神經膠質母細胞瘤（GBM）是原發型惡性腦腫瘤，在2016年世界衛生組織（WHO）定義為第四級的膠質瘤，目前標準治療神經膠質母細胞瘤病人的方式，是以手術摘除後給予放射線及化療藥物輔助治療。帝盟多（TMZ）是口服烷基化試劑和神經膠質母細胞瘤的標準化療藥劑。帝盟多會甲基化去氧核醣核酸（DNA）並影響其複製機制進而導致細胞死亡。在神經膠質母細胞瘤的治療中會產生帝盟多的抗性。醛脫氫酶1（ALDH1）屬醛脫氫酶酵素家族之一，負責催化細胞內醛的氧化，進而成為神經膠質母細胞瘤治療的主要障礙之一。醛脫氫酶1直接參與神經膠質母細胞瘤治療抗性的產生，並在化療期間受自噬調節。自噬有助於癌細胞克服癌症惡化過程中的壓力。到目前為止，少有論文研究神經膠質母細胞瘤在治療抵抗過程涉及自噬和上皮-間質轉化（EMT）的過程。上皮-間質轉化或所謂的間質分化（MD）過程使上皮細胞轉化為間質細胞。間質分化的可逆性顯示環境信號與侵襲性腫瘤表型有強烈相關性。在化學抗性和惡性進展的過程中，腫瘤微環境有著關鍵作用。最近的研究發現，細胞外基質硬度的提高可獨立促進神經膠質母細胞瘤的侵襲。因此，我們假設當神經膠質母細胞瘤接受微環境的物理環境改變時，ALDH1、自噬和間質分化可能在帝盟多誘導的抗性過程中發生改變。本實驗我們使用了U87MG及1306MG兩種人類神經膠質母細胞瘤細胞株，並探討兩種細胞株的不同。首先，結果發現在具有帝盟多抗性的神經膠質母細胞瘤細胞株中ALDH1、自噬和間質分化都有增強的表現。然後我們建立了兩種不同硬度的聚丙烯酰胺（PA）凝膠來培養親代神經膠質母細胞瘤細胞株和具有帝盟多抗性的神經膠質母細胞瘤細胞株。我們使用原子力顯微鏡（AFM）測量建立的PA凝膠及控制組的玻片，其量測結果硬的凝膠為10.5 kPa、軟的為0.1 kPa及玻片為181.8 kPa。在軟凝膠的柔軟度相當於人類腦組織的軟度，然而在硬凝膠的硬度相當於人類皮膚或肌肉組織。我們觀察到兩種皆具有帝盟多抗性的神經膠質母細胞瘤（TIR-GBM）細胞株在軟凝膠中培養後，細胞形態從紡錘狀變為圓形，並且尺寸變得更小。在硬凝膠中培養發現，具有抗性的U87MG細胞面積皆顯著變小；在具有抗性的1306MG方面，細胞型態則有顯著拉長。最後，我們證明了具有帝盟多抗性的神經膠質母細胞瘤對於環境的反應不同。在親代細胞系中，自噬標記物Beclin和LC3顯示低表達水平，然而，它在抗性細胞系中增加。當TIR-U87MG在軟凝膠上生長時，LC3陽性細胞增加，然而，TIR-1306MG中陽性細胞的數量減少。當TIR細胞系在硬凝膠中生長時，自噬標記物沒有顯著的差異。當具有TMZ抗性的U87MG在軟凝膠中生長時，ALDH1，自噬，間充質標記物 - 纖連蛋白和波形蛋白的螢光強度增強，但在硬凝膠中未發現。當TIR-U87MG在軟凝膠中生長時，在螢光強度ALDH1、自噬、間質標誌-的都有增強的現象，在硬凝膠中則無此現象。此外，我們發現在上皮標誌 β-catenin的螢光強度降低，而Fibronectin和Vimentin則增加。結果顯示當環境變為軟凝膠時，某些細胞表型會發生變化，ALDH1、自噬、間質標誌-Fibronectin和Vimentin的螢光強度在TIR-1306MG呈現下降趨勢，且在硬介質也有一樣的發現。在TIR-1306MG培養在軟凝膠後，β-catenin表現顯著增加，但在硬介質無此現象。這些結果也清楚地表明，當U87MG與1306MG相比時，對培養的微環境表現出不同的反應。總結，當神經膠質母細胞瘤在化學抗性的軟環境或硬環境中生長時，確實產生ALDH1不同的表現並且可能促使或削弱自噬及間質分化的細胞反應。

Glioblastoma (GBM) is the primary malignant brain tumor. In 2016, the World Health Organization (WHO) define GBM as the grade IV glioma. The current standard of care for GBM patients are surgical resection followed by adjuvant radiation therapy and chemotherapy. Temozolomide (TMZ) is the oral alkylating agent and GBM chemotherapy standard agent. TMZ methylates DNA and interferes with replication that results in cell death. Resistance to TMZ is one of the major barrier for GBM therapy. Aldehyde dehydrogenase 1 (ALDH1) is a member of the ALDH enzyme family, which catalyzes the oxidation of intracellular aldehydes. ALDH1 is directly involved in therapy resistance of GBM, and is regulated by autophagy during chemotherapy. Autophagy helps cancer cells to overcome the stressful conditions during cancer progression. Up to data, a few papers show that in the development of therapeutic resistant GBM involve the autophagy and epithelial-mesenchymal transition (EMT) processes. EMT or so called mesenchymal differentiation (MD) process in gliomas that makes epithelial cells transform to mesenchymal cells. The reversibility of MD suggests that environmental signals strongly correlate with aggressive tumor phenotypes. Tumor microenvironment