Investigation of Surface Glycans on Glioblastoma multiforme and Its Stem Cells

多形性神經膠母細胞瘤被世界衛生組織定義為第四級的星狀細胞瘤，是最常見也是最惡性的成人腦瘤。根據統計，即使接受過手術切除、放射線治療和化學治療，病人的中位存活時間仍只有14.6個月，癒後情況相當差。因此，針對此腫瘤發展一個新的治療方式是相當重要的。我們在17株多型性神經膠母細胞瘤細胞株上利用流式細胞技術分析其表面醣分子之表現，結果顯示其中12株具有階段特異性胚胎抗原-4(SSEA-4)之表現。而高效能薄層層析免疫染色與基質輔助雷射脫附游離質譜分析結果確定了SSEA-4分子的存在與結構。我們利用免疫組織化學染色法分析病人檢體顯示著有百分之六十九的病人其腦癌細胞具有SSEA-4的表現。相比之下，正常人類大腦組織則鮮少有SSEA-4的表現。此外，免疫組織化學染色結果指出SSEA-4的表現與星狀細胞瘤惡化程度有著成正比的關係。接著，我們利用SSEA-4的單株抗體(MC813-70)可以在體外誘發補體依賴型細胞毒殺作用在多型性神經膠母細胞瘤細胞上而引發細胞死亡。並且在實驗老鼠動物上，SSEA-4的單株抗體能有效的抑制腫瘤的生長。進一步的實驗指出SSEA-4不僅表現在多型性神經膠母細胞瘤，我們甚至觀察到SSEA-4表現在許多不同癌症的細胞株上，因此我們認為針對SSEA-4發展治療抗體與疫苗是一個有潛力的方向。而在另一方面，我們成功地產生一群具有較高幹細胞特性的癌細胞群。而為了去找尋多形性神經膠母細胞瘤幹細胞上是否具有特定分子的表現，我們利用抗體染色去進行多種醣類的比較，這包括了醣蛋白和醣脂質。在這些被偵測的醣分子中，我們發現一神經節苷脂GD2大量表現在多形性神經膠母細胞瘤幹細胞上，更多的實驗將會闡明GD2與多形性神經膠母細胞瘤幹細胞的關係。Glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and aggressive brain tumor in adults. Despite advances in medical managements, the survival rate of GBM patients remains poor, suggesting that identification of GBM-specific targets for therapeutic development is urgently needed. Analysis of several glycan antigens on GBM cell lines revealed that 12 out of 17 GBM cell lines were positive for stage-specific embryonic antigen-4 (SSEA-4), and the identity of SSEA-4 was confirmed by high performance thin-layer chromatography immunostaining and mass spectrometry. Immunohistochemical staining confirmed that 38/55 (69%) of human GBM specimens, but not normal brain tissue, were SSEA-4-positive, and correlated with high-grade astrocytoma. MC813-70, an SSEA-4 specific monoclonal antibody, was found to induce complement-dependent cytotoxicity against SSEA-4hi GBM cell lines in vitro, and suppressed GBM tumor growth in mice. Since SSEA-4 is expressed on GBM and many other types of cancers, but not on normal cells, it could be a target for development of therapeutic antibodies and vaccines. On the other hand, we successfully generated a population of GBM cells with higher stemness and tumorigenicity. In order to discover the specific markers expressed on GBM stem cells, we utilized a panel of glycan-related antibodies to profile the expression of glycan epitopes, including glycoproteins or glycolipids. Among these glycans, the expression of GD2 was found to be elevated on stem-like cell population. Further study would illustrate the relationship between GD2 and GBM stem cells.口試委員會審定書…………………………………………………………………........i 誌謝………………………………………………..…..………………………………...ii 摘要……………………………………………………..…..…………………………..iii Abstract…………………………………...……………………………………………..iv Table of Contents………………………………………………………………………...v List of Figures…………………………………………………………………………viii List of Supplementary Figures………………………………………………………….ix List of Tables……………………………………………………………………………ix Abbreviations…………………………………………………………………………….x CHAPTER1: INTRODUCTION…………………………………………..…………….1 1.1 Perspective……………………………………………………………………...2 1.2 Overview of glioblastoma multiforme…………………………………………2 1.3 Glycosylation…………………………………………………………………...6 1.4 Glycosphingolipids………………………………………………………..……7 1.5 Globo-series GSLs……………………………………………………………...9 1.6 Glycans in cancers………………………………………………………….....10 1.7 Glioma-associated GSLs………………………………………………….......13 1.8 Target therapy of GBM………………………………………………………..14 1.9 GBM stem cells……………………………………………………………….15 1.10 Significance and rationale…………………………………………………...17 CHAPTER 2: MATERAILS AND METHODS………………………………………..18 2.1 Reagents………………………………………………………………………19 2.2 Flow Cytometry………………………………………………………………19 2.3 Cell Culture……………………………………………………………….......20 2.4 Immunofluorescent Staining………………………………………………….21 2.5 Immunohistochemistry……………………………………………………….21 2.6 Glycan Array Fabrication…………………………………………………….22 2.7 Antibody Binding Assay……………………………………………………...22 2.8 Sialidase Treatment…………………………………………………………...23 2.9 Extraction of Glycosphingolipids…………………………………………….23 2.10 High-Performance Thin-Layer Chromatography…………………………...24 2.11 TLC Immunostaining………………………………………………………..25 2.12 MALDI-MS Profiling and MS/MS Analysis………………………………..25 2.13 Complement-Dependent Cytotoxicity Assay…………………………….....26 2.14 In vivo Tumor Growth……………………………………………………....26 2.15 Neurosphere culture…………………………………………………………27 2.16 Quantitative real- time polymerase chain reaction……………………….....28 2.17 SDS-PAGE and western blot………………………………………………..28 CHAPTER 3: RESULTS…………………………………………………………….....30 3.1 Flow cytometric analysis of glycan epitopes revealed MC813-70 binds to GBM cell lines…………………………………………………………………………...31 3.2 Examination of MC813-70 specificity by glycan microarray………………32 3.3 Verification of MC813-70 staining on GBM cell lines by HPTLC immunostaining…………………………………………………………………...33 3.4 Sialidase treatment confirms the structure of MC813-70 antigen as an α2,3-sialyl globo-series GSL……………………………………………………...34 3.5 Analysis of DBTRG gangliosides by mass spectrometry indicates the presence of SSEA-4 glycolipid………………………………………………………….......35 3.6 Expression of SSEA-4 in GBM tissues……………………………………….36 3.7 MC813-70 Mediates CDC against GBM Cell Lines………………………….37 3.8 MC813-70 Suppresses Brain Tumor Growth in Vivo…………………….......38 3.9 Expression of SSEA-4 in Various Cancer Cell Lines…………………………38 3.10 Cells maintained in in vitro neurosphere culture expressed higher levels of stemness genes…………………………………………………………………….40 3.11 Neurosphere cells show higher potential of self-renewal and tumorogenicity…………………………………………………………………….41 3.12 Expression of GD2 is increased in neurosphere cells………………………..41 CHAPTER 4: DISCUSSION…………………………………………………………..43 CHAPTER 5: FIGURES……………………………………………………………….50 CHAPTER 6: SUPPLEMENTARY FIGURES………………………………………...74 CHAPTER 7: TABLES………………………………………………………………...77 CHAPTER 8: REFERENCES………………………………………………………….85 APPENDIX…………………………………………………………………………...106 The manuscript published on-line in Proceedings of the National Academy of Sciences of the United States of Americ