臺灣博碩士論文加值系統

「外膜囊泡」是革蘭氏陰性細菌的外膜自然釋放出的胞外囊泡。此一種奈米顆粒大小的外膜囊泡含有豐富多樣的病原相關分子模式，其中包括格蘭氏陰細菌特有的內毒素「脂多醣」。近年來，許多研究利用外膜囊泡能夠引發先天性和適應性免疫反應的特性，將其廣泛應用在癌症免疫療法之領域。然而，外膜囊泡中作為誘導免疫反應與抑制腫瘤生長的關鍵成分，目前仍需更多詳細研究。在本篇研究中，我們系統地研究了脂多醣免疫原性對外膜囊泡抗腫瘤作用的影響。在我們的研究結果中顯示，脂多醣免疫原性的變化並不會影響外膜囊泡誘導抗原呈現細胞成熟的能力。然而，脂多醣免疫原性對外膜囊泡的抗腫瘤效應有著關鍵作用，而其中可能的原因為影響其招募白血球浸潤到腫瘤之中的能力有關。為了進一步增強對抗腫瘤的免疫反應，我們將外膜囊泡療法與化療藥物奧沙利寶結合使用，預期利用奧沙利寶誘導癌細胞引發免疫原性細胞死亡，並且協同外膜囊泡療法達到增強對抗腫瘤之免疫反應。此外我們嘗試將不同的類鐸受體激動劑表達在外膜囊泡上，預期能進一步增加其引發免疫反應對抗癌症之能力，儘管此策略在本次研究中未能產生預期的效果，但此種成功改造外膜囊泡的方式，仍然保有許多研究發展的可能性。總結，本篇研究揭示了脂多醣之免疫原性對於外膜囊泡建立免疫系統對抗癌症功效的重要性。

Outer membrane vesicles (OMVs) are nano-meter-sized exosomes naturally derived from the outer membrane of Gram-negative bacteria. They contain diverse pathogen-associated molecular patterns (PAMPs), with a particular enrichment of lipopolysaccharides (LPS). Recently, OMV-based applications have gained wide interest as cancer immunotherapy since they have been reported to provoke innate and adaptive immunity against tumors. However, the key component of OMVs that induces cancer immunity still remains largely unknown. In this study, we systemically investigated the impact of LPS immunogenicity on the anti-tumor effects of OMVs. Our results showed that variations in LPS immunogenicity did not affect the ability of OMVs to induce the maturation of antigen-presenting cells (APCs). However, we observed that the immunogenicity of LPS played a crucial role in the anti-tumor effects of OMVs, possibly by influencing the recruitment of tumor-infiltrating leukocytes. To further enhance the immune response against tumors, we combined the OMV-based therapy with the chemotherapeutic drug oxaliplatin, an inducer of immunogenic cell death (ICD), to synergistically boost immunity against the tumor. Moreover, we explored the possibility of enhancing OMV-based immune therapy by engineering additional Toll-like receptor agonists onto OMVs. Although this approach did not yield the desired outcome, the potential of engineered OMV-based therapy with other therapeutic proteins still holds great promise and warrants further investigation. Together, our findings shed light on the role of LPS immunogenicity in the recruitment of tumor-infiltrating leukocytes and its impact on the anti-tumor effects of OMVs. These insights contribute to a better understanding of OMVs as potential immunotherapeutic agents.

誌謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS iv
Chapter 1 Introduction 1
1.1 Outer membrane vesicles (OMVs) 1
1.2 The contribution of OMVs to bacterial survival and pathogenesis 2
1.3 Interactions between OMVs and Toll-like receptors 4
1.3.1 Toll-like receptors 4
1.3.2 PAMPs on OMVs 8
1.4 OMV biomedical applications 10
1.4.1 Bacterial vaccines 10
1.4.2 Vaccine adjuvants 11
1.4.3 Drug delivery vehicles 12
1.4.4 Cancer immunotherapy agents 14
1.5 Double-edge roles in OMVs cancer immunotherapy 15
1.5.1 Pro- and anti-tumor effects of TLR signaling 15
1.5.2 Pros and cons of LPS in cancer immune therapy 17
1.6 LPS-detoxified E. coli strains 18
1.6.1 Attenuated LPS derived from ∆mabB mutants 19
1.6.2 LPS precursor lipid IVa derived from endotoxin-free ClearColi strain 19
1.7 Chemotherapy drug: Oxaliplatin 19
Chapter 2 Materials and Methods 21
2.1 Plasmid construct 21
2.2 Cell line culture 21
2.3 Western blot 22
2.4 OMV purification 22
2.5 OMV characterization (particle size and zeta potential) 23
2.6 SEAP assay 23
2.7 Splenocytes isolation 24
2.8 Antigen-presenting cell maturation assay 24
2.9 Splenocytes proliferation assay 25
2.1 Mouse anti-tumor experiments 26
2.11 Cytometric Bead Array (CBA) assay 26
2.12 Tumor-infiltrating leukocytes (TILs) flow cytometry analysis 27
2.13 IVIS experiment 28
Chapter 3 Results 29
3.1 The characteristics of OMVs secreted from E. coli BL21(DE3) mutants 29
3.2 The OMVs induce the maturation of antigen-presenting cells 32
3.3 The OMVs stimulate T cell clonal expansion ex vivo 34
3.4 The LPS immunogenicity is crucial for anti-tumor effects of OMVs 38
3.5 The LPS immunogenicity of OMVs plays an important role on recruitment of tumor-infiltrating leukocytes 42
3.6 OMV distribution and tumor targeting efficiency in vivo 46
3.7 Approaches for enhancement of OMVs cancer immunotherapy with oxaliplatin 50
3.7.1 The anti-tumor effects of combination therapy with oxaliplatin and OMVs 51
3.7.2 The alteration of tumor-infiltrating leukocytes with oxaliplatin and ∆Lpp OMVs combination therapy 56
3.8 Approaches for anti-tumor effects improvement through engineered the TLR agonists on OMVs 59
3.8.1 Engineered BL21(DE3) ∆Lpp OMVs with flagellin 60
3.8.2 Engineered BL21(DE3) ∆Lpp ∆msbB OMVs carried with flagellin 63
3.8.3 Engineered ClearColi OMVs with flagellin 66
3.8.4 Engineered ClearColi OMVs with bacterial TLR4 agonists 70
Chapter 4 Discussion 73
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