plays important role in the development of chemoresistance and in malignant progression. The recent studies showed that elevated extracellular matrix stiffness independently foster GBM aggression. Thus, we hypothesize that when GBM grows in altered microenvironment with different stiffness, ALDH1, autophagy, and MD maybe altered in parental and TMZ-induced resistant GBM (TIR-GBM) cell lines. In this experiment, we used U87MG and 1306MG human GBM cell lines to develop TIR-GBM. First, we found that autophagy and MD markers were enhanced in TIR-GBM cell lines. Then we established two different stiffness polyacrylamide (PA) gels to culture parental and TIR-GBM cell lines. By using atomic force microscope (AFM) to measure gel stiffness and control glass, we demonstrated that the stiffness of hard matrix was 10.5 kPa, soft PA gel was 0.1 kPa, and glass was 181.8 kPa. The soft matrix softness was similar to human brain tissue, and the hard substrate stiffness was similar to human skin or muscle tissue. We observed that TIR-GBM cell lines cultured on soft substrate, morphology of the cells changed from spindle to round shape and have smaller size. In hard substrate, the cellular area of resistant U87MG became smaller, and length of resistant 1306MG became longer. Finally, we demonstrated that TIR-U87MG showed different responses with microenvironment. In parental cell lines, autophagy markers Beclin and LC3 showed low expression levels, however, it was increased in resistant cell lines. When TIR-U87MG grew on soft gel, LC3 positive cells were increased, however, numbers of positive cells in TIR-1306MG were reduced. There was no significant alternation in autophagy markers when TIR cell lines grew on hard gel. When U87MG with TMZ-resistance was grew on a soft matrix, the fluorescence intensity of ALDH1, autophagy, mesenchymal markers-Fibronectin and Vimentin were enhanced, but it not found in hard gels. On the contrary, epithelium marker β-catenin, fluorescence intensity was reduced. However, Fibronectin and Vimentin were increased. The results indicated when environment changed to soft gel certain cellular phenotypes are altered, such as ALDH1, autophagy, Fibronectin and Vimentin intensity were decreasd in TIR-1306MG. It also found in hard gels. β-catenin was significant increase after TIR-1306MG cultured on soft matrix, but it not found in hard gels. These results also clearly demonstrated that when U87MG compared with 1306MG showed different responses to the cultured microenvironments. In summary, when GBM grow on soft or hard environmental in chemoresistant, it indeed resulted in changes in ALDH1 expression, and may either promote or diminished autophagy and MD processes.

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