter To evaluate iPS-NSC tumor killing effects in the situation close to patient conditions, we established a highly metastatic and invasive glioblastoma model in mouse using experimental lung metastasis (ELM) assay By enriching metastatic cells from highly homogenous glioma cells through tail vein injection and pulmonary lesion collection, invasive 2M1 cell line was created and showed characteristics of high grade glioma such as pseudo-palisading necrosis, infiltrating tumors and multiple foci As current xenograft mouse models for human GBM failed to recapitulate the human disease owing to slow growth and invasiveness, the invasive model developed here provided feasible solution to investigate anti-glioma 133    activity not only by NSCs but also by other agents in upcoming research Following the production of 2M1 cells, we performed in vivo therapeutic effect experiment with intratumoral iPS-NSCs The outcome from survival analysis showed that injected iPS-NSCs inhibited the growth of 2M1 cells in brain tissue This suggests that iPS-NSCs would function similarly to hESC-NSCs Interestingly, when experimenting with iPS-NSC migration towards 4T1 breast cancer cells in vitro by Boyden chamber assay, we discovered that breast cancer did attract iPS-NSCs Moreover, further investigations demonstrated a high accumulation of iPSNSCs in tumor at mammary fatpad region Thus, we proposed that iPS-NSCs should be exploited for breast cancer inhibition Combining with HSV-tk gene system, our iPSNSCs, indeed proved that they could delay 4T1 breast cancer development This data provides direct in vivo evidence of tumor tropism of systemically injected human iPS cell -NSCs for breast cancer using dual color imaging and demonstrates a cancer gene therapy application of these cells Collectively, this thesis has demonstrated that pluripotent stem cells would be attractive alternatives to produce uniform cellular vehicles on a large scale for cancer treatment However, it should be noted that this study does not cover the perspective of probable tumorigenesis from pluripotent cell sources It is well known that ESCs and iPS cells form teratoma in transplanted mice This can be a big safety concern if there are undifferentiated cells remaining after differentiation from pluripotent stem cells Hence, further research should be done to develop a method for complete removal of undifferentiated cells prior to clinical application Another future direction is to generate regulation systems that function in these derived cells to prevent off-target expression of 134    a therapeutic gene in non-tumor regions and minimize possible side effects Furthermore, downstream studies to explain underlying molecular mechanism of migratory capabilities by derived stem cells need to be done as this understanding would help to enhance stem cell migration towards tumor Along with recent FDA approval of two clinical trials for human ESC derivatives, results from this thesis opens up new opportunities for more effective cancer treatment 135    Appendix Analyses from REMBRANDT database showed the clinical importance of genes overexpressed in 2M1 CDH11 CHI3L1 136 HGF LAMA4 137 PGF S100A4 138 LOX ANGPT1 139 FN1 JAG1 140 ... Ng W H , Wang S 4 .Human embryonic stem cell- derived mesenchymal stem cells as cellular delivery vehicles for prodrug gene therapy of glioblastoma Human gene therapy , Volume 22 , Issue 11 (2011)... Pluripotent stem cells 19 20 20 1.2.1.1.1 Embryonic stem cells 20 1.2.1.1.2 Induced pluripotent stem cells 21 1.2.1.2 Adult stem cells 23 1.2.1.2.1 Mesenchymal stem cells 23 1.2.1.2.2 Neural stem cells... source for MSCs and NSCs in suicide gene therapy 113 5.1.1 Baculoviruses as gene therapy vectors for human cancer 113 5.1.2 HESC- derived MSCs and NSCs as gene delivery vehicles for glioma therapy