臺灣博碩士論文加值系統

過繼性細胞治療依賴有效的抗原呈現，以啟動並維持T細胞免疫反應。因此，腫瘤來源抗原的製備方式是影響樹突細胞活化及後續免疫反應的重要因素。雖然冷凍-解凍及超音波破碎等傳統方法廣泛應用於腫瘤裂解物的製備，光熱策略則提供了一種替代方法，透過施加可控制的熱刺激，可能保留或增強腫瘤來源材料的免疫原性。
本研究建立並評估了一種以聚乙醇幾丁聚醣-聚吡咯(GCS-Ppy)奈米粒子為基礎的光熱平台，探討其利用E.G7-OVA細胞（一種源自EL4且表現卵清蛋白的腫瘤細胞株）產生具免疫刺激能力之腫瘤裂解物的潛力。GCS-Ppy 奈米粒子展現出適當的物理化學特性，在808 nm近紅外光照射下具有優異的光熱轉換能力，並可與腫瘤來源材料共同被骨髓來源樹突細胞有效攝取。經最佳化光熱處理條件後，溫和加熱（45–48°C）所產生的腫瘤裂解物，相較於高溫處理（>60°C），具有較佳的免疫刺激特性，可更有效促進樹突細胞成熟，並提升成熟標誌及抗原呈現相關分子的表現。
此外，由GCS-Ppy光熱平台所產生的腫瘤裂解物可進一步促進下游T細胞免疫反應，並相較於傳統裂解物製備方法展現較持續性的T細胞增殖能力。雖然傳統裂解物在早期可誘導較強的樹突細胞活化標誌表現，但光熱產生的裂解物則呈現不同的免疫反應時程，其特徵為可維持較長時間的T細胞增殖。綜合而言，本研究結果顯示奈米粒子輔助光熱腫瘤裂解物製備是一項具有潛力的策略，可調控抗原呈現並增強樹突細胞介導的 T 細胞活化，為未來過繼性細胞治療的應用奠定基礎。

Adoptive cell therapy (ACT) relies on effective antigen presentation to initiate and sustain T-cell responses. Therefore, the method used to generate tumor-derived antigens represents an important factor that influences dendritic cell activation and subsequent immune responses. In comparison to conventional lysate generation methods, photothermal strategies provide an alternative approach by introducing controlled thermal stress that may preserve or enhance immunogenic properties of tumor-derived materials.
In this study, a glycol chitosan-polypyrrole (GCS-Ppy)-based photothermal platform was developed and evaluated for its ability to generate immunostimulatory tumor lysates using E.G7-OVA cells, an EL4-derived tumor cell line expressing ovalbumin. GCS-Ppy nanoparticles demonstrated suitable physicochemical characteristics, efficient photothermal conversion under 808 nm near-infrared irradiation, and successful uptake by bone marrow-derived dendritic cells (BMDCs) together with tumor-derived materials. Optimization of photothermal parameters revealed that mild heating conditions (45-48oC) generated tumor lysates with enhanced immunostimulatory properties compared with higher temperature conditions (>60oC), as demonstrated by increased expression of dendritic cell maturation markers and antigen presentation-associated molecules.
Furthermore, GCS-Ppy-generated tumor lysates promoted downstream T-cell responses, demonstrating sustained T- cell expansion compared with conventional lysate preparation methods. Although conventional lysate induced stronger early dendritic cell activation markers, photothermal-generated lysates exhibited a distinct temporal immune response profile characterized by prolonged T-cell expansion. Together, these findings demonstrate that nanoparticle-assisted photothermal tumor lysate generation represents a potential strategy for modulating antigen presentation and improving dendritic cell-mediated T-cell activation, providing a foundation for future applications in adoptive cell therapy.

Table of Contents
Acknowledgements ii
Abstract 1
摘要 2
Table of Contents iii
Table of Figures v
Chapter 1 Introduction 1
1.1. Cancer Immunotherapy and the Role of T Cells 3
1.2. Expanding T Cells 3
1.3. Quality Antigens with Dendritic Cells 4
1.4. Thermal-Generated Lysates 4
1.5. Nanoparticle Platform Design 6
1.6. Experimental Design 7
Chapter 2 Materials and Methods 10
2.1. Reagents and Materials 10
Nanomaterial Synthesis Reagents 10
Cell Culture Reagents and Antibodies 10
Cell Lines and Experimental Animals 10
2.2. Equipment and Instrumentation 11
2.3. Methods 11
Nanoparticle Synthesis and Characterization 11
Dynamic Light Scattering (DLS) 11
UV-Visible Spectroscopy 12
Photothermal Characterization 12
Cell Culture and Viability Assays 12
Nanoparticle Cytotoxicity Assay 12
Immune Cell Isolation and Differentiation 12
Tumor Lysate and Antigen Presentation 13
Dendritic Cell Functional Assays 13
CD8 T Cell Functional Assays 14
Chapter 3 Results 15
3.1. Characterization of GCS-Ppy Nanoparticles 15
3.2. Nanoparticle Cytotoxicity and Target Cell Lysis 15
3.3. Dendritic Cells Phenotypic Selection 16
3.4. Evaluation of E.G7-OVA Lysate Acquisition by BMDCs 17
3.5. Optimization of Photothermal Treatment Temperature for BMDC Activation 18
3.6. Optimization of Photothermal Treatment Duration for BMDC Activation 19
3.7. Comparison of Immunostimulatory Properties of E.G7-OVA Lysates Generated by Different Preparation Methods 20
3.8. T-Cell Culture Optimization 20
3.9. Time-Dependent IFN-γ Production by T Cells Stimulated with Optimized E.G7-OVA Lysates 21
3.10. Time-Dependent Evaluation of T Cell Expansion Following E.G7-OVA Lysate-Loaded BMDC Stimulation 22
Chapter 4 Discussion 23
Chapter 5 Conclusion 25
References 26
Figures 31

Table of Figures
Figure 1. Experimental design for in vivo vaccination and ex vivo antigen-specific CD8+ T cell
expansion............................................................................................................................................... 31
Figure 2. Particle Size Distribution of Glycol Chitosan-Polypyrrole Nanoparticles ............................ 32
Figure 3. Photoconversion potential of GCS-Ppy ................................................................................. 33
Figure 4. Cytotoxicity evaluation of GCS-Ppy nanoparticles ............................................................... 34
Figure 5. Viability of cells following various treatments ...................................................................... 35
Figure 6. Comparison of Flt3L- and GM-CSF generated bone marrow-derived dendritic cells activity
............................................................................................................................................................... 36
Figure 7. BMDC uptakes nanoparticles and lysates.............................................................................. 37
Figure 8. Confocal microscopy analysis of fluorescently labeled E.G7-OVA lysate uptake by BMDCs
............................................................................................................................................................... 38
Figure 9. Evaluation of BMDCs activation following stimulation with E.G7-OVA lysates generated by
different photothermal treatment temperatures ..................................................................................... 39
Figure 10. Evaluation of BMDC activation following stimulation with E.G7-OVA lysates generated by
different photothermal treatment durations ........................................................................................... 40
Figure 11. Evaluation of BMDC activation following stimulation with E.G7-OVA lysates generated by
different preparation methods ............................................................................................................... 41
Figure 12. Morphological observation of splenocyte expansion under different culture conditions .... 42
Figure 13. Time-dependent IFN-γ production by T cells stimulated with E.G7-OVA lysate-loaded
BMDCs ................................................................................................................................................. 43
Figure 14. T Cells Expansion (Day 9) ................................................................................................... 44
Figure 15. T Cells Expansion (Day 12) ................................................................................................. 45

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