Signal transducers and activator of transcription 3 (STAT3)是位於細胞質中的轉錄因子，經由特定的細胞激素活化後會轉入到細胞核內並引起下游訊號傳導，進而使細胞產生和癌化相關的細胞行為。研究顯示STAT3常常被發現活化在腫瘤微環境中的癌症細胞和免疫細胞，在腫瘤邊界幫助癌細胞抵禦免疫反應並且進行逃脫。因此STAT3被認為是潛力的抗癌藥物開發標的，但臨床上尚無STAT3的標靶藥被開發使用。FTY720是一種多發性硬化症的臨床免疫抑制藥物，新進研究發現FTY720也可以透過多重細胞訊息路徑抑制腫瘤細胞生長。我們先前的研究發現，FTY720可以透過抑制STAT3的活化，降低小鼠肺癌形成。因此在本研究中，我們想探討FTY720對腫瘤微環境中的免疫細胞的改變，同時開發FTY720的奈米藥物去提升FTY720的抗癌效果與改善腫瘤的治療。為了探討奈米藥物對腫瘤微環境免疫細胞的影響，我們首先建立了兩種不同模式的異種移植小鼠，分別為將路易斯氏肺癌(Lewis lung carcinoma LLC)細胞株注射到免疫健全小鼠背部皮下的模式以及將細胞株注射到左側大腿皮下的模式。在背部皮下的模式中，奈米藥物以尾靜脈注射，透過血液循環與高滲透長滯留效應(EPR effect)達到腫瘤微環境中；在左側大腿皮下的模式中，奈米藥物透過股動脈注射，不經全身血液循環而直接到達腫瘤微環境。我們比較了兩種異種移植模式中腫瘤微環境的不同與FTY720對腫瘤微環境各種免疫細胞的影響，我們發現巨噬細胞的極化分型(polarization)、殺手T細胞(Cytotoxic T cell)、輔助型T細胞(T-Helper Cell)和自然殺手細胞(Natural killer cell)分佈均受影響，但FTY720單獨使用對治療腫瘤進程的效果有限。因此，我們利用PLGA與FePt兩種不同性質奈米材料，開發新型FTY720奈米藥物作為改善腫瘤免疫微環境和腫瘤進程的策略。我們發現PLGA製程奈米藥物(PLGA-FTY720)有較大流失率，因此後續使用鐵鉑(FePt)奈米顆粒作為FTY720的載體(FePt@FTY720)。初步測定FePt@FTY720在體外細胞的耐受性後，我們進一步比較藥物在奈米化前後對腫瘤微環境免疫細胞的影響。在靜脈與動脈注射給予FTY720奈米藥物的模式中，我們都發現FTY720可以增強腫瘤微環境對FePt奈米材質的吸收。於體內動物實驗和體外細胞實驗中，利用組織染色和流式細胞儀進行免疫細胞定量分析，我們發現FePt@FTY720相較未包埋奈米化FePt的FTY720，對促進腫瘤生長的M2巨噬細胞分化有較佳的抑制效果，而對於抑制腫瘤的M1型巨噬細胞有增加的現象。在動物實驗中，我們也發現促進腫瘤生長的調節性T細胞於FePt@FTY720 處理後24小時開始有下降的趨勢。此外我們也發現體外處理FePt@FTY720奈米藥物對於T淋巴球細胞活化所釋放的干擾素-γ分泌有抑制的效果。在本篇研究中，我們成功合成以鐵鉑奈米顆粒為載體的FTY720藥物，並且建立了適合的異種移植小鼠動物模式，探討奈米藥物對腫瘤微環境免疫細胞改變，研究中我們釐清了FTY720奈米藥物和腫瘤免疫細胞的相關性，此外，我們開發的FePt@FTY720奈米藥物，可以有效減少FTY720對動物體的副作用、提升藥物進入腫瘤的效果與免疫抑制的作用，由於鐵鉑奈米具放射線增敏劑的特性，本研究在FePt@FTY720對免疫細胞的探討，將有助後續FePt@FTY720合併放射線治療或免疫治療對於改善癌症進程的研究。

Signal transducers and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor that is translocated into nucleus upon cytokine stimulation or oncogenic activation. Activated STAT3 is often found in tumor cells and inflammatory cells particularly on the invasive edge of tumors microenvironment, suggesting STAT3 regulates processes that are crucial for tumor progression in the microenvironment. Therefore, STAT3 is considered as a novel cancer drug target. We previously found that inhibition of STAT3 activation by S1PR1 antagonist (FTY720) decreased lung cancer formation in animal model. In this study, we plan to clarify the role of FTY720 on changes of immune cells of tumor microenvironment and develop novel FTY720 nanodrugs to improve cancer treatment. We first established two of the Lewis lung carcinoma (LLC1) cells xenograft tumor models in immunocompetent mice. In subcutaneous flank tumor model, administrated anti-cancer drugs could reach to tumor sites through EPR effects via intravenous injection. And subcutaneous thigh tumor model was established to investigate the effects that anti-cancer drugs could be delivered directly to the tumor site without whole blood circulation. We compared the immune cells composition in the tumor microenvironment of these two models. We found macrophage polarization and T cell population were affected by FTY720 treatment, however, FTY720 treatment alone did not significantly suppress tumor growth in the subcutaneous flank tumor model. Therefore, we then development FTY720 nanodrugs by using PLGA or FePt nanoparticle to improve drug efficacy and cancer treatment. We found PLGA-FTY720 nanodrug was difficult to scale up, therefore, Fept@FTY720 nanodrugs was used for further study. We found that FTY720 could enhance FePt nanoparticle uptake in the tumor microenvironment and M1 macrophage population was elevated by FePt@FTY720 treatment. On the contrary, M2 macrophage population was reduced by FePt@FTY720 treatment, indicating the immune suppression function of FePt@FTY720 in tumor microenvironment. In this study, we have successfully synthesized FePt@FTY720 nanodrugs and established suitable xenograft tumor model for investigating immune cells change by nanodrugs. In this study, we improved drug efficacy of FTY720 on immune suppression function and reduced the FTY720 side effect by FePt nanoparticle. We further clarified the relationship between FePt@FTY720 nanodrug and immune cells changes. Since FePt material has radio sensitization characteristic, combination therapeutic of FePt@FTY720 with radiotherapy or immune therapy were worthy to be investigated.

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