大腸癌為全球常見之惡性腫瘤之一，現行治療方式如手術、放射治療及化療雖可有效抑制腫瘤生長，然而腫瘤復發及免疫逃脫仍為臨床上重要挑戰。近年來，誘導免疫系統辨識並清除腫瘤細胞的策略逐漸受到重視，其中免疫原性細胞死亡(immunogenic cell death， ICD)被認為是活化抗腫瘤免疫反應的關鍵機制。本研究延續先前所開發之細菌膜塗層奈米粒子(BM-ICPNPs)，探討其在光熱、光動及化學治療聯合作用下誘導免疫反應之能力。透過近紅外光照射，BM-ICPNPs可促使靛氰綠產生光熱與光動效應，同時促進喜樹鹼進行化學治療，進一步誘導腫瘤細胞死亡。在免疫機制分析方面，本研究針對ICD相關指標進行評估，結果顯示BM-ICPNPs處理後可顯著提升calreticulin(CRT)表現並促進高遷移率族蛋白1(HMGB1)釋放，顯示其具有誘導免疫原性細胞死亡之能力。此外，利用RAW 264.7巨噬細胞進行免疫活化分析，結果顯示BM-ICPNPs可提升誘導型一氧化氮合酶(inducible nitric oxide synthase， iNOS)表現，顯示其可能促進免疫細胞活化並參與後續抗腫瘤免疫反應。另一方面，粒子穩定性分析與細菌膜染色結果證實奈米粒子具有良好結構穩定性及成功包覆細菌質膜；細胞存活分析亦顯示其具備顯著之細胞毒殺效果。未來將進一步利用FITC標記奈米粒子進行吞噬能力分析，以評估BM-ICPNPs誘導免疫細胞活化後，是否能提升免疫細胞對目標物之辨識與吞噬能力。綜合上述結果，BM-ICPNPs不僅具備直接殺傷腫瘤細胞之能力，亦可透過誘導免疫原性細胞死亡及調節免疫細胞活化，促進抗腫瘤免疫相關反應，顯示其於大腸癌後天免疫調控應用上具有潛力，未來有望發展為結合多重治療策略之新型奈米治療平台。;Colorectal cancer is one of the most common malignant diseases worldwide. Current treatments， including surgery， radiotherapy， and chemotherapy， can effectively inhibit tumor growth. However， tumor recurrence and immune evasion remain major clinical challenges. In recent years， strategies that activate the immune system to recognize and eliminate tumor cells have gained increasing attention. Among them， immunogenic cell death (ICD) is considered a key mechanism for triggering anti-tumor immune responses. In this study， we further investigated the previously developed bacterial membrane-coated nanoparticles (BM-ICPNPs) and evaluated their ability to induce immune responses through combined photothermal therapy， photodynamic therapy， and chemotherapy. Upon near-infrared (NIR) irradiation， BM-ICPNPs enable indocyanine green (ICG) to generate photothermal and photodynamic effects， while simultaneously facilitating the chemotherapeutic effect of camptothecin (CPT)， thereby inducing tumor cell death. For the analysis of immune-related mechanisms， ICD-associated markers were evaluated. The results demonstrated that BM-ICPNPs treatment significantly enhanced calreticulin (CRT) expression and promoted the release of high mobility group box 1 (HMGB1)， indicating their potential to induce immunogenic cell death. Furthermore， immune activation analysis using RAW 264.7 macrophages revealed that BM-ICPNPs increased the expression of inducible nitric oxide synthase (iNOS)， suggesting that BM-ICPNPs may promote immune cell activation and contribute to subsequent anti-tumor immune responses. Moreover， particle stability analysis and bacterial membrane staining confirmed the structural stability of the nanoparticles and successful coating of the bacterial plasma membrane. Cell viability analysis further demonstrated the significant cytotoxic effect of BM-ICPNPs toward tumor cells. In future studies， FITC-labeled nanoparticles will be utilized to evaluate phagocytic activity， determining whether BM-ICPNPs-induced immune cell activation can enhance immune cell recognition and phagocytosis of target substances. Taken together， BM-ICPNPs not only exhibit direct tumor-killing ability but also promote anti-tumor immune-related responses through induction of immunogenic cell death and modulation of immune cell activation. These findings suggest that BM-ICPNPs hold potential for colorectal cancer immunomodulatory applications and may serve as a novel nanotherapeutic iii platform integrating multiple therapeutic strategies.